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(a) Efficiency of MAP DNA isolation in faecal pellets using selected isolation kits: ZR Quick-DNA Fecal Soil Microbe Microprep Kit (Zymo Research, Tustin, CA, USA), QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany), QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany), Power Fecal DNA Kit (QIAGEN, Hilden, Germany), DNeasy Power Soil Kit (QIAGEN, Hilden, Germany), NucleoSpin DNA Stool (Macherey-Nagel, Düren, Germany), Gen Elute Stool DNA Isolation Kit (Sigma-Aldrich, St. Louis, MO, USA), Innu PREP Stool DNA kit (Analytik Jena, Berlin, Germany), MagMAX™ Total Nucleic Acid Isolation Kit (Applied Biosystems by Thermo Fisher Scientific, Vilnius, Lithuania), Nuclisens Magnetic Extraction Reagents (Biomérieux, Marcy-l'Étoile, France), MagVet Mycobacterium paratuberculosis Isolation Kit (LSI, Lissieu, France), and ID Gene Mag Paratuberculosis Extraction Kit (ID vet Genetics, Grabels, France). Error bars represent the 90th percentile and standard deviation for median and mean, respectively. (b) Efficiency of MAP DNA isolation in faecal samples using selected isolation kits; for detailed isolation kit information, see (a). Error bars represent the 90th percentile and standard deviation for median and mean, respectively.
Source publication
Timely and reliable detection of animals shedding Mycobacterium avium subsp. paratuberculosis (MAP) should help to effectively identify infected animals and limit infection transmission at early stages to ensure effective control of paratuberculosis. The aim of the study was to compare DNA extraction methods and evaluate isolation efficiency using...
Contexts in source publication
Context 1
... DNA isolated from faecal pellets was found to provide higher isolation efficiencies than DNA isolated directly from faeces for all kits tested (Figure 2). Half of the tested isolation kits achieved efficiencies in the order of tens of percent, and three kits had efficiencies in the order of units of percent. ...
Context 2
... Materials: The following are available online at http://www.mdpi.com/1996-1944/13/22/5112/s1. Figure S1: Distribution of DNA purity values of selected isolation kits for MAP DNA isolation from milk; Figure S2: Distribution of DNA purity values of selected isolation kits for MAP DNA isolation from faeces; Table S1: List of isolation kits used for isolation of MAP DNA from milk; Table S2: List of isolation kits used for isolation of MAP DNA from faeces; Table S3: List of isolation kits used for automatic magnetic separation of MAP DNA from milk and faeces; Table S13: Concentration and purity of nucleic acids isolated from milk; Table S14: Concentration and purity of nucleic acid isolated from faecal pellets; Table S15: Concentration and purity of nucleic acid automatically isolated from milk. ...
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Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis (MAP) does not produce glycopeptidolipids (GPL) on the surface of the cell wall but a lipopentapeptide called L5P (also termed Lipopeptide-I or Para-LP-01) characterized in C-type (bovine) strains. This lipopeptide antigen contains a pentapeptide core, D-Phenylalanine-N-methyl-L-...
Mycobacterium avium subsp. paratuberculosis (MAP) is the pathogen responsible for paratuberculosis or Johne’s Disease (JD) in ruminants, which is responsible for substantial economic losses worldwide. MAP transmission primarily occurs through the fecal-oral route, and the introduction of an MAP infected animal into a herd is an important transmissi...
Background
Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease (or paratuberculosis), a chronic wasting disease of ruminants and other animals resulting from granulomatous enteritis. There are increasing concerns that MAP is zoonotic. The prevalence of Johne’s disease is increasing worldwide. In an attempt to control an epidemi...
The Irish Johne's Control Programme (IJCP) provides a long-term approach to the voluntary control of Johne's disease (JD) in Ireland, strongly supported by Irish cattle industry leadership. It leverages the establishment of Animal Health Ireland for control of animal diseases not regulated by the European Union. The IJCP has four objectives: facili...
Background
Johne’s disease is a chronic granulomatous enteritis of cattle and other ruminants of economic, animal and public health significance, caused by Mycobacterium avium subsp. paratuberculosis. It is endemic in UK, but there is currently limited information in Northern Ireland. To address this gap, for the first time surveillance data were u...
Citations
... Compared with other commercially available kits or automatized extraction methods, organic solvent extraction is the most sensitive and efficient nucleic acids extraction method available [27,[30][31][32][33], and the phenol-chloroform method is considered as the gold standard for mycobacterial nucleic acids extraction [34]. As the method involves the use of volatile and hazardous chemicals, there is a need for specialized equipment and skilled personnel. ...
Mycobacteria can be one of the main contaminants of biological products, and their presence can have serious consequences on patients’ health. For this reason, the European Pharmacopoeia mandates the specific testing of biological products for mycobacteria, a critical regulatory requirement aimed at ensuring the safety of these products before they are released to the market. The current pharmacopeial reference, i.e., microbial culture method, cannot ensure an exhaustive detection of mycobacteria due to their growth characteristics. Additionally, the method is time consuming and requires a continuous supply of culture media, posing logistical challenges. Thus, to overcome these issues, pharmaceutical industries need to consider alternative non-microbiological techniques to detect these fastidious, slow-growing contaminating agents. This review provides an overview of alternative methods, which could be applied within a quality control environment for biological products and underlines their advantages and limitations. Nucleic acid amplification techniques or direct measurement of mycobacteria stand out as the most suitable alternatives for mycobacterial testing in biological products.
... Compared with other commercially available kits or automatized extraction methods, organic solvent extraction is the most sensitive and efficient nucleic acids extraction method available [26,[29][30][31][32], and the phenol-chloroform method is considered as the gold standard for mycobacterial nucleic acids extraction [33]. As the method involves the use of volatile and hazardous chemicals, there is a need for specialized equipment and skilled personnel. ...
Mycobacteria can be one of the main contaminants of biological products, and their presence can have serious consequences on patients’ health. Thus, specific testing is necessitated by regulations such as the European Pharmacopoeia. The current pharmacopeial reference i.e., microbial culture method cannot ensure an exhaustive detection of mycobacteria due to their growth characteristics. Additionally, the method is time-consuming and requires a continuous supply of culture media, which could be further challenging. Thus, to overcome these challenges, pharmaceutical industries need to consider alternative non-microbiological techniques to detect these fastidious, slow-growing contaminating agents. This review provides an overview of alternative methods, which could be applied within a quality control environment of biological products, and underlines advantages and limitations of these methods. Nucleic acid amplification techniques or direct measurement of mycobacteria stands out as the most suitable alternatives for mycobacterial testing in biological products.
... The 'hook effect' may be due to the errors during denaturation and annealing cycles of amplification [42], bringing false-negative results. For both assays, the lower signal detected in serum samples compared to TE is due to not fully complete recovery of RNA from serum samples that typically affect the column extraction kit [43]. From the analyses in serum samples, we can conclude that off-target miRNAs or other biomolecules do not interfere with signal transduction of microgels assay giving false results in real samples. ...
Low abundance, small size, and sequence similarities render microRNA (miRNAs) detection challenging, particularly in real samples, where quantifying weakly expressed miRNAs can be arduous due to interference of more abundant molecules. The standard quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires multiple steps, thermal cycles, and costly enzymatic reactions that can negatively affect results. Here we present a direct, precise, enzyme-free assay based on microgels particles conjugating molecular beacons (MB) capable of optically detecting low abundant miRNAs in real samples. We validate the applicability of microgels assay using qRT-PCR as a reference technology. As a relevant case, we chose miR-103-3p, a valuable diagnostic biomarker for breast cancer, both in serum samples and MCF7 cells. As a result, microgels assay quantifies miRNA molecules at room temperature in a single step, 1 h (vs. 4 hrs for qRT-PCR) without complementary DNA synthesis, amplification, or expensive reagents. Microgels assay exhibits femtomolar sensitivity, single nucleotide specificity, and a wide linear range (102-107 fM) (wider than qRT-PCR), with low sample consumption (2 μL) and excellent linearity (R2= 0.98). To test the selectivity of the microgel assay in real samples, MCF7 cells were considered where the pool of 8 other miRNAs were further upregulated with respect to miRNA 103-3p. In such complex environments, microgels assay selectively detects the miRNA target, mainly due to MB advanced stability and specificity as well as high microgel antifouling properties. These results show the reliability of microgels assay to detect miRNAs in real samples.
... An RNA/DNA purification protocol consists of four steps: effective tissue disruption, denaturation of nucleoprotein complexes, inactivation of nucleases, and removal of contaminants. Methods allowing nucleic acid extraction can be divided into solution-based, which is currently rarely used, column-based or involving magnetic separation [1,2]. Current trends in molecular biology include increasing automation of the extraction process and options such as all-in-one commercial kits, allowing simultaneous extraction of RNA and DNA often along with protein [1]. ...
Optimized and reliable DNA/RNA extraction protocols are a vital tool in clinical practice in the context of molecular testing. Here, we present our successful attempt to enhance the quantity of RNA isolated from clinical specimens, which we originally found challenging (breast and testis). We compared several purification methods with special focus on two AllPrep system-based protocols (QIAGEN). Our data suggest that addition of proteinase K may markedly increase RNA and, in some cases, also DNA yield. The extraction kit used, AllPrep DNA/RNA/miRNA universal kit, provides RNA amounts comparable with the phenol-chloroform extraction method; however, part of the final yield consisted of small RNAs, visible as a thick band in the bioanalyzer gel-like image (5S peak). The 5S peak, albeit in some cases dominating the bioanalyzer image, plays only a small role in RT-qPCR analysis, and Qubit or NanoDrop measurements can still be used as a reliable estimate of starting amounts of mRNA for downstream analyses. In conclusion, we showed that implementing a protocol containing a step of proteinase K digestion markedly increases RNA yield. The AllPrep DNA/RNA/miRNA Universal Kit can be successfully used for simultaneous extraction of DNA and total RNA, irrespective of the tissue of origin, and does not present inconveniences related to phenol-chloroform extraction.
... Six different DNA extraction protocols, some of which have been used in previous studies (Gao et al., 2007;Husakova et al., 2020;Lima et al., 2018;Mayer, 2018;Oikonomou et al., 2012) Gao et al. (2007). Ten milliliters of the initial volume of the quarter milk sample were heated for 10 min at 95°C in a water bath and then cooled in ice water for 10 min. ...
The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony‐forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α‐ and β‐diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols.
... This process comprises lysing target cells to liberate nucleic acids, binding the nucleic acid selectively to a silica membrane, washing away non-bound particulates and inhibitors, and elution of the nucleic acid, resulting in purified nucleic acid in an aqueous solution (Diefenbach et al., 2018). This principle has been demonstrated to be effective at increasing the DNA concentration in milk and stool samples during the DNA extraction process (Husakova et al., 2020). ...
... The magnetic beads are then collected magnetically, and the biomarkers captured on their surface are released into a more amenable reaction buffer (Sasso et al., 2012;Shields et al., 2012;Bordelon et al., 2013). In comparison to the spin column approach, the extraction of DNA using magnetic beads has been widely reported to be successful (Husakova et al., 2020), including eDNA studies (Shahraki et al., 2019;Wood et al., 2019). The findings of PCR amplification for metazoan primers indicated that no sample was not amplified successfully (Fig. 3). ...
Environmental DNA (eDNA) monitoring has gained popularity in the last decade as one of the most sensitive and cost-effective monitoring methods. However, information regarding the type of DNA extraction used still needs to be studied, especially for metazoan in fresh water samples. This parameter is also critical for a project's experimental design. This study aims to compare the effectiveness of two extraction kits between DNeasy® Blood & Tissue Kit (Qiagen) silica column-based and ZymoBIOMICS 96 MagBead DNA Kit (Zymo Research) magnetic bead-based. The quantity of DNA extracts was measured using a spectrophotometer at 260/280 nm. Following that, we continued the metazoa PCR procedure. Qiagen has higher mean value of DNA concentration (88.48 ng/μl) than Zymo (20.89 ng/μl). For DNA purity, Zymo has higher mean value of DNA purity (1.84) than the Qiagen (1.59). However, both kits were equally successful in amplifying universal metazoan primers. We recommend that the use of these types of kits appears to be the least important consideration. Other important factors that may have a major impact on DNA extraction such as water volume, membrane type, sampling strategy need to be investigated in freshwater samples.
... Six different DNA extraction protocols, some of which have been used in previous studies (Gao et al., 2007;Husakova et al., 2020;Lima et al., 2018;Mayer, 2018;Oikonomou et al., 2012) Gao et al. (2007). Ten milliliters of the initial volume of the quarter milk sample were heated for 10 min at 95°C in a water bath and then cooled in ice water for 10 min. ...
Teat disinfection is a recommended preventive tool to improve udder health and to prevent new intramammary infections. However, side effects are discussed, such as bacterial selection of less-susceptible bacteria with the application of certain teat disinfectants. The objective of this study was to assess the species composition and bacterial in vitro susceptibility by means of an interventive trial. For this purpose, 3 different postmilking teat treatments (disinfection with 0.215% chlorhexidine or 3.5% lactic acid, or control group with no dipping) were applied to 28 cows in a 6-d intervention approach using a split-udder design. Milk samples were taken before and after intervention. Bacteria were cultured and differentiated to species or genus level by MALDI-TOF mass spectrometry. Minimum inhibitory concentrations (MIC) were determined, and MIC changes over time were recorded. Susceptibilities to chlorhexidine and lactic acid were compared between species of the genera Staphylococcus, Streptococcus, Corynebacterium, and others. Species composition changed during the intervention. Under the treatment of chlorhexidine and lactic acid, the proportion of coagulase-negative staphylococci (CNS) decreased. An increased proportion of species belonging to the genus Corynebacterium was observed especially under the application of lactic acid. Although both teat disinfectants were basically effective, isolates differed in their susceptibility to both teat disinfectants. Populations of CNS, Staphylococcus aureus, and Corynebacterium spp. showed significantly lower absolute MIC values for chlorhexidine. Compared with other species, Corynebacterium spp. showed the lowest susceptibility for chlorhexidine as well as for lactic acid. A significant increase in MIC values after 6 d of intervention was observed with the lactic acid treatment in all isolates, as well as in CNS. This increase can be interpreted as either adaptation of isolates or displacement of more-susceptible species by less-susceptible species. Further studies using long-term intervention might reveal more pronounced effects on MIC values and species composition.
... In order to apply PCR methods and the aforementioned sequencing approaches for microbiome analysis, the extraction of good quality DNA from faecal samples is pivotal for microbiome analysis [84]. A number of studies have compared a wide variety of commercial kits and protocols in order to determine the most sensitive and specific kit for carrying out molecular detection of MAP [84][85][86][87]. Extraction of genomic DNA from MAP is difficult due to its unique lipid rich cell wall, mentioned earlier in this review, making it difficult to lyse. ...
... Inefficient removal of contaminants, such as humic acids and complex polysaccharides, may result in PCR inhibition. DNA extraction efficiency has been compared using commercially available DNA extraction kits based on both magnetic separation and silica columns [87]. It was found that silica column based methods were superior to magnetic separation methods for both milk and faecal microbial DNA isolation. ...
Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne’s disease in ruminants. As an infectious disease that causes reduced milk yields, effects fertility and, eventually, the loss of the animal, it is a huge financial burden for associated industries. Efforts to control MAP infection and Johne’s disease are complicated due to difficulties of diagnosis in the early stages of infection and challenges relating to the specificity and sensitivity of current testing methods. The methods that are available contribute to widely used test and cull strategies, vaccination programmes also in place in some countries. Next generation sequencing technologies have opened up new avenues for the discovery of novel biomarkers for disease prediction within MAP genomes and within ruminant microbiomes. Controlling Johne’s disease in herds can lead to improved animal health and welfare, in turn leading to increased productivity. With current climate change bills, such as the European Green Deal, targeting livestock production systems for more sustainable practices, managing animal health is now more important than ever before. This review provides an overview of the current knowledge on genomics and detection of MAP as it pertains to Johne’s disease.
... • Faecal samples from the healthy controls were collected on Bio-Me filter cards organised by HUNT4. Three 6 mm discs were punched out from each sample filter card, and microbial DNA was extracted using a Microbiome MagMAX Ultra kit (Thermo Fisher Scientific, Waltham, MA, USA) [28] essentially following the manufacturer's recommendations on KingFisher™ Flex (ThermoFisher Scientific). The bacterial cell wall was disrupted using a VWR Star Beater at maximum settings for 2 min. ...
Background:
An abnormal faecal microbiota could be a causal factor for disease. This study evaluated a new method for faecal microbiota analysis in subjects with obesity and irritable bowel syndrome.
Methods:
The study had a matched case-control design. Forty-six subjects with morbid obesity (defined as BMI > 40 or >35 kg/m2 with obesity-related complications) of whom 23 had irritable bowel syndrome (IBS), were compared with 46 healthy volunteers. The faecal microbiota was analysed with Precision Microbiome Profiling (PMP™) which quantified 104 bacteria species. The primary aim was comparisons between the cases and controls.
Results:
Two men and 44 women with a mean age of 43.6 years were included in each of the groups; BMI in the groups was (mean and SD) 41.9 (3.5) and 22.5 (1.5) kg/m2, respectively. Seventeen bacterial species showed statistically significant differences between the groups after adjusting for multiple testing. In a post hoc analysis, the sensitivity and specificity were 78%. Alpha diversity was lower in the group with obesity. In subjects with morbid obesity, no clinically significant differences were seen between subjects with and without IBS or from before to six months after bariatric surgery.
Conclusions:
The results encourage further evaluation of the new microbiome profiling tool.
... In an attempt to reduce time to result, (real-time) PCR based techniques have been established and introduced in routine diagnostics (7)(8)(9). The performance of PCR based methods depends largely on the efficacy of the protocol used for nucleic acid extraction from clinical samples (10,11). The detection rate is reduced when samples with low bacterial load are tested (12,13). ...
Analysis of volatile organic compounds (VOCs) is a novel approach to accelerate bacterial culture diagnostics of Mycobacterium avium subsp. paratuberculosis (MAP). In the present study, cultures of fecal and tissue samples from MAP-infected and non-suspect dairy cattle and goats were explored to elucidate the effects of sample matrix and of animal species on VOC emissions during bacterial cultivation and to identify early markers for bacterial growth. The samples were processed following standard laboratory procedures, culture tubes were incubated for different time periods. Headspace volume of the tubes was sampled by needle trap-micro-extraction, and analyzed by gas chromatography-mass spectrometry. Analysis of MAP-specific VOC emissions considered potential characteristic VOC patterns. To address variation of the patterns, a flexible and robust machine learning workflow was set up, based on random forest classifiers, and comprising three steps: variable selection, parameter optimization, and classification. Only a few substances originated either from a certain matrix or could be assigned to one animal species. These additional emissions were not considered informative by the variable selection procedure. Classification accuracy of MAP-positive and negative cultures of bovine feces was 0.98 and of caprine feces 0.88, respectively. Six compounds indicating MAP presence were selected in all four settings (cattle vs. goat, feces vs. tissue): 2-Methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, heptanal, isoprene, and 2-heptanone. Classification accuracies for MAP growth-scores ranged from 0.82 for goat tissue to 0.89 for cattle feces. Misclassification occurred predominantly between related scores. Seventeen compounds indicating MAP growth were selected in all four settings, including the 6 compounds indicating MAP presence. The concentration levels of 2,3,5-trimethylfuran, 2-pentylfuran, 1-propanol, and 1-hexanol were indicative for MAP cultures before visible growth was apparent. Thus, very accurate classification of the VOC samples was achieved and the potential of VOC analysis to detect bacterial growth before colonies become visible was confirmed. These results indicate that diagnosis of paratuberculosis can be optimized by monitoring VOC emissions of bacterial cultures. Further validation studies are needed to increase the robustness of indicative VOC patterns for early MAP growth as a pre-requisite for the development of VOC-based diagnostic analysis systems.
