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Standardization of diagnostic PCR for the detection of foodborne pathogens
Abstract
In vitro amplification of nucleic acids using the polymerase chain reaction (PCR) has become, since its discovery in the 1980s, a powerful diagnostic tool for the analysis of microbial infections as well as for the analysis of microorganisms in food samples. However, despite its potential, PCR has neither gained wide acceptance in routine diagnostics nor been widely incorporated in standardized methods. Lack of validation and standard protocols, as well as variable quality of reagents and equipment, influence the efficient dissemination of PCR methodology from expert research laboratories to end-user laboratories. Moreover, the food industry understandably requires and expects officially approved standards. Recognizing this, in 1999, the European Commission approved the research project, FOOD-PCR (http://www.PCR.dk), which aims to validate and standardize the use of diagnostic PCR for the detection of pathogenic bacteria in foods. The present review focuses on the harmonization procedure and standardization criteria for detection of foodborne pathogens by PCR. The progress of standardization so far and future perspectives of diagnostic PCR are discussed.
... Traditional culture methods have long been considered the most reliable way to isolate and identify foodborne bacterial pathogens [5]. These methods involve several steps, including enrichment, plating, and confirmation through various tests. ...
... However, it can be limited by various factors such as the presence of certain substances in the food matrix [6]. Furthermore, if a pathogen is detected using PCR, traditional confirmation procedures must still be followed [5]. Removing inhibitory chemicals from samples is an important step in preparing them for PCR-based detection of foodborne pathogens. ...
... Regarding the sensitivity of these techniques, some works recognize real-time PCR as the most sensitive technique [14,15] and others the nPCR [16,17]. Multiple factors, such as the selection of the gene to be amplified, the primers used, the nature of the sample, and the DNA extraction method, influence the results of molecular techniques [24]. In this work, nPCR and real-time PCR developed using the same primers (internal primers of nPCR were used for real-time PCR) had the same sensitivity (LOD, 30 plasmid DNA standard copies/reaction). ...
The aim of the present study was to compare the performance of a nested polymerase chain reaction (nPCR) and a real-time PCR based on the amplification of the HlyA gene from Listeria monocytogenes using a plasmid DNA standard. Nested PCR was developed with an internal amplification control (IAC). Both techniques were validated in soft cheese samples by comparing their results with the results of the microbiological reference method ISO 11290–1:2017. Cheese samples artificially contaminated with 3.5 to 3,500 UFC/25 g were processed by ISO 11290–1:2017 and, at several times of culture, DNA samples were extracted. All cheeses contaminated with L. monocytogenes were positive for the microbiological method 96 h post contamination and for nPCR and real-time PCR 48 h post contamination. At this time, the HlyA gene was amplified in all contaminated samples. Both molecular techniques showed the same sensitivity, 30 copies/reaction or 3.5 UFC/25 g, when plasmid DNA standard or artificially contaminated cheese samples were used. Finally, eighty soft cheese samples obtained from local retail stores and tested by three methods were negative, indicating a 100% concordance in results. The development of an nPCR with IAC reinforces the reliability of the negative results without increasing the costs of the reaction. Besides, nPCR showed less sensitivity to the presence of inhibitory substances in the reaction. The use of one of these molecular techniques could be easily coupled to the microbiological method, serving as a screening method in the food industry for hygiene monitoring and early identification of contaminated foods.
... To reduce the risks and control disease progression, a diverse range of methods have been developed for pathogen detection, including culture methods and molecular methods, such as those based on nucleic acid amplification (e.g., polymerase chain reaction (PCR)) [9][10][11] and the enzyme-linked immunosorbent assay (ELISA) [12]. Although they are the most frequently used methods for pathogen detection, they show many disadvantages that prevent efficient health security and sanitary control. ...
The rapid and sensitive detection of pathogenic bacteria is becoming increasingly important for the timely prevention of contamination and the treatment of infections. Biosensors based on nucleic acid aptamers, integrated with optical, electrochemical, and mass-sensitive analytical techniques, have garnered intense interest because of their versatility, cost-efficiency, and ability to exhibit high affinity and specificity in binding bacterial biomarkers, toxins, and whole cells. This review highlights the development of aptamers, their structural characterization, and the chemical modifications enabling optimized recognition properties and enhanced stability in complex biological matrices. Furthermore, recent examples of aptasensors for the detection of bacterial cells, biomarkers, and toxins are discussed. Finally, we explore the barriers to and discuss perspectives on the application of aptamer-based bacterial detection.
... RT-qPCR has proven itself as an useful tool for demonstrating alterations in gene expression in life sciences, agriculture and medicine. In addition to its use in research, many diagnostic applications have been developed, including microbial quantification, gene dosage determination, identification of transgenes in genetically modified foods, risk assessment of cancer recurrence, and applications for forensic use (Schnerr et al., 2001;Ahmed, 2002;Bernard et Wittwer, 2002;Mackay et al., 2002;Auer, 2003;Malorny et al., 2003;Mayer et al., 2003;Petit et al., 2003;Gachon et al., 2004;Bustin et Mueller, 2005;Morling, 2009). Ever since the method was introduced in 1992 ( Higuchi et al., 1992;Higuchi et al., 1993), there has been a strong increase in the number of publications reporting RT-qPCR data, which invariably use diverse reagents, protocols, analysis methods and reporting formats. ...
Gene expression analysis by quantitative reverse transcription polymerase chain reaction (RT-qPCR) allows accurate and sensitive measurment of gene expression levels. However, a series of steps needs to be taken to ensure the relevance, accuracy, correct interpretation and repeatability of the RT-qPCR experiment. We describe here a simple experiment of determining relative gene expression for caspase 8 gene in chicken chondrocytes treated with an apoptosis inducing compound 5-fluorouracil. We use this example to point out some important guidelines in setting up a gene expression study in tissue cells, analyzing and interpreting the results and reporting on the findings.
... To overcome these limitations, molecular, biological, biochemical and immunological techniques have been applied for the rapid and specific detection of L. monocytogenes in food (Manzano et al. 1998; Almeida and Almeida 2000; Solve et al. 2000). Various methods involving DNA probes and polymerase chain reaction (PCR) have been suggested (Scheu et al., 1998; de Boer and Beumer, 1999; Malorny et al., 2003). Direct detection of L. monocytogenes in food samples is a major goal for these methods, but it is difficult to obtain. ...
The detection of Listeria monocytogenes in food samples using enrichment and PCR is described. The objectives were to determine whether the type of enrichment medium has the influence on the results obtained with PCR and to evaluate proposed method by analysing naturally contaminated food samples. Comparative evaluation of different enrichment media for bacteria of the genus Listeria (half Fraser - HF, buffered peptone water - BPW, triptic soy yeast extract broth - TSBYE, universal preenrichment broth - UPB) showed that only the UPB enabled detection of L. monocytogenes in artificially contaminated food samples after 24h of incubation. The PCR-based method gave equal results as standard cultural method by analysis of naturally contaminated food samples. Described PCR-based procedure is comparable to some other PCR-based methods of L. monocytogenes detection in foods and shows promise as a rapid, routine method which requires 30-72 h, whereas cultural methods require 96-120 h.
... Traditional visual monitoring is the most commonly used method for evaluating plant diseases [16]. In addition, molecular detection methods, such as PCR, are also among the standard methods, as they have a high sensitivity and precision [17]. However, both methods come with several disadvantages. ...
Fusarium spp. are important pathogens on cereals, capable of causing considerable yield losses and significantly reducing the quality of harvested grains due to contamination with mycotoxins. The European Union intends to reduce the use of chemical-synthetic plant protection products (csPPP) by up to 50% by the year 2030. To realize this endeavor without significant economic losses for farmers, it is crucial to have both precise early detection of pathogens and effective alternatives for csPPP. To investigate both the early detection of Fusarium head blight (FHB) and the efficacy of selected biological control agents (BCAs), a pot experiment with spring wheat (cv. ‘Servus’) was conducted under semi-field conditions. Spikes were sprayed with different BCAs prior to inoculation with a mixture of F. graminearum and F. culmorum conidia. While early detection of FHB was investigated by hyperspectral imaging (HSI), the efficiency of the fungal (Trichoderma sp. T10, T. harzianum T16, T. asperellum T23 and Clonostachys rosea CRP1104) and bacterial (Bacillus subtilis HG77 and Pseudomonas fluorescens G308) BCAs was assessed by visual monitoring. Evaluation of the hyperspectral images using linear discriminant analysis (LDA) resulted in a pathogen detection nine days post inoculation (dpi) with the pathogen, and thus four days before the first symptoms could be visually detected. Furthermore, support vector machines (SVM) and a combination of LDA and distance classifier (DC) were also able to detect FHB symptoms earlier than manual rating. Scoring the spikes at 13 and 17 dpi with the pathogen showed no significant differences in the FHB incidence among the treatments. Nevertheless, there is a trend suggesting that all BCAs exhibit a diminishing effect against FHB, with fungal isolates demonstrating greater efficacy compared to bacterial ones.
... Classic as well as modernized tools for disease and pathogen surveillance and diagnosis exist [7], but most of these are time consuming, laborious, or are less sensitive. Traditional methods that are based on the identification of a plant disease or pest attack through phenotypical symptoms are simpler to perform but sample preparation and analysis are laborious and fail to produce quantitative results. ...
Agriculture plays a pivotal role in food security and food security is challenged by pests and pathogens. Due to these challenges, the yields and quality of agricultural production are reduced and, in response, restrictions in the trade of plant products are applied. Governments have collaborated to establish robust phytosanitary measures, promote disease surveillance, and invest in research and development to mitigate the impact on food security. Classic as well as modernized tools for disease diagnosis and pathogen surveillance do exist, but most of these are time-consuming, laborious, or are less sensitive. To that end, we propose the innovative application of a hybrid imaging approach through the combination of confocal fluorescence and optoacoustic imaging microscopy. This has allowed us to non-destructively detect the physiological changes that occur in plant tissues as a result of a pathogen-induced interaction well before visual symptoms occur. When broccoli leaves were artificially infected with Xanthomonas campestris pv. campestris (Xcc), eventually causing an economically important bacterial disease, the induced optical absorption alterations could be detected at very early stages of infection. Therefore, this innovative microscopy approach was positively utilized to detect the disease caused by a plant pathogen, showing that it can also be employed to detect quarantine pathogens such as Xylella fastidiosa.
... The use of conventional culture-based methods is inexpensive, however it can lack sensitivity [12], as well as the possibility of false negative results because of the viable but nonculturable state of existence [13]. The Polymerase Chain Reaction (PCR) has proven to be one of the reliable and highly sensitive microbiological methods for the detection and identification of pathogenic bacteria [14]. PCR tests have been developed for the detection of pathogens in water, food and other environmental samples [15]. ...
The use of polluted water for irrigation is prominent in urban and peri-urban farms in Meknes (Morocco). However, this water may contain many pathogens that cause serious infections. Consumption of contaminated vegetables could be a significant public health problem. Salmonella is a bacteria responsible for multiple body infections, including gastroenteritis, fevers, and more severe symptoms. This study aims to determine the prevalence of Salmonella in urban and periurban farms of Meknes. A total of seventy samples (17 Water, 11 soil, and 42 vegetables) were collected and analyzed using a combination of conventional and molecular methods. The results showed that irrigation water was the most contaminated with Salmonella, followed by soil samples (45.5%) and vegetables (16.6%). This study shows a high prevalence of Salmonella in irrigation water, which can be a source of contamination of crops. It raises public health concerns as farmers and consumers are at risk of infection.
... There are many biorecognition components to choose from, from antibodies to aptamers to imprinted polymers [12][13][14]. Enzyme-linked immunosorbent assay (ELISA) [15] and polymerase chain reaction (PCR) [16,17] have been extensively studied for the detection of infections. ...
Electrochemical biosensors are a family of biosensors that use an electrochemical transducer to perform their functions. In recent decades, many electrochemical biosensors have been created for pathogen detection. These biosensors for detecting infections have been comprehensively studied in terms of transduction elements, biorecognition components, and electrochemical methods. This review discusses the biorecognition components that may be used to identify pathogens. These include antibodies and aptamers. The integration of transducers and electrode changes in biosensor design is a major discussion topic. Pathogen detection methods can be categorized by sample preparation and secondary binding processes. Diagnostics in medicine, environmental monitoring, and biothreat detection can benefit from electrochemical biosensors to ensure food and water safety. Disposable and reusable biosensors for process monitoring, as well as multiplexed and conformal pathogen detection, are all included in this review. It is now possible to identify a wide range of diseases using biosensors that may be applied to food, bodily fluids, and even objects’ surfaces. The sensitivity of optical techniques may be superior to electrochemical approaches, but optical methods are prohibitively expensive and challenging for most end users to utilize. On the other hand, electrochemical approaches are simpler to use, but their efficacy in identifying infections is still far from satisfactory.
... There are now three distinct molecular tests that have been used. All of them are based on either proteins or nucleic acids: polymerase chain reaction (PCR), Enzymelinked immunosorbent assay (ELISA), and other traditional methods like colony counting, fluorescence in situ hybridization (FISH), and flow cytometric detection (F.C.M.) immunologybased approach are used to assess apparatus for pathogen detection [7,8]. However, these methods are costly, time-consuming, and labor-intensive, hindering the early stage, cost-effective and rapid pathogen diagnosis necessary for diversified crops. ...
Plant pathogens massively affect crop productivity and are one of the significant challenges in attaining sustainable development goals related to agriculture, food production and addressing hunger issues. Conventional techniques of generic seasonal chemical spraying severely damage the environment and human health. On the contrary, nanomaterials-based biosensors have emerged as economical, efficient, selective, prompt and precise strategies for plant pathogen and disease diagnosis. The integration of nano-biosensors with artificial intelligence, internet-of-things, cloud computing, drones, and 5G communication has recently raised the paradigm of internet-of-nano-things inspired intelligent plant-diagnostic biosensors. This prospect highlights these modern-age plant-pathogenbiosensors for shaping smart and 5th-generation agricultural practice
... In order to confirm that the isolated strains belong to the genus Salmonella, partial amplification by PCR and subsequent sequencing of the 16S rRNA gene were performed, a technique that has greatly facilitated the identification of bacteria, especially in the case of fastidious pathogens (Kolbert and Persing 1999;Stackebrandt et al. 1992) A second test was carried out to confirm the Salmonella genus by detecting the invA gene by PCR, commonly being used to detect Salmonella due to its known role in the invasion process (Malorny et al. 2003). ...
Salmonellosis is one of the most common foodborne illnesses in the world. The irrational use of antibiotics in medicine and in animal nutrition has greatly favored the emergence and spread of resistant strains of non-typhoid Salmonella. This study aims the determination of the prevalence of Salmonella in bivalve mollusks in Northern Morocco, as well as the molecular typing and antibiotic susceptibility testing of the strains isolated from positive samples. In total, 150 samples from shellfish composed of mussels (Mytilus galloprovincialis), clams (Callista chione and Ruditapes descussatus) and oysters (Magallana gigas). Isolated Salmonella were characterized by Molecular techniques PCR, MLST and MLVA, phylogenetically grouped by MLSA, and susceptibilities were determined for 30 antimicrobial drugs using microdilution method by the BD Phoenix Automated Microbiology System. Prevalence of Salmonella enterica subsp. enterica was 12.67%, grouped in four serovars identified as Chester, Hadar, Typhimurium and Kentucky. Five different MLST STs (sequence types) were detected, ST1954 being the most common, which was mostly found in Chester isolates. Forty-two percent of the isolates showed resistance to more than one antibiotic, especially trimethoprim, sulfa drugs, quinolones and β-lactam. There was a marked change in the serovars and antimicrobial resistance profiles of the Salmonella isolates in this study compared to those in previous studies.
... Conventional methods for bacterial pathogens detection in foods are generally based on identification of bacteria using selective culture media by their morphological, biochemical and immunological characteristics [45]. Polymerase Chain Reaction (PCR) based methods have been identified as a powerful diagnostic tool for the detection of pathogenic microorganisms [30]. Therefore, the objective of the current study was to determine the level of salmonella and E.coli contamination in meat and poultry meat cuts by convention method and PCR technique. ...
A total of 100 random samples of chicken (thigh and breast) and red meat cuts (mutton and beef shoulders) were collected from different poulterer’s and butcher’s shops at Cairo, El- Kalyobia and El-Gharbia governorates to detect level of Salmonella and E.coli contamination. The obtained results indicated that salmonella organisms were isolated from the examined samples of chicken thigh, chicken breast, mutton and beef with percentages of 16%, 16%, 8% and 8% respectively. Moreover, the isolated Salmonellae could be serologically identified as S. Typhimurium (28%), S. Enteritidis (16%) and S. Haifa (4%). On the other hand, the percentages of isolated E. coli from the examined samples of chicken thigh, chicken breast, mutton and beef were 16%, 12%, 28%and 12% respectively. Moreover, the results cleared that PCR is an ideal method for identification of Salmonella spp. as it was effective, less labor and more sensitive as well as reduces effort and time. Out of 10 strains of different serotypes of Salmonella isolated from chicken (thigh and breast), mutton and beef by traditional method, 4 strains were positive in m-PCR for Salmonella from which, one strain was identified as S. Typhimurium. As well as out of 10 strains of different serotypes of E .coli isolated from chicken (thigh and breast), mutton and beef shoulders, 2 strains were positive in m-PCR. E.coli O55: K59 (B5) and E.coli O119: K69 (B14) isolated from thigh and breast, respectively, which were positive for elt gene (labile toxin).
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... Conventional methods for bacterial pathogens detection in foods are generally based on identification of bacteria using selective culture media by their morphological, biochemical and immunological characteristics [45]. Polymerase Chain Reaction (PCR) based methods have been identified as a powerful diagnostic tool for the detection of pathogenic microorganisms [30]. Therefore, the objective of the current study was to determine the level of salmonella and E.coli contamination in meat and poultry meat cuts by convention method and PCR technique. ...
A total of 100 random samples of chicken (thigh and breast) and red meat cuts (mutton and beef shoulders) were collected from different poulterer’s and butcher’s shops at Cairo, El- Kalyobia and El-Gharbia governorates to detect level of Salmonella and E.coli contamination. The obtained results indicated that salmonella organisms were isolated from the examined samples of chicken thigh, chicken breast, mutton and beef with percentages of 16%, 16%, 8% and 8% respectively. Moreover, the isolated Salmonellae could be serologically identified as S. Typhimurium (28%), S. Enteritidis (16%) and S. Haifa (4%). On the other hand, the percentages of isolated E. coli from the examined samples of chicken thigh, chicken breast, mutton and beef were 16%, 12%, 28%and 12% respectively. Moreover, the results cleared that PCR is an ideal method for identification of Salmonella spp. as it was effective, less labor and more sensitive as well as reduces effort and time. Out of 10 strains of different serotypes of Salmonella isolated from chicken (thigh and breast), mutton and beef by traditional method, 4 strains were positive in m-PCR for Salmonella from which, one strain was identified as S. Typhimurium. As well as out of 10 strains of different serotypes of E .coli isolated from chicken (thigh and breast), mutton and beef shoulders, 2 strains were positive in m-PCR. E.coli O55: K59 (B5) and E.coli O119: K69 (B14) isolated from thigh and breast, respectively, which were positive for elt gene (labile toxin).
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... In vitro amplification of DNA using PCR has become a powerful and fast analytical tool for the analysis of microbes in food samples (Malorny et al., 2003). The emergence of PCR has revolutionized the process of microbiological analyses toward the identification of the targeted microbial DNA. ...
Introduction:
The number of severe food offenses being perpetrated by a group of organized criminals has been an increasing concern for food safety and environmental forensic professionals all over the world. Food adulteration is a significant concern globally because it is directly involved with consumers’ health, lifestyle, religion, and economic issues. Food adulteration, either intentional or accidental, is a common type of food fraud affecting consumers as well as all stakeholders involved in food production and supply chain worldwide. However, the majority of the incidences remain undetected and unreported, as most of them do not cause direct health harm. The food fraud has been estimated to cause an annual financial loss of more than $40 billion to the food industry (Bo¨hme et al., 2019). Authentication of food ingredients to detect substitution or adulteration either entirely or partially by a cheaper one and sometimes by a harmful component is significant food safety and quality issues. Literature reviews reflect that food forgery is a common phenomenon all over the world. Over the last years, mislabeling of meat products was found to be 78.3% in
Malaysia, 37% in Europe (Walker et al., 2013), 22% in Turkey (Ayaz et al., 2006), 19.4% in the United States, 15% in Switzerland (Ali et al., 2014), (Hsieh et al., 1995), and 8% in the United Kingdom (Chuah et al., 2016). Moreover, chemically treated meat is often found in big markets worldwide, such as China, where chemically treated rat meat was sold as a lamb (Ahamad et al., 2017) or chemically treated pork sold as beef (Tan, 2013). Consumers are therefore increasingly concerned
regarding food safety since, in addition to religious belief, some animal species might also pose potential threats due to zoonotic diseases. Legislation has been introduced
worldwide to ensure the authenticity of the food products all over the distributionchain and thus eliminating food fraud and adulteration as well as mislabeling (Bo¨hme et al., 2019). The US Food and Drug Administration has introduced a rule that requires that food companies develop mitigation plans for protecting food against intentional adulteration under the Food Safety Modernization Act. This rule aims at minimizing intentional adulteration and protects consumers’ rights, religious beliefs, and public health (Mermelstein, 2018). For complying with these regulations, there is an urgent need for reliable and sensitive analytical tools that will verify and ensure all the ingredients included in a food product so that consumers may be assured that the qualities and features match the declaration in labels. Numerous food analytical methodologies have been applied in food forensic science for authentication of food products, including protein and DNA-based techniques.
Among all these detection methods, researchers mainly focus on the DNA-based polymerase chain reaction (PCR) assays because of high specificity, sensitivity, reliability, and reproducibility. The DNA-based methods are useful tools in all scientific domains as they utilize the unique characteristics of DNA molecule such as codon degeneracy,
thermal stability, abundance in most cells as well as intraspecies conservation and interspecies polymorphism (Ali et al., 2014; Mafra et al., 2008). The stability of biomarkers throughout animal species detection is a critical factor for the success of the procedure, particularly meat products that have been processed and exposed in
extremely high temperatures during their treatment. Unlike protein-based methods, which are unsuitable, as proteins denature under high heat, DNA biomarkers exhibit higher stability under different processing conditions (Mane et al., 2012). Furthermore, due to the presence of multiple DNA copies in the cell, a low quantity of sample is adequate for species detection in DNA-based methods (Gupta et al., 2011). Also, DNA carries more information than proteins due to the degeneracy of the genetic code and the existence of large noncoding regions (Pereira et al., 2008). DNA-based methods are therefore advantageous over other detection techniques as they provide specificity, rapidity, lower sensitivity, and cost-effectiveness. Likewise, the challenges posed to public health and food safety by foodborne
pathogens cannot always be encountered with the use of culture-based methods. There is an increased demand for quick, accurate, and sensitive, methodologies for pathogen detection in foods. DNA-based molecular assays have demonstrated considerable advantages in this field in terms of sensitivity, specificity, and speed. This chapter aims at providing an overview of current DNA-based techniques applicable in food forensic sciences to identify animal species origin as well as detection of foodborne pathogens focusing on their merits and limitations.
... Diagnostic specificity is defined as a measure of the degree to which the method is affected by nontarget components present in a sample, which may result in false positive responses. Diagnostic sensitivity is defined as a measure of the degree to detect the target pathogen in the sample, which may result in false negative responses (Malorny et al., 2003). Too low sensitivity often leads to false negatives. ...
Every year huge crop losses occur due to different pathogens and disease. The traditional method of pathogen detection, which is still in practice, through visual examination is not always precise. Early detection of plant pathogens prior to severe infection is very crucial which is possible through molecular diagnostic approaches and nucleic acid-based tests. As the genetic materials are the ultimate information storage sites in living organism, their exploration through the use of nanotechnology provides the path forward for the three Ds of genomic analysis of pathogens: Diversity, Detection, and Disease diagnosis. Molecular detection method is not only precise and accurate but also faster and easier approach. Pathogen detection through PCR based tests, microarray technology, multiplexing, gene sequencing, genetic markers play a pivotal role in timely detection of causatives and take proper action to prevent the pandemic in plant population and safeguard against possible risks and famine. It is of utmost importance to prioritize such methods to detect plant pathogens, to increase our understanding of ecology and epidemiology and to prevent the spread of inoculum prior to disease spread. The application of novel diagnostic methods to inoculum detection will guide towards better understanding of the temporal and spatial dynamics of epidemic development, and open up new opportunities for disease forecasting and management.
... As a result, biorecognition elements widely vary, including antibodies, aptamers, and imprinted polymers. Several comprehensive reviews have been written on pathogen detection using high-throughput, well plate-based bioanalytical techniques (Alahi and Mukhopadhyay 2017;Lazcka et al. 2007;Zourob et al. 2008), such as enzyme-linked immunosorbent assay (ELISA) (Law et al. 2015) and polymerase chain reaction (PCR) (Klein 2002;Malorny et al. 2003), which remain the gold standards for pathogen detection. Few reviews, however, have focused on emerging label-free biosensors for pathogen detection, which provide useful characteristics for applications in process monitoring, environmental monitoring, and precision agriculture. ...
Recent advances in electrochemical biosensors for pathogen detection are reviewed. Electrochemical biosensors for pathogen detection are broadly reviewed in terms of transduction elements, biorecognition elements, electrochemical techniques, and sensor performance. Transduction elements are discussed in terms of electrode material and form factor. Biorecognition elements for pathogen detection, including antibodies, aptamers, and imprinted polymers, are discussed in terms of availability, production, and immobilization approach. Emerging areas of electrochemical biosensor design are reviewed, including electrode modification and transducer integration. Measurement formats for pathogen detection are classified in terms of sample preparation and secondary binding steps. Applications of electrochemical biosensors for the detection of pathogens in food and water safety, medical diagnostics, environmental monitoring, and bio-threat applications are highlighted. Future directions and challenges of electrochemical biosensors for pathogen detection are discussed, including wearable and conformal biosensors, detection of plant pathogens, multiplexed detection, reusable sensors for process monitoring applications, and low-cost, disposable biosensors.
... Of the numerous serotypes of E. coli that have been shown to produce Shiga toxin (Stx), E. coli 0157:H7 and E. coli 0157:NM (non-motile) are most frequently implicated in human disease. Polymerase Chain Reaction (PCR) based methods have been identified as a powerful diagnostic tool for the detection of pathogenic microorganisms (Malorny et al., 2003). Compared to other methods of detection, these methods are rapid, highly specific and sensitive in the identification of target organisms (Wang et al., 2007).PCR can be applied on fixed tissues (frozen or formalin fixed) reducing the potential dangers involved in transport and handling of specimens with live virulent pathogens (Reinoso et al., 2004). ...
... Polymerase Chain Reaction (PCR) based methods were identified as a powerful diagnostic tool for the detection of pathogenic microorganisms (Malorny et al., 2003). Compared to other methods of detection, these methods were rapid, highly specific and sensitive in the identification of target organisms (Wang et al., 2007). ...
... Conventional methods for bacterial pathogens detection in foods are generally based on identification of bacteria using selective culture media by their morphological, biochemical and immunological characteristics [45]. Polymerase Chain Reaction (PCR) based methods have been identified as a powerful diagnostic tool for the detection of pathogenic microorganisms [30]. Therefore, the objective of the current study was to determine the level of salmonella and E.coli contamination in meat and poultry meat cuts by convention method and PCR technique. ...
A total of 100 random samples of chicken (thigh and breast) and red meat cuts (mutton and beef shoulders) were collected from different poulterer's and butcher's shops at Cairo, El-Kalyobia and El-Gharbia governorates to detect level of Salmonella and E.coli contamination. The obtained results indicated that salmonella organisms were isolated from the examined samples of chicken thigh, chicken breast, mutton and beef with percentages of 16%, 16%, 8% and 8% respectively. Moreover, the isolated Salmonellae could be serologically identified as S. Typhimurium (28%), S. Enteritidis (16%) and S. Haifa (4%). On the other hand, the percentages of isolated E. coli from the examined samples of chicken thigh, chicken breast, mutton and beef were 16%, 12%, 28%and 12% respectively. Moreover, the results cleared that PCR is an ideal method for identification of Salmonella spp. as it was effective, less labor and more sensitive as well as reduces effort and time. Out of 10 strains of different serotypes of Salmonella isolated from chicken (thigh and breast), mutton and beef by traditional method, 4 strains were positive in m-PCR for Salmonella from which, one strain was identified as S. Typhimurium. As well as out of 10 strains of different serotypes of E .coli isolated from chicken (thigh and breast), mutton and beef shoulders, 2 strains were positive in m-PCR. E.coli O 55 : K 59 (B 5) and E.coli O 119 : K 69 (B 14) isolated from thigh and breast, respectively, which were positive for elt gene (labile toxin).
... Identifying such viruses in food samples is technically challenging because of the low genomic copy numbers (10 2 -10 6 ) or virus particles per gram of the contaminated food samples (Polo et al. 2015) as well as the effects of complex food sample matrices and inhibitors (Malorny et al. 2003;Mikel et al. 2016;Niesters 2002;Stals et al. 2012). Therefore, sensitive, specific, rapid, high-throughput, and reliable molecular assays, such as reverse transcription polymerase chain reaction (RT-PCR) or real-time RT-PCR, have been widely adopted in the surveillance of foodborne viruses (Zhang et al. 2015); however, potential for false-negative results remains a concern with any molecular assay. ...
Foodborne viruses are a global threat to food safety. Real-time reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used method to detect viral RNA in food. Armored RNA (AR) prepared using the MS2 phage system is a successful positive control for detecting foodborne viruses and is an important quality control process when using real-time RT-PCR. In this study, we report a novel technology for preparing AR using bacteriophage Qβ and compare its stability with AR prepared using the MS2 phage system for packaging norovirus detection target RNA. AR could be successfully and efficiently produced using the developed bacteriophage Qβ system. Two types of AR–AR-QNoV prepared using the Qβ system and AR-MNoV prepared using the MS2 system—were stored at different temperatures for different durations. After incubating at − 20 °C for 360 days, the copy numbers of AR-QNoV and AR-MNoV decreased by 8.9% and 35.9%, respectively. After incubating at 4 °C for 60 days, the copy numbers of AR-QNoV and AR-MNoV decreased by 12.0% and 38.9%, respectively. After incubating at 45 °C, the copy numbers of AR-QNoV decreased by 71.8% after 5 days, whereas those of AR-MNoV decreased by 92.9% after only 4 days. After 5 days, AR-MNoV could not be detected using real-time RT-PCR. There was a significant difference in copy numbers decrease rate between AR-QNoV and AR-MNoV at three different temperatures (P < 0.05 ). Therefore, AR prepared using the new bacteriophage Qβ system is more stable than the traditional AR, making the developed strategy a good candidate for AR preparation and quality control.
... Routine identification and enumeration of E. coli in food sample by traditional methods are laborious and time consuming, and sometimes may lead to unpredicted results (Malorny et al. 2003). The PCR-based detection methods have become the valuable and effective tool to detect pathogenic bacteria in different food matrices (Hines 2000) because of their rapid and accurate results. ...
Purpose
Numerous outbreaks of foodborne diseases through fresh agricultural produce urge research to assess the source of entry of pathogens to the produce that compromise microbiological safety. In the present investigation, the entry of shiga-like toxin-producing Escherichia coli O157:H7 in a fresh vegetable production system was assessed by microbiological and molecular approaches.
Methods
Five major vegetables, viz., beetroot, cabbage, carrot, onion, parsley, and potato, being cultivated routinely in the Western Guats of South India (The Nilgiris), were assessed for the prevalence of E. coli O157:H7. The fresh produce, rhizosphere soil, and water resources were sampled and the total coliforms and E. coli counts were assessed by plate count method and the O157:H7 by polymerase chain reaction targeting shiga-like toxin gene (stx1).
Results
The results revealed that all the vegetables collected from the fields had high levels of total coliforms (3 log CFU per g) with high proportions of E. coli (1–2 log CFU per g). The prevalence of O157:H7 among the E. coli isolates in these vegetables ranged from 0 to 5.8%. However, the prevalence of O157:H7 in rhizosphere soil of these vegetables was relatively high (1.6 to 42.5%). The water used for irrigation and washing the produce (carrot) also showed the presence of O157:H7. The real-time quantitative PCR (qPCR)–based detection of stx1 revealed that the O157:H7 prevalence in these vegetables and their rhizosphere soil were in higher magnitude than the counts by culturable method. The rhizosphere soil and water samples had higher O157:H7 CFU equivalents than fresh produce.
Conclusions
It is evident that the soil as well as the irrigation and process water got contaminated with feces, which are assumed to be the primary source and cause for the entry of O157:H7 to the fresh vegetable. Hence, good agronomical practices and good hygiene post-harvest practices have to be imposed in the vegetable production system to avoid the pathogen entry.
... Culture-based methods and PCR-based methods are the main methods of Salmonella detection at present (Bhutta 2006;Malorny et al. 2004;Malorny et al. 2003). Testing based on culturing generally takes several days and relies on experienced operators. ...
Salmonella is a type of serious foodborne pathogenic bacterium that threatens the food safety worldwide. In this study, a fluorescence probe-based recombinase polymerase amplification analytical method (exo RPA) was developed for the sensitive and specific detection of Salmonella enterica (S. enterica). The primers and probe for this method were designed based on the iroB gene of the bacterium. The exo RPA reaction could detect as low as 10² copies of template DNA per reaction or 2.2 × 10³ CFU/mL of S. enterica, and SPSS probit regression analysis indicated the limit of detection (LOD) at 95% probability of the exo RPA assay was 2.0 × 10³ CFU/mL. Moreover, the exo RPA assay could successfully differentiate S. enterica from all the tested non-Salmonella strains. Importantly, this method is highly reliable; the DNA from other bacteria or food complex matrices has no obvious effect on the S. enterica exo RPA. Besides, positive signal could be obtained from the chicken or egg sample inoculated with 2.2 × 10¹ CFU/mL of S. enterica after 4- to 6-h enrichment at 36 °C. Therefore, this study describes a rapid test method for S. enterica, which is expected to be beneficial for monitoring trace S. enterica in real food products.
... Also, environmental factors are associated with variation in gene expression of microorganisms that might affect the results of biochemical tests. Furthermore, feasible but non-cultivable cells are not detected through conventional culture procedures (MALORNY & al. [20]). More rapid techniques have been developed for recognising Salmonella in foods such as electrical methods, nucleic acid probes and immunoassays (JENÍKOVÁ & al. [21]), but there are still problems with their sensitivity and specificity. ...
A study was conducted to investigate the prevalence of Salmonella spp. in processed raw frozen and fresh poultry meat sold in retail markets and shops of Quetta, Pakistan. A total of 200 samples (100 each of fresh and frozen meat samples) were randomly collected
from retailers during the period of March to September 2016. Out of 200 samples tested, 66 (33%) were found to be contaminated with Salmonella spp. The contamination rate of frozen samples was 30% as compared to 36% of fresh meat samples. Out of 66 Salmonella
positive samples, the most prevalent sero-groups identified were S. enteritidis (43.9%) followed by S. typhimurium (30.3%), S. gallinarum (16.6%), S. pullorum (7.6%) and S. typhi (1.5%). Our findings highlight the magnitude of Salmonella contamination in chicken meat sold in the city of Quetta and demonstrate the efficacy of biochemical characterization and typing through multiplex PCR. The results indicate an urgent need for applying proper food hygiene practices in the Quetta region to reduce the incidence of foodborne diseases.
... Conventional methods for bacterial pathogens detection in foods are generally based on identification of bacteria using selective culture media by their morphological, biochemical and immunological characteristics [45]. Polymerase Chain Reaction (PCR) based methods have been identified as a powerful diagnostic tool for the detection of pathogenic microorganisms [30]. Therefore, the objective of the current study was to determine the level of salmonella and E.coli contamination in meat and poultry meat cuts by convention method and PCR technique. ...
A total of 100 random samples of chicken (thigh and breast) and red meat cuts (mutton and beef shoulders) were collected from different poulterer's and butcher's shops at Cairo, El-Kalyobia and El-Gharbia governorates to detect level of Salmonella and E.coli contamination. The obtained results indicated that salmonella organisms were isolated from the examined samples of chicken thigh, chicken breast, mutton and beef with percentages of 16%, 16%, 8% and 8% respectively. Moreover, the isolated Salmonellae could be serologically identified as S. Typhimurium (28%), S. Enteritidis (16%) and S. Haifa (4%). On the other hand, the percentages of isolated E. coli from the examined samples of chicken thigh, chicken breast, mutton and beef were 16%, 12%, 28%and 12% respectively. Moreover, the results cleared that PCR is an ideal method for identification of Salmonella spp. as it was effective, less labor and more sensitive as well as reduces effort and time. Out of 10 strains of different serotypes of Salmonella isolated from chicken (thigh and breast), mutton and beef by traditional method, 4 strains were positive in m-PCR for Salmonella from which, one strain was identified as S. Typhimurium. As well as out of 10 strains of different serotypes of E .coli isolated from chicken (thigh and breast), mutton and beef shoulders, 2 strains were positive in m-PCR. E.coli O 55 : K 59 (B 5) and E.coli O 119 : K 69 (B 14) isolated from thigh and breast, respectively, which were positive for elt gene (labile toxin).
Background and Aims
Nosocomial infections caused by Salmonella spp. are common in veterinary facilities. The early identification of high-risk patients and sources of infection is important for mitigating the spread of infections to animal patients and humans. This study investigated the occurrence of Salmonella spp. among patients at a veterinary academic hospital in South Africa. In addition, this study describes the environmental factors that contribute to the spread of Salmonella spp. in the veterinary facility.
Materials and Methods
This study used a dataset of Salmonella-positive animals and environmental samples submitted to the bacteriology laboratory between 2012 and 2019. The occurrence of Salmonella isolates at the veterinary hospital was described based on source, month, season, year, and location. Proportions and 95% confidence intervals were calculated for each variable.
Results
A total of 715 Salmonella isolates were recorded, of which 67.6% (483/715) came from animals and the remainder (32.4%, 232/715) came from environmental samples. The highest proportion (29.2%) of Salmonella isolates was recorded in 2016 and most isolates were reported in November (17.4%). The winter season had the lowest (14.6%) proportion of isolates reported compared to spring (31.3%), summer (27.8%), and autumn (26.4%). Salmonella Typhimurium (20.0%) was the most frequently reported serotype among the samples tested, followed by Salmonella Anatum (11.2%). Among the positive animal cases, most (86.3%) came from equine clinics. Most reported isolates differed based on animal species with S. Typhimurium being common in equines and S. Anatum in bovines.
Conclusion
In this study, S. Typhimurium emerged as the predominant strain in animal and environmental samples. Equines were the most affected animals; however, Salmonella serotypes were also detected in the production animals. Environmental contamination was also a major source of Salmonella species in this study. To reduce the risk of transmission, strict infection prevention and control measures (biosecurity) must be implemented.
Major health events caused by pathogenic microorganisms are increasing, seriously jeopardizing human lives. Currently PCR and ITA are widely used for rapid testing in food, medicine, industry and agriculture. However, due to the non-specificity of the amplification process, researchers have proposed the combination of nucleic acid amplification technology with the novel technology CRISPR for detection, which improves the specificity and credibility of results. This paper summarizes the research progress of nucleic acid amplification technology in conjunction with CRISPR/Cas technology for the detection of pathogens, which provides a reference and theoretical basis for the subsequent application of nucleic acid amplification technology in the field of pathogen detection.
Salmoneloza in listerioza sta alimentarni infekciji zato so hitre in zanesljive metode določanja bakterij rodu Salmonella in vrste Listeria monocytogenes pogoj za zagotavljanje varne hrane. Metode na osnovi PCR imajo teoretične prednosti v primerjavi s tradicionalnimi mikrobiološkimi metodami, vendar morajo biti pred rutinsko uporabo ustrezno vrednotene. V prispevku je predstavljeno vrednotenje metode PCR za sočasno določitev bakterij rodu Salmonella in vrste L. monocytogenes v živilih kot primer validacije alternativne kvalitativne metode glede na referenčne metode. Ustrezna izvedba metode PCR je hitrejša od standardnih mikrobioloških metod, ima enako relativno točnost, relativno občutljivost in relativno specifičnost ter pomeni dobro osnovo za nadaljnje medlaboratorijsko vrednotenje.
A total number of 100 samples from ten random broiler chicken carcasses (breast and thigh) were collected from an automatic poultry slaughtering plant in Ismailia city, Egypt. The mean values of Enterobacteriacae count were 5.9x104±9.7x103 cfu/g and 7.1x 104 ± 1.1x104 cfu/g for chicken breast and thigh samples respectively. The prevalence of E.coli were 12% and 9% breast and thigh samples examined, respectively. They are serologically identified as 33.35 and 22.2% O 157 :H 7 (EHEC) , 16.6% and 11.1% O114:H21 (EPEC), 16.6% and 33.3 %O127:H6 (ETEC) , 0% and 0% O126 (ETEC) and 33.3% and 0% O26 (EHEC) for breast and thigh samples, respectively. The incidence of E.coli O 157 :H 7 was 100% in both serological and PCR methods from biochemical positive E.coli samples. Culture is specific and cheap whereas PCR is sensitive and expensive, hence, we recommend both culture and molecular methods, which improve sensitivity and specificity, to enhance detection of foodborne pathogens including E.coli.
A total number of 100 samples from ten random broiler chicken carcasses (breast and thigh) were collected from an automatic poultry slaughtering plant in Ismailia city, Egypt. The mean values of Enterobacteriacae count were 5.9x104±9.7x103 cfu/g and 7.1x 104 ± 1.1x104 cfu/g for chicken breast and thigh samples respectively. The prevalence of E.coli were 12% and 9% breast and thigh samples examined, respectively. They are serologically identified as 33.35 and 22.2% O 157 :H 7 (EHEC) , 16.6% and 11.1% O114:H21 (EPEC), 16.6% and 33.3 %O127:H6 (ETEC) , 0% and 0% O126 (ETEC) and 33.3% and 0% O26 (EHEC) for breast and thigh samples, respectively. The incidence of E.coli O 157 :H 7 was 100% in both serological and PCR methods from biochemical positive E.coli samples. Culture is specific and cheap whereas PCR is sensitive and expensive, hence, we recommend both culture and molecular methods, which improve sensitivity and specificity, to enhance detection of foodborne pathogens including E.coli.
Parasitic diseases are among neglected disease of human and animals, especially in tropical and sub tropical regions. In the era of artificial intelligence, the novel biosensing diagnostic platform is needed for an early control measure implementation. This goal can be successfully achieved by onsite application of electrochemical biosensors. They are being developed towards point of care diagnostics; however commercial availability is scanty. The recent developments during last one decade in terms of the electrode surface modification for rapid diagnosis of important emerging/re‐emerging parasites is presented. The information would help future improvement in the electrochemical biosensing of parasites.
Background:
Salmonella in chicken, specially, the motile salmonellae, causes the food chain unsafe from farm to table and is considered a significant global threat to public health.
Aims:
The present study was carried out for molecular detection of Salmonellae in commercial poultry using PCR.
Methods:
The study was conducted for eight months, from July 2019 to February 2020, and a total of 26 poultry farms, including 15 broiler and 11-layer farms, were visited individually. Pooled faecal samples were obtained from the sheds. A total of 189 necropsy cases were examined for gastrointestinal lesions. Isolation and identification of the organism were done using microbe culture method, and the molecular characterization was performed via PCR targeting invA and ent genes.
Results:
The prevalence of salmonellosis in the broiler and layer farms was recorded at 20.0% and 45.4%, respectively, through the traditional gold standard culture method. From 189 necropsy birds, salmonellosis was recorded at 1.58% dead cases. Molecular detection of Salmonella isolates by PCR targeting invA gene was confirmed in 13.33% of the broiler farms and 36.3% of the layer farms. Further detection of Salmonella enteritidis was performed by PCR targeting ent gene by which 11.11% positivity was determined.
Conclusion:
This study, focused on the Salmonella prevalence, highlighted the zoonotic importance of the bacterium in the commercial poultry farms, which can subsequently be dispersed into the human food chain causing harmful health effects.
Marker-assisted selection or marker aided selection (MAS) is a process whereby a marker (morphological, biochemical or one based on DNA/RNA variation) is used for indirect selection of a genetic determinant or determinants of a trait of interest (i.e., productivity, disease resistance, abiotic stress tolerance, and/or quality). This process is used in plant and animal breeding
Die DNA liegt in einer genau definierten Kopienzahl pro Zelle vor und ist chemisch und physikalisch relativ stabil, wodurch auch die Analyse von stark verarbeiteten Produkten, die beispielsweise thermisch behandelt wurden, möglich ist. Grundsätzlich muss vor jeder molekularbiologischen Analyse die zugrunde liegende DNA aus dem zu untersuchenden Lebensmittel isoliert, d. h. von störenden Begleitstoffen befreit werden. Im Rahmen der Lebensmittelanalytik spielen besonders PCR-basierte Methoden eine Rolle.
Increasingly frequent outbreaks of bacteria-caused illness heightened the need to develop a rapid, portable, sensitive, and reliable method for bacteria detection. Herein, a nanozyme Ps-Pt was synthesized by growing Pt nanoparticles (Pt) on the surface of carboxyl functionalized polymer nanospheres (Ps). The Pt endowed the Ps-Pt with ultrahigh peroxidase-like catalytic activity. Meanwhile, carboxyl groups enabled Ps-Pt to attach streptavidin via covalent binding, which showed higher activity and stability than physical adsorption. To get immune Ps-Pt (IPs-Pt), biotin-antibody was attached to the streptavidin modified Ps-Pt. The two-step binding method enabled the IPs-Pt to own more antibodies and possess high stability. Based on the immune nanozyme, a simple, sensitive colorimetric assay was reported for Salmonella typhimurium (S. typhi) detection. With the assay, as low as 100 CFU/mL S. typhi could be distinguished from the blank by measuring the absorbance, and the generated color change of 1000 CFU/mL S. typhi could be seen clearly with the naked eye. Additionally, the assay has been successfully applied to the detection of S. typhi in spiked milk, Luria-Bertani broth, and serum samples. The specificity and sensitivity of the IPs-Pt-based sensor demonstrated its feasibility in practical applications. Most importantly, the results of 24 single-blind milk samples demonstrated the high sensitivity and 100% accuracy of the assay, indicated that the developed method could detect S. typhi in food samples.
This work focuses on the combination of molybdenum disulfide (MoS2) and à la carte functionalized carbon nanodots (CNDs) for the development of DNA biosensors for selective and sensitive detection of pathogens. MoS2 flakes prepared through liquid-phase exfoliation, serves as platform for thiolated DNA probe immobilization, while thionine functionalized carbon nanodots (Thi-CNDs) are used as electrochemical indicator of the hybridization event. Spectroscopic and electrochemical studies confirmed the interaction of Thi-CNDs with DNA. As an illustration of the pathogen biosensor functioning, DNA sequences from InIA gen of Listeria monocytogenes bacteria and open reading frame sequence (ORF1ab) of SARS-CoV-2 virus were detected and quantified with a detection limit of 67.0 fM and 1.01 pM, respectively. Given the paradigmatic selectivity of the DNA hybridization, this approach allows pathogen detection in the presence of other pathogens, demonstrated by the detection of Listeria monocytogenes in presence of Escherichia Coli. We note that this design is in principle amenable to any pathogen for which the DNA has been sequenced, including other viruses and bacteria. As example of the application of the method in real samples it has been used to directly detect Listeria monocytogenes in cultures without any DNA Polymerase Chain Reaction (PCR) amplification process.
The presented study is focused on the development of electrochemical genosensor for detection of tox gene fragment of toxigenic Corynebacterium diphtheriae strain. Together with our previous studies it fulfils the whole procedure for fast and accurate diagnostic of diphtheria at its early stage of infection with the use of electrochemical methods. The developed DNA sensor potentially can be used in more sophisticated portable device. After the electrochemical stem-loop probe structure optimization the conditions for real asymmetric PCR (aPCR) product detection were selected. As was shown it was crucial to optimize the magnesium and organic solvent concentrations in detection buffer. Under optimal conditions it was possible to selectively detect as low as 20.8 nM of complementary stand in 5 minute or 0.5 nM in 30 minutes with sensitivity of 12.81 and 0.24 1⋅μM⁻¹ respectively. The unspecific biosensor response was elucidated with the use of new electrode blocking agent, diethyldithiocarbamate. Its application in electrochemical genosensors lead to significant higher current values and the biosensor response even in conditions with magnesium ion depletion. The developed biosensor selectivity was examined using samples containing genetic material originated from a number of non-target bacterial species which potentially can be present in the human upper respiratory tract.
We have developed a virtual filter that quickly and efficiently captures and detects pathogenic bacteria in large amounts of water. The virtual filter comprised magnetic nanoparticle chains (MNCs) obtained by cross-linking alginate-coated magnetic nanoparticles (MNPs). When the MNC solution in a disposable plastic tube was exposed to an external magnetic field, the MNCs were aligned along the magnetic field lines, forming a filter similar to a whale's baleen filtering system. A Halbach ring that increased the magnetic field inside the ring was used as the source of an external magnetic field. The Halbach ring produced a more uniform and denser alignment of MNCs than any other ring array. To demonstrate the performance of the virtual filter comprising MNCs, Escherichia coli (E. coli) O157-spiked water was injected into the virtual filter comprising polyethyleneimine-coated MNCs, and the concentration of E. coli O157 captured by electrostatic interaction was determined using qPCR analysis. The bacterial capture efficiency using MNCs was 90% at a flow rate of 5 mL/min, and the detection limit after 10 min of preconcentration of bacteria was 10 cfu/mL, which is 100 times lower than that obtained using qPCR alone.
It has seen increasing development of reliable, robust, and flexible biosensors for rapid food-safety analysis in the past few decades. Recently, functional nucleic acid-based biosensors have attracted attention because of their programmability, bottom-up characteristics, and structural switches. However, few systematic reviews devoted to categorizing the potential of DNA nanostructures and devices were found for detecting food contaminants. Hence, the applications of functional nucleic acid-based biosensors were reviewed for analyzing food contaminants, including foodborne pathogen bacteria, biotoxins, heavy metals, and et al. In addition to categorizing the various biosensors, multiple signal readout strategies, such as optical, electrochemical, and mass-based signals were also examined. Finally, the future changes and potential opportunities, as well as practical applications of functional nucleic acid-based biosensors were discussed.
A novel protein adsorbent was developed by the surface modification of porous rGO-polysaccharide sphere composites with Coomassie Brilliant Blue G250 (CBB-G250). The advanced protein adsorbent combined selectively with proteins from crude bacteria lysate within 10 min, while deoxyribonucleic acid (DNA) was not adsorbed. Thus, protein and DNA in complicated biological samples were separated very quickly and easily during mild mixing treatment with the surface-functionalized rGO-polysaccharide sphere composites. The rGO in the porous sphere composites plays a key role as a linker in the adsorption of CBB-G250 on the composites and makes the CBB-G250 dye bind protein selectively from crude samples. It was confirmed that selective protein adsorption was only possible when the CBB-G250 dye had a chance of interaction with rGO. In addition, the CBB-G250-functionalized rGO-polysaccharide sphere composites could capture whole pathogens because of binding to the surface proteins of the pathogens. Therefore, the surface-functionalized rGO-polysaccharide sphere composites are good candidates for application in simple and efficient methods for the separation and purification of DNA in crude pathogen lysate and for the removal of whole pathogens by selective binding with proteins in the area of rapid pathogen detection and diagnosis.
Organic approaches of pest and plant disease control using pesticides need to be seriously evaluated to counter the negative
impact of high agrochemical input in conventional agriculture. This approach is possible through utilization of vermiwash and
vermicompost extract, products derived from vermiculture and vermicomposting process, respectively. The products contain
high variation of compounds rich in beneficial microorganisms, nutrients, vitamins, and growth hormones that serve as biofertilizer and biocontrol agents against diseases and pests. Liquid forms of vermicompost derivatives are more efficient compared to solid forms due to its ability to reach the target area on plant above ground through foliar application and rhizosphere part of plant underground through soil drench. Vermicompost derivatives can be solely used or mixed with solid vermicompost, fertilizer, or any organic material in soil to achieve the best result not only for pest and disease control but more importantly for soil health and plant growth.
A simple, rapid, highly sensitive, and quantitative fluorescence-based immunochromatographic test (ICT) was successfully developed for the determination of Escherichia coli O157:H7 in food samples. Europium (III) chelated nanoparticles (EuNPs) conjugated to monoclonal antibodies specific to the O-specific polysaccharide fractions of E. coli O157:H7 were used as the fluorescence reporter. The fluorescence was visually observable within 15 min under dark conditions with an ultraviolet light source, and a test-strip reader was used for quantitative analysis. Under optimal conditions, the visual limit of detection (LOD) of the strip was 10⁴ CFU/mL, and the LOD for quantitative detection was as low as 5 × 10² CFU/mL with the test-strip reader. No cross-reaction was observed in six E. coli strains with different somatic (O) antigens and five other major foodborne pathogenic strains, thus indicating the good specificity of this method. Additionally, the detection sensitivity of E. coli O157:H7 was improved to 1 CFU/mL of the original bacterial content by pre-incubation for 8 h in meat (beef, pork, and chicken) and 6 h in milk samples. The average recovery of E. coli O157:H7 spiked in pork was 84.5–103%, thus indicating the accuracy and reliability of the novel test strips. The developed EuNP-ICT strip assay is highly sensitive, specific, and replicable. This detection system provides a tool that can potentially be used to detect E. coli O157:H7 and other pathogenic bacteria in food samples.
E. coli O157 is the most commonly identified serotype among Shiga like-toxin E. coli infections. Current reference methodologies to detect these bacteria are reliable but take several days to provide a definitive result, as they require selective enrichment, molecular detection of the bacteria, followed by plate confirmation. Even alternative methods need extensive sample enrichment in order to increase the amount of bacteria to detectable numbers, resulting in “next-day detection” methodologies, as it is not possible to reach the final result within the same day of the start of the analysis. In the current study, the sample treatment was optimized in such a way that after a short enrichment (3 h) followed by an enzymatic treatment, DNA extraction and amplification by real-time PCR, it was possible to detect down to 3.9 and 3.3 CFU/25 g of ground beef and leafy greens respectively, achieving a combined LOD95 of 3.6 CFU/25 g. This sample treatment optimization allowed us to develop a real “same-day detection” method, being this a major advantage over other previously published methodologies. Additionally, the analysis of spiked samples provided excellent results (90%) for the relative sensitivity, specificity and accuracy; along with the positive and negative predictive values. Finally, the Cohen's k also obtained a value of 1, indicating that the results obtained with the novel method were in “almost complete concordance” with the expected values. It is worth to highlight that the performance parameters fulfill the requirements of the NordVal regulation for the validation of alternative methods.
The study was preformed for investigating of Salmonella from meat, and compared Vidas UP Salmonella (SPT) with the traditional methods of isolation for Salmonella , were examined 42 meat samples (Beef and Chicken) from the Local and Imported From local markets in the city of Baghdad from period December 2013 -February 2014 the samples were cultured on enrichment and differential media and examined samples Vidas, and confirmed of isolates by cultivation chromgenic agar, biochemical tests ,Api20 E systeme , In addition serological tests , and the serotypes determinate in the Central Public Health Laboratory / National Institute of Salmonella The results showed the contamination in imported meat was more than in the local meat 11.9% and 2.3 %, respectively, also recorded the highest isolation of Salmonella from chicken meat compared with beef meat 9.5% and 4.7%, respectively, where isolated 3 serotype (7.14%) from imported chicken meat include S.typhimurium S.entertidis and S.livingstone at16.6% for each of them and 1 serotype (2.3%) isolate from local chicken meat include S.hadar 16.6%. The study recorded for the first time isolate of S. living stone in Iraq from imported chicken. While isolated 2 serotype (4.7%) isolate from imported beef included S. dublin and S. antum at 16.6% each of them .the sensitivity of antibiotics , was studies the results showed that it was resistant to each of Trimethprime Cetazidime Ampicillin Chloromphenicol, Ceftriaxone, and Amoxicillin (100, 66.6 ,33.3, 16.6 , 16.6 , and 16.6 ) % respectively , but sensitivite to Imipenem and Ciprofloxacin at 100% and confirmed the results that the Vidas technique better than traditional methods for speed and accuracy of their results.
Conditions influencing the survival of Campylobacter jejuni in the natural aquatic environment have been determined. Release of Campylobacter spp. into natural waters by animal hosts is postulated to play a key role in the maintenance of viability and transmission of the organism in the environment. Laboratory flask microcosms containing filter-sterilized stream water were used to test C. jejuni for the ability to remain viable in simulated natural systems. The microcosms were compared with the biphasic and shaking broth procedures used routinely for growth of Campylobacter spp. in the research laboratory. The stream-water microcosms were analyzed to determine effects of temperature and aeration on the survival of a well-characterized C. jejuni strain isolated originally from a human campylobacteriosis patient. Morphological characteristics were evaluated by phase-contrast microscopy and scanning or transmission electron microscopy. Survival curves were quantified on the basis of plate counts, epifluorescent microscopy, optical density measurements, and direct viable counts associated with protein synthesis in the absence of DNA replication. A significant difference was observed between results of direct enumeration, i.e., direct viable counts or acridine orange direct counts, and those from spread plate cultures. In all cases, increasing temperature of cultivation resulted in decreased recoverability on laboratory media, due possibly to an increased metabolic rate, as analyzed by CO2 evolution in the presence of radiolabeled glutamate. Stream water held at low temperature (4 degrees C) sustained significant numbers of campylobacters for greater than 4 months. Microcosms, aerated with shaking, exhibited logarithmic decline in recoverable C. jejuni, while stationary systems underwent a more moderate rate of decrease to the nonculturable state.(ABSTRACT TRUNCATED AT 250 WORDS)
The 5' nuclease PCR assay detects the accumulation of specific PCR product by hybridization and cleavage of a double-labeled fluorogenic probe during the amplification reaction. The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. An increase in reporter fluorescence intensity indicates that the probe has hybridized to the target PCR product and has been cleaved by the 5'-->3' nucleolytic activity of Taq DNA polymerase. In this study, probes with the quencher dye attached to an internal nucleotide were compared with probes with the quencher dye attached to the 3'-end nucleotide. In all cases, the reporter dye was attached to the 5' end. All intact probes showed quenching of the reporter fluorescence. In general, probes with the quencher dye attached to the 3'-end nucleotide exhibited a larger signal in the 5' nuclease PCR assay than the internally labeled probes. It is proposed that the larger signal is caused by increased likelihood of cleavage by Taq DNA polymerase when the probe is hybridized to a template strand during PCR. Probes with the quencher dye attached to the 3'-end nucleotide also exhibited an increase in reporter fluorescence intensity when hybridized to a complementary strand. Thus, oligonucleotides with reporter and quencher dyes attached at opposite ends can be used as homogeneous hybridization probes.
We have developed a novel "real time" quantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe (i.e., TaqMan Probe). This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude). Real-time quantitative PCR is extremely accurate and less labor-intensive than current quantitative PCR methods.
Campylobacter jejuni is a pathogenic, microaerophilic, gram-negative, mesophilic bacterium. Three strains isolated from humans with enteric campylobacteriosis were able to survive at high population levels (10(7) cells ml-1) as viable-but-nonculturable (VBNC) forms in microcosm water. The VBNC forms of the three C. jejuni strains were enumerated and characterized by using 5-cyano-2,3-ditolyl tetrazolium chloride-4',6-diamino-2-phenylindole staining. Cellular volume, adenylate energy charge, internal pH, intracellular potassium concentration, and membrane potential values were determined in stationary-phase cell suspensions after 48 h of culture on Columbia agar and after 1 to 30 days of incubation in microcosm water and compared. A notable increase in cell volume was observed with the VBNC state; the average cell volumes were 1.73 microliter mg of protein-1 for the culturable form and 10.96 microliter mg of protein-1 after 30 days of incubation in microcosm water. Both the internal potassium content and the membrane potential were significantly lower in the VBNC state than in the culturable state. Culturable cells were able to maintain a difference of 0.6 to 0.9 pH unit between the internal and external pH values; with VBNC cells this difference decreased progressively with time of incubation in microcosm water. Measurements of the cellular adenylate nucleotide concentrations revealed that the cells had a low adenylate energy charge (0.66 to 0.26) after 1 day of incubation in microcosm water, and AMP was the only nucleotide detected in the three strains after 30 days of incubation in microcosm water.
In 1997, the Foodborne Diseases Active Surveillance Program (FoodNet) conducted active surveillance for culture-confirmed cases of Campylobacter, Escherichia coli O157, Listeria, Salmonella, Shigella, Vibrio, Yersinia, Cyclospora, and Cryptosporidium in five Emerging Infections Program sites. FoodNet is a collaborative effort of the Centers for Disease Control and Prevention's National Center for Infectious Diseases, the United States Department of Agriculture's Food Safety and Inspection Service, the Food and Drug Administration's Center for Food Safety and Applied Nutrition, and state health departments in California, Connecticut, Georgia, Minnesota, and Oregon. The population under active surveillance for foodborne infections was approximately 16.1 million persons or roughly 6% of the United States Population. Through weekly or monthly contact with all clinical laboratories in these sites, 8,576 total isolations were recorded: 2,205 cases of salmonellosis, 1,273 cases of shigellosis, 468 cases of cryptosporidiosis, 340 of E. coli O157:H7 infections, 139 of yersiniosis, 77 of listeriosis, 51 of Vibrio infections, and 49 of cyclosporiasis. Results from 1997 demonstrate that while there are regional and seasonal differences in reported incidence rates of certain bacterial and parasitic diseases, and that some pathogens showed a change in incidence from 1996, the overall incidence of illness caused by pathogens under surveillance was stable. More data over more years are needed to assess if observed variations in incidence reflect yearly fluctuations or true changes in the burden of foodborne illness.
Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional diagnostic testing for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable
state. Nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, we
present a 5′-nuclease PCR assay for quantitative detection of C. jejuni and describe its evaluation. A probe including positions 381121 to 381206 of the published C. jejuni strain NCTC 11168 genome sequence was identified. When this probe was applied, the assay was positive for all of the isolates
of C. jejunitested (32 isolates, including the type strain) and negative for all other Campylobacter spp. (11 species tested) and several other bacteria (41 species tested). The total assay could be completed in 3 h with a
detection limit of approximately 1 CFU. Quantification was linear over at least 6 log units. Quantitative detection methods
are important for both research purposes and further development of C. jejuni detection methods. In this study, we used the assay to investigate to what extent the PCR signals generated by heat-killed
bacteria interfere with the detection of viable C. jejuni after exposure at elevated temperatures for up to 5 days. An approach to the reduction of the PCR signal generated by dead
bacteria was also investigated by employing externally added DNases to selectively inactivate free DNA and exposed DNA in
heat-killed bacteria. The results indicated relatively good discrimination between exposed DNA from dead C. jejuni and protected DNA in living bacteria.
A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica isolates, including 100 problematic "rough" isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonella strains by resulting in positive end-point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [DeltaRn], >0. 6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (DeltaRn, < or =0.5). All 100 rough Salmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at -20 degrees C. Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method for identification of Salmonella, particularly in large reference laboratories.
Intercomparisons of PCR-based data between laboratories require an assurance of assay reproducibility. We performed an interlaboratory study to investigate the contribution made by a variety of thermal cyclers to PCR performance as measured by interblock reproducibility and intrablock repeatability.
Two standardized assays designed to minimize the introduction of non-thermal-cycler-dependent variations were evaluated by 18 laboratories in the United Kingdom, using 33 thermal cyclers of various makes and models. We used a single-product (590 bp) PCR, established in our laboratory as a robust and specific reaction. The second reaction, a multiproduct random amplified polymorphic DNA (RAPD) PCR, was known to be more susceptible to small changes in block temperature and was therefore considered a way of assessing block uniformity with respect to temperature. Assay repeatability data were analyzed with respect to temperature calibration status, the type of temperature control mechanism, thermal cycler age, and the presence of oil overlay or heated lid systems.
All (100%) of the laboratories produced the correct target for the single-product PCR assay, although substantial variation in yield in replicate reactions was observed in 9.4% of these. The RAPD reaction generated results that varied extensively both within the same block and between different thermal cyclers. For eight replicates of a positive sample, 88% intrablock repeatability was demonstrated in calibrated thermal cyclers, which decreased to 63% in noncalibrated instruments.
Irrespective of the make and model of thermal cycler, temperature-calibrated instruments consistently generated more repeatable RAPD data than noncalibrated instruments. Guidelines are offered on optimizing and monitoring thermal cycler performance.
The aim of this study was to introduce the use of a peptide nucleic acid (PNA)-thiazole orange conjugate for real-time monitoring of PCR. When the so-called light-up probes hybridize sequence-specifically to the PCR product, an increase in the fluorescent signal is obtained. It was found that the light-up probe can quantitatively measure the amount of DNA or intact bacterial cells in the reaction mixture, without interfering with the PCR amplification. A linear detection range of at least 4 log units was obtained without optimization of the system. The detection limit of this light-up assay per reaction mixture was 0.4 pg genomic Yersinia enterocolitica DNA.
Campylobacter jejuni is a pathogenic, microaerophilic, gram-negative, mesophilic bacterium. Three strains isolated from humans with enteric campylobacteriosis were able to survive at high population levels (10 ⁷ cells ml ⁻¹ ) as viable-but-nonculturable (VBNC) forms in microcosm water. The VBNC forms of the three C. jejuni strains were enumerated and characterized by using 5-cyano-2,3-ditolyl tetrazolium chloride–4′,6-diamino-2-phenylindole staining. Cellular volume, adenylate energy charge, internal pH, intracellular potassium concentration, and membrane potential values were determined in stationary-phase cell suspensions after 48 h of culture on Columbia agar and after 1 to 30 days of incubation in microcosm water and compared. A notable increase in cell volume was observed with the VBNC state; the average cell volumes were 1.73 μl mg of protein ⁻¹ for the culturable form and 10.96 μl mg of protein ⁻¹ after 30 days of incubation in microcosm water. Both the internal potassium content and the membrane potential were significantly lower in the VBNC state than in the culturable state. Culturable cells were able to maintain a difference of 0.6 to 0.9 pH unit between the internal and external pH values; with VBNC cells this difference decreased progressively with time of incubation in microcosm water. Measurements of the cellular adenylate nucleotide concentrations revealed that the cells had a low adenylate energy charge (0.66 to 0.26) after 1 day of incubation in microcosm water, and AMP was the only nucleotide detected in the three strains after 30 days of incubation in microcosm water.
Traditional biochemical and immunochemical methods for the detection of micro-organisms in food have been supplemented by a number of DNA-based methods during the last decade. Besides the development of direct hybridization techniques, emphasis has been laid onin vitroamplification methods.In vitromethods such as the self-sustained sequence replication (3SR) or the nucleic acid sequence based amplification (NASBA), the Q-beta replicase amplification and the ligase chain reaction (LCR) have had, until now, only limited practical relevance for food monitoring and control. The most developedin vitroamplification method is the polymerase chain reaction (PCR) that allows rapid and selective identification of micro-organisms. Because PCR is increasingly raising interest as a detection method in food hygiene and control, an overview of PCR-systems for the detection of bacteria and viruses in food is provided with this paper. Although the PCR method has advantages (particular specificity, sensitivity) it also has limitations, one of which is its inability to allow differentiation between viable and non-viable micro-organisms. Furthermore, the inhibition of PCR by food-derived inhibitors can result in false negative results. Possible reasons for PCR inhibition and ways to differentiate between viable and dead micro-organisms are discussed in this review.
The decontamination of meat and poultry carcasses can help to reduce human foodborne infections. However, process hygiene to prevent contamination should never be neglected. Some examples of methods to decontaminate carcasses and meat products are discussed in this article.
Faster methods for the detection of foodborne microbial pathogens are needed. The polymerase chain reaction (PCR) can amplify specific segments of DNA and is used to detect and identify bacterial genes responsible for causing diseases in humans. The major features and requirements for the PCR are described along with a number of important variations. A considerable number of PCR-based assays have been developed, but they have been applied most often to clinical and environmental samples and more rarely for the detection of foodborne microorganisms. Much of the difficulty in implementing PCR for the analysis of food samples lies in the problems encountered during the preparation of template DNAs from food matrices; a variety of approaches and considerations are examined. PCR methods developed for the detection and identification of particular bacteria, viruses, and parasites found in foods are described and discussed, and the major features of these reactions are summarized.
DNA-hybridization and the polymerase chain reaction (PCR) are techniques commonly used to detect pathogenic bacteria. In this paper, the use of these techniques for detection of Salmonella, E. coli, V. cholerae, non-O1 Vibrio, Yersinia enterocolitica, Campylobacter, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, and C. botulinum is reviewed with emphasis on application in food microbiology. In food control, DNA-techniques have most often been used in a 'culture confirmation' fashion, i.e. bacteria are enriched and sometimes even purified by traditional culture procedures and thereafter identified by the use of DNA-based methods. The most desirable approach is, however, to detect organisms directly in the food, but major problems remain to be solved before this can be routinely performed.
Detection of pathogens in contaminated food products by PCR can result in false-positive data due to the amplification of DNA from nonviable cells. A new method based on reverse transcription-PCR (RT-PCR) amplification of mRNA for the specific detection of viable Listeria monocytogenes was developed. The expression of three L. monocytogenes genes, iap, hly, and prfA, was examined to determine a suitable target for amplification of RT-PCR. Total RNA from L. monocytogenes was isolated, and following DNase treatment, the RNA was amplified by both RT-PCR and PCR with primers specific for the three genes. Amplicon detection was accomplished by Southern hybridization to digoxigenin-labeled gene probes. The levels of expression of these three genes differed markedly, and the results indicated that the iap gene would provide a good target for development of a specific method for detection of viable L. monocytogenes based on RT-PCR amplification. After a 1-h enrichment, the 371-bp iap-specific product was detected with a sensitivity of ca. 10 to 15 CFU/ml from pure culture. Detection of the 713-bp hly-specific amplicon was ca. 4,000 times less sensitive after 1 h, whereas detection of the 508-bp prfA product showed the lowest level of sensitivity, with detection not observed until after a 5-h enrichment period. The amplification of the iap mRNA was specific for L. monocytogenes. Overall, the assay could be completed in ca. 54 h. The use of RT-PCR amplification for the detection of viable L. monocytogenes was validated in artificially contaminated cooked ground beef. Following a 2-h enrichment incubation, the iap-specific amplification product could be detected in a cooked meat sample that was originally inoculated with ca. 3 CFU/g. These results support the usefulness of RT-PCR amplification of mRNA as a sensitive method for the specific detection of viable L. monocytogenes and indicate that this method may prove useful in the detection of this pathogen in ready-to-eat, refrigerated meat products.
The technology and applications of microarrays of immobilized DNA or oligonucleotides are reviewed. DNA arrays are fabricated by high-speed robotics on glass or nylon substrates, for which labeled probes are used to determine complementary binding allowing massively parallel gene expression and gene discovery studies. Oligonucleotide microarrays are fabricated either by in situ light-directed combinational synthesis or by conventional synthesis followed by immobilization on glass substrates. Sample DNA is amplified by the polymerase chain reaction (PCR), and a fluorescent label is inserted and hybridized to the microarray. This technology has been successfully applied to the simultaneous expression of many thousands of genes and to large-scale gene discovery, as well as polymorphism screening and mapping of genomic DNA clones.
The relationship between the detection of mRNA and cellular viability in Escherichia coli was investigated in cells killed by heat or ethanol. Reverse transcription-PCR (RT-PCR) methods were developed for detecting mRNA from rpoH, groEL, and tufA genes. mRNA from all three genes was detected immediately after the cells had been killed by heat or ethanol but gradually disappeared with time when dead cells were held at room temperature. In heat-killed cells, some mRNA targets became undetectable after 2 to 16 h, whereas after ethanol treatment, mRNA was still detected after 16 h. In contrast, 16S rRNA was detected by RT-PCR in all samples containing dead cells and did not disappear during a subsequent incubation of 16 h at room temperature. Of the different types of nucleic acid, mRNA is the most promising candidate for an indicator of viability in bacteria, but its persistence in dead cells depends on the inactivating treatment and subsequent holding conditions.
In recent years the growing interest in quantitative applications of the polymerase chain reaction (PCR) has favoured the development of a large number of assay procedures suitable for this purpose. In this paper we review some basic principles of quantitative PCR and in particular the role of reference materials and calibrators and the different strategies adopted for nucleic acid quantification. We focus on two methodological approaches for quantitative PCR in this review: competitive PCR and real-time quantitative PCR based on the use of fluorogenic probes. The first is one of the most common methods of quantitative PCR and we discuss the structure of the competitors and the various assay procedures. The second section is dedicated to a recent promising technology for quantitative PCR in which the use of fluorogenic probes and dedicated instrumentation allows the development of homogeneous methods. Assay performance of these methods in terms of practicability and reliability indicates that these kinds of technologies will have a widespread use in the clinical laboratory in the near future.
Functional genomics is currently the most effective approach for increasing the knowledge at the molecular level of metabolic and adaptive processes in whole cells. High-throughput technologies, such as DNA microarrays, and improved two-dimensional electrophoresis methods combined with tandem mass-spectroscopy, supported by bioinformatics, are useful tools for food biotechnology, which depends on detailed knowledge of the properties of food microbes (and pathogens) in their industrial, food and consumer environments. Genomics of food microbes, based on rapidly emerging genome sequence information, generates valuable knowledge that can be used for metabolic engineering, improving cell factories and development of novel preservation methods. Furthermore, pre- and probiotic studies, characterization of stress responses, studies of microbial ecology and, last but not least, development of novel risk assessment procedures will be facilitated.
A reverse transcription-polymerase chain reaction (RT-PCR) method was developed for detecting mRNA from the sefA gene of Salmonella enteritidis. Detection of target mRNA was examined from cells grown in buffered peptone water at different temperatures (37, 25 and 15 degrees C) and pH (5.5, 7.2 and 8.5). The results revealed that the levels of transcription of the sefA gene differed depending upon the physiological state of the cells. This affected the sensitivity of the RT-PCR assay. When the assay was evaluated for the detection of S. enteritidis PT4 in artificially contaminated minced beef and whole egg samples, an enrichment step was used (buffered peptone water, pH 7.2, 37 degrees C, 16 h) to increase the sensitivity of the assay. In the presence of the normal background flora of each food type, it was possible to detect ten cells of S. enteritidis PT4 after a 16-h enrichment using the RT-PCR assay, with a total testing time of 28 h. Unlike the PCR test for the sefA gene that was tested in parallel, the RT-PCR assay did not detect nonviable (heat-inactivated) S. enteritidis PT4 cells. The results supported the usefulness of RT-PCR as a method for the detection of viable microorganisms.
The stability of mRNA in prokaryotes depends on multiple factors and it has not yet been possible to describe the process of mRNA degradation in terms of a unique pathway. However, important advances have been made in the past 10 years with the characterization of the cis-acting RNA elements and the trans-acting cellular proteins that control mRNA decay. The trans-acting proteins are mainly four nucleases, two endo- (RNase E and RNase III) and two exonucleases (PNPase and RNase II), and poly(A) polymerase. RNase E and PNPase are found in a multienzyme complex called the degradosome. In addition to the host nucleases, phage T4 encodes a specific endonuclease called RegB. The cis-acting elements that protect mRNA from degradation are stable stem-loops at the 5' end of the transcript and terminators or REP sequences at their 3' end. The rate-limiting step in mRNA decay is usually an initial endonucleolytic cleavage that often occurs at the 5' extremity. This initial step is followed by directional 3' to 5' degradation by the two exonucleases. Several examples, reviewed here, indicate that mRNA degradation is an important step at which gene expression can be controlled. This regulation can be either global, as in the case of growth rate-dependent control, or specific, in response to changes in the environmental conditions.
Since its introduction in the mid-80s, polymerase chain reaction (PCR) technology has been recognised as a rapid, sensitive and specific molecular diagnostic tool for the analysis of micro-organisms in clinical, environmental and food samples. Although this technique can be extremely effective with pure solutions of nucleic acids, it's sensitivity may be reduced dramatically when applied directly to biological samples. This review describes PCR technology as a microbial detection method, PCR inhibitors in biological samples and various sample preparation techniques that can be used to facilitate PCR detection, by either separating the micro-organisms from PCR inhibitors and/or by concentrating the micro-organisms to detectable concentrations. Parts of this review are updated and based on a doctoral thesis by Lantz [1] and on a review discussing methods to overcome PCR inhibition in foods [2].
A real-time quantitative polymerase chain reaction (PCR) assay for direct detection and enumeration of Listeria monocytogenes and Listeria innocua was developed and applied to artificially contaminated milk samples. The iap gene present in both species was used as a target for amplification of a 175-bp (L. monocytogenes) and a 309-bp (L. innocua) fragment. To ensure that L. monocytogenes and L. innocua are specifically detectable, tests were carried out using 42 L. monocytogenes strains and 33 L. innocua strains belonging to different serovars. Specificity was also confirmed using 22 bacterial strains not belonging to the genus Listeria, including closely related bacteria. In addition to specificity, the reported assay is characterized by a wide dynamic range of quantification and a high sensitivity, as we could detect as few as six copies of the iap gene per PCR using purified DNA as template. When applied to direct detection and quantification of L. monocytogenes in milk, the more rapid real-time quantitative PCR assay was as sensitive as the traditional plate count method, but real-time quantitative PCR-derived iap gene copy numbers were one to two logs higher than colony-forming units obtained by the plate count method.
Advances in molecular diagnostic technologies have enabled genetic testing in single closed-tube reactions. The purpose of this review is to highlight some of the platforms and technologies currently available for the homogeneous detection of targets and the application of the technologies in the clinical setting. Validation issues surrounding the technologies, which may need to be addressed before they can become widely accepted, will also be discussed.
This review discusses the principles of several of the major technologies available for performing homogeneous genetic analyses. Publications arising from the application of the technologies in a wide range of clinical areas are used to highlight and compare the potential advantages and shortcomings of the various technologies.
This review is descriptive and focuses on three areas: the technologies available for performing homogeneous analysis, the clinical applications where the technologies are being used, and validation issues surrounding the acceptance of the technologies in the general clinical setting.
This review intends to give the reader a greater understanding of the various technologies available for performing homogeneous genetic testing in the clinical laboratory. Through insight into the principles and performance characteristics underlying these technologies, the end user can evaluate their value and limitations in the clinical diagnostic setting.
Four enrichment PCR protocols for detecting unlysed cells of pathogenic Yersinia enterocolitica were studied. First, the probability of detecting Y. enterocolitica cells of known concentrations by a multiplex PCR assay was determined, and it was found to follow a logistic regression model. From this model, the probability of detecting Y enterocolitica at a specific concentration could be estimated; for example, the detection probability of 10(4) CFU/ml was estimated to be 85.4%. The protocols were evaluated on enrichment cultures inoculated with 10(2) CFU/ml Y. enterocolitica and 10(2)-10(6) CFU/ml of a defined background flora. For each protocol, the time for sample withdrawal and the presence of background flora were studied with respect to PCR detection. The optimal point in time of sample withdrawal was found to be different for each protocol employed. Early detection was favoured by concentrating the target cells, and the most rapid PCR detection of Y. enterocolitica was achieved with enrichment in Yersinia-PCR-compatible-enrichment (YPCE) medium for 3 h at 25 degrees C, followed by a centrifugation prior to PCR analysis. For detection of Y. enterocolitica in the presence of high concentrations (10(6) CFU/ml) of background flora, a long incubation time followed by density centrifugation and a dilution step was most successful. The protocol that gave the most reliable PCR detection in the presence of 10(6) CFU/ml background flora included 24 h incubation in Yersinia-selective-enrichment (YSE) broth at 25 degrees C, followed by Percoll density centrifugation, and a 100 times dilution prior to PCR analysis.
Detection of mRNA by reverse transcription-PCR as an indicator of viability in Escherichia coli cells
- Sheridan
Preliminary FoodNet data on the incidence of foodborne illnesses—selected sites, United States, 2000
- Anonymous