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Cross-reactivity testing of the mPCR with closely related organisms. Lane 1: 100 bp DNA ladder, Lane 2: Burkholderia pseudomallei, Lane 3: Burkholderia mallei, Lane 4: Y. pestis, Lane 5: Y. pseudotuberculosis, Lane 6: Bacillus anthracis, Lane 7: Bacillus cereus, Lane 8: Brucella mellitus, Lane 9: Multiplex positive control. Lane 10: Y. enterocolitica.
Source publication
Background & objectives:
Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei and Brucella species are potential biowarfare agents. Classical bacteriological methods for their identification are cumbersome, time consuming and of potential risk to the handler.
Methods:
We describe a sensitive and specific multiplex polymerase chain reacti...
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Biosensors are analytical devices combining a physical sensor with a part of biological origin providing sensitivity and selectivity toward analyte. Biological warfare agents are infectious microorganisms or toxins with the capability to harm or kill humans. They can be produced and spread by a military or misused by a terrorist group. For example,...
Citations
... Y. pestis is responsible for plague, a zoonotic disease, with dissemination of the infection occurring through direct contact with infected animals, animal tissue, bodily fluids, ingestion of the meat from infected animals, drinking contaminated water, bites from arthropods or inhalation of infectious droplets [2]. Different methodologies have been reported for the detection of Y. pestis, offering a more rapid alternative to the gold standard technique of culture-based methods, whilst having the same or even higher sensitivity, including the use of molecular techniques [3][4][5][6][7], immunochemistry [8][9][10] or the use of nanoconstructs [11]. However, these techniques cannot be deployed for use in situ at the point of need and in order to reduce the impact of this potential biowarfare agent, an accurate, rapid, inexpensive and easy-to-use analytical tool is essential. ...
Due to the extreme infectivity of Yersinia pestis it poses a serious threat as a potential biowarfare agent, which can be rapidly and facilely disseminated. A cost-effective and specific method for its rapid detection at extremely low levels is required, in order to facilitate a timely intervention for containment. Here, we report an ultrasensitive method exploiting a combination of isothermal nucleic acid amplification with a tailed forward primer and biotinylated dNTPs, which is performed in less than 30 minutes. The polymerase chain reaction (PCR) and enzyme linked oligonucleotide assay (ELONA) were used to optimise assay parameters for implementation on the LFA, and achieved detection limits of 45 pM and 940 fM using SA-HRP and SA-polyHRP, respectively. Replacing PCR with isothermal amplification, namely recombinase polymerase amplification, similar signals were obtained (314 fM), with just 15 minutes of amplification. The lateral flow detection of the isothermally amplified and labelled amplicon was then explored and detection limits of 7 fM and 0.63 fg achieved for synthetic and genomic DNA, respectively. The incorporation of biotinylated dNTPs and their exploitation for the ultrasensitive molecular detection of a nucleic acid target has been demonstrated and this generic platform can be exploited for a multitude of diverse real life applications.
... Developments of rapid identification systems for Y. pestis could provide a much faster diagnosis and identify those who require escalation to hospital for definitive treatment, and could be utilized in the prehospital setting to triage patients. [81][82][83][84] A dipstick system has been developed for detection of the F1 antigen in humans, and another immuno-strip (PLA-dipstick) that can detect F1-negative plague rapidly in asymptomatic humans under field conditions. 85,86 This could prove extremely valuable in the prehospital and hospital settings to guide management, and requirement for post exposure prophylaxis. ...
Yersinia pestis is the causative agent of plague and is considered one of the most likely pathogens to be used as a bioweapon. In humans, plague is a severe clinical infection that can rapidly progress with a high mortality despite antibiotic therapy. Therefore, early treatment of Y. pestis infection is crucial. This review provides an overview of its clinical manifestations, diagnosis, treatment, prophylaxis, and protection requirements for the use of clinicians.
We discuss the likelihood of a deliberate release of plague and the feasibility of obtaining, isolating, culturing, transporting and dispersing plague in the context of an attack aimed at a westernized country. The current threat status and the medical and public health responses are reviewed. We also provide a brief review of the potential prehospital treatment strategy and vaccination against Y. pestis. Further, we discuss the plausibility of antibiotic resistant plague bacterium, F1-negative Y. pestis, and also the possibility of a plague mimic along with potential strategies of defense against these. An extensive literature search on the MEDLINE, EMBASE, and Web of Science databases was conducted to collate papers relevant to plague and its deliberate release. Our review concluded that the deliberate release of plague is feasible but unlikely to occur, and that a robust public health response and early treatment would rapidly halt the transmission of plague in the population. Front-line clinicians should be aware of the potential of a deliberate release of plague and prepared to instigate early isolation of patients. Moreover, front-line clinicians should be weary of the possibility of suicide attackers and mindful of the early escalation to public health organizations.
... In previous reports, a non-competitive IAC with two pairs of primers being complementary to both the target DNA and non-target DNA was used to indicate PCR inhibition. However, the inclusion of additional primer sets in the reaction invariably results in a higher probability of mispriming and primer dimerization [32]. It is difficult to avoid the interference among primer sets and reaction conditions for all primer sets must be optimized. ...
Various constituents in food specimens can inhibit the PCR assay and lead to false-negative results. An internal amplification control was employed to monitor the presence of false-negative results in PCR amplification. In this study, the objectives were to compare the real-time PCR-based method by introducing a competitive internal amplification control (IAC) for the detection of Escherichia O157:H7 with respect to the specificity of the primers and probes, analytical sensitivity, and detection limits of contamination-simulated drinking water. Additionally, we optimized the real-time fluorescent PCR detection system for E. coli O157:H7. The specificity of primers and probes designed for the rfbE gene was evaluated using four kinds of bacterial strains, including E. coli O157:H7, Staphylococcus aureus, Salmonella and Listeria monocytogenes strains. The real time PCR assay unambiguously distinguished the E. coli O157:H7 strains after 16 cycles. Simultaneously, the lowest detection limit for E. coli O157:H7 in water samples introducing the IAC was 104 CFU/mL. The analytical sensitivity in water samples had no influence on the detection limit compared with that of pure cultures. The inclusion of an internal amplification control in the real-time PCR assay presented a positive IAC amplification signal in artificially simulated water samples. These results indicated that real-time fluorescent PCR combined with the IAC possessed good characteristics of stability, sensitivity, and specificity. Consequently, the adjusted methods have the potential to support the fast and sensitive detection of E. coli O157:H7, enabling accurate quantification and preventing false negative results in E. coli O157:H7 contaminated samples.
... A multiplex PCR for the simultaneous detection of Burkholderia species have been reported previously [18][19][20] but was not evaluated in clinical samples. Other studies have reported multiplex PCR for the detection of B. pseudomallei along with Brucella species, Bacillus anthracis and Yersinia pestis [21] but the assay was not evaluated in clinical samples. We previously showed the usefulness of multiplex PCR for the detection of B. pseudomallei along with S. Typhi and Mycobacterium tuberculosis in the buffy coat samples compared to blood culture [22]. ...
Enteric fever, caused by infection with Salmonella Typhi or Paratyphi A, B, or C (typhoidal Salmonella) and melioidosis are among the most common bacterial causes of acute febrile illness in tropical and subtropical countries. The diseases are widely spread even in developed countries mainly affecting travellers returning from endemic areas. Melioidosis is an increasingly recognized fatal septicemic infection also mimicking tuberculosis. These pathogens are largely underreported due to lack of sensitive and specific diagnostic tools. In this study, an inhouse multiplex real-time PCR assay was developed for the simultaneous detection of Salmonella Typhi, S. Paratyphi A and B. pseudomallei using specific primers. The inhouse developed assay was evaluated on buffy coat and serum samples collected from patients with acute febrile illness at four different centres. The assay had a detection limit of less than 1 genome copy for S. Typhi and S. Paratyphi A and 18 genome copies per 10 µl of PCR input for B. pseudomallei. Among the 1101 samples tested by multiplex real-time PCR, one (0.09%) sample was positive for S. Typhi. One sample was positive for blood culture identified as S. Typhi but negative by real-time PCR. The samples were negative for S. Paratyphi A and B. pseudomallei. The multiplex real-time assay would be highly useful as a diagnostic aid for the syndromic approach and comprehensive diagnosis of enteric fever and melioidosis. The assay could improve the overall diagnostic capability and be a useful tool during outbreak investigations.
... Multiplex PCR technique (mPCR) was developed in 1988 by Chamberlain et al. (1988) and reported to be highly effective for detecting various types of the disease agent in one reaction. This technique is more economical than a single PCR technique for use less chemical reagent in the process of DNA fragments amplification (Batra et al. 2013). Ekawasti et al. (2014) has used mPCR technique to detect T. evansi from haematophagus flies (Tabanus sp, Stomoxys sp and Hipobosca sp) as vector of T. evansi. ...
Trypanosoma evansi is a Hemoflagella parasite that infects cattle and is known as the agents of Surra. Several other Trypanosoma species are pathogenic to mammals: T. equiperdum, T. b. rhodesiense, T. b. gambiense, T. vivax, T. congolense. Some of these species is quite difficult to be distinguished morphologically with T. evansi through conventional techniques (thin blood smear). Molecular technique by polymerase chain reaction (PCR) is reported to have the ability to identify, characterize and diagnose Trypanosomes accurately. However, a single PCR used is relatively expensive because it takes at least two or more pairs of primers to determine T. evansi. The purpose of this study is to develop T. evansi species identification techniques by multiplex PCR/mPCR (the three pairs of primer in one reaction) that takes the relatively fast and inexpensive. A total of 32 isolates T.evansi were obtained from Bblitvet Culture Collection (BCC) and the Department of Parasitology BBLitvet used in this study. Isolates represent isolates from endemic areas and Surra outbrake isolated from 1988-2014. DNA extraction performed on each isolates, including isolates Bangkalan 87 which has been purified as a positive control. Primers used are specific for T. evansi, the ITS-1, Ro Tat 1.2 VSG and ESAG 6/7. Before running mPCR, each primer is optimized by using a single PCR. The results showed that the three primers can be combined in a single reaction with mPCR technique and amplify each DNA fragment target perfectly, so identified 32 isolates as T. evansi. This technique can be applied in the field with a lower cost and faster time.
... Additionally, B. pseudomallei has earned the nickname "the great mimicker" due to its wide repertoire of clinical manifestations which often prompt misdiagnosis. Several rapid detection/diagnostic biochemical methods have been introduced; yet problems have been reported such as the misidentification of B. pseudomallei as B. cepacia [19][20][21][22][23]. Thus research has shifted focus toward molecular methods, but these too have had their problems, primarily assay specificity and sensitivities which are less than 100% [24,25]. ...
... Figure 1 shows a schematic of the target regions in both species. All oligonucleotides used in the PCR assays for B. mallei and B pseudomallei are listed in Table 2 [20,30] were later incorporated in the assays and produced a 1500 bp product to act as a control. The universal primers were IAC-16S-F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and IAC-rDNA-R (5 -ACGGCTACCTTGTTACGACTT-3 ). ...
The etiologic agents for melioidosis and glanders, Burkholderia mallei and Burkholderia pseudomallei respectively, are genetically similar making identification and differentiation from other Burkholderia species and each other challenging. We used pyrosequencing to determine the presence or absence of an insertion sequence IS407A within the flagellin P (fliP) gene and to exploit the difference in orientation of this gene in the two species. Oligonucleotide primers were designed to selectively target the IS407A-fliP interface in B. mallei and the fliP gene specifically at the insertion point in B. pseudomallei. We then examined DNA from ten B. mallei, ten B. pseudomallei, 14 B. cepacia, eight other Burkholderia spp., and 17 other bacteria. Resultant pyrograms encompassed the target sequence that contained either the fliP gene with the IS407A interruption or the fully intact fliP gene with 100% sensitivity and 100% specificity. These pyrosequencing assays based upon a single gene enable investigators to reliably identify the two species. The information obtained by these assays provides more knowledge of the genomic reduction that created the new species B. mallei from B. pseudomallei and may point to new targets that can be exploited in the future.
Natural environment serves as a reservoir for Burkholderia cepacia complex organisms, including the highly transmissible opportunistic human pathogen B. cenocepacia. Currently, there is a lack of an effective and quantitative method for B. cenocepacia detection in fresh food and other environmental niches. A quantitative real-time PCR (qPCR) detection method for B. cenocepacia bacteria was established in this study and validated using artificially inoculated fresh vegetable samples. Genome-wide comparative methods were applied to identify target regions for the design of species-specific primers. Assay specificity was measured with 12 strains of closely related Burkholderia bacteria and demonstrated the primer pair BCF6/R6 were 100% specific for detection of B. cenocepacia. The described qPCR assay evaluated B. cenocepacia with a 2 pg μl-1 limit of detection and appropriate linearity (R2 = 0.999). In 50 samples of experimentally infected produce (lettuce, onion, and celery), the assay could detect B. cenocepacia as low as 2.6 × 102 cells in each sample equal to 1 g. The established qPCR method quantitatively detects B. cenocepacia with high sensitivity and specificity, making it a promising technique for B. cenocepacia detection and epidemiological research on B. cepacia complex organisms from fresh vegetables.
Plague is an acute bacterial infection caused by Yersinia pestis. The three major clinical forms of plague are bubonic, pneumonic and septicemic, which have high case-mortality rates. Therefore, rapid and reliable diagnostic tools are crucial. Currently, bacteriological means and traditional serological assays are used for detecting infection with Y. pestis. However, such methods have their limitations. Polymerase chain reaction (PCR) is one of the most useful tools for rapid diagnosis of plague. The present review introduced the main PCR techniques and their applications for detecting and confirmation of Y. pestis. The advantages and disadvantages of the different PCR methods were also summarized.
The development of a gold nanoparticle aptamer assay is persued for rapid and sensitive determination of histamine in foodstuffs, which could be deployed for on-site use. The assay is based on a histamine-specific aptamer and gold nanoparticles and the salt-induced aggregation of the particles in the presence of histamine indicated by the color change from red to blue. Gold nanoparticle size, salt type, and concentration as well as aptamer concentration were optimized, and using optimum conditions, a limit of detection of 8 nM (~ 0.05 mg/kg) was obtained. Finally, the aptamer AuNP assay was applied to the determination of histamine in quality control fish samples. The histamine levels of these samples had previously been determined using HPLC and commercial ELISA kits by numerous independent laboratories and a good correlation was obtained. The developed AuNP assay is rapid, sensitive, and reproducible.
Graphical abstract
Anthrax is a zoonotic disease and its lethality is due to two secreted exotoxins; lethal toxin and edema toxin. The receptor for anthrax toxin is called Anthrax Toxin Receptor (ATR). Two distinct cellular Anthrax toxin receptors, ANTXR1 (also known as tumour endothelial marker 8; TEM8) and ANTXR2 (also known as capillary morphogenesis protein 2; CMG2) have been identified. TEM8 and CMG2 both are ubiquitous in nature. Apart from their function as anthrax toxin receptor, their ubiquitous presence is suggestive of their physiological role. TEM8 is preferentially expressed in blood vessel of tumours and in vasculature indicating its probable role during angiogenesis and regulation of neovasculature. CMG2 is present in capillary cells and is associated with capillary morphogenesis. Both the TEM8 and CMG2 are present in different isoforms, share homology in amino acid residues and apart from their role in angiogenesis regulation, are also involved in interaction with extracellular matrix. Mutation in TEM8 result a condition appeared to alter physical characters in form of growth retardation, alopecia, pseudoanodontia, and progressive visual impairment known as GAPO syndrome. Mutation in CMG2 result in autosomal recessive disorder in humans called Hyaline Fibromatosis Syndrome (IFS) and infantile systemic hyalinosis (ISH). Because of role in physiological functions and participation as toxin receptor, these receptors could be target for several curative therapies both for the anthrax disease as well as for receptor associated physiological disorders. This review presents a detailed insight into isoforms, functions, diseases and therapeutic implications of anthrax toxin receptors.
