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Foodborne pathogens present serious concerns to human health and can even lead to fatalities. Microbial forensic science thus plays an important role in consumer protection, food security, and even in litigation. The gold standard for pathogen identification – bacterial culture – is costly and time-consuming. A cheaper and quicker alternative will benefit both forensic science and medical diagnosis. In this study, we developed and validated a molecular-based method termed ‘multiplex-direct PCR assay’ to simultaneously detect three common foodborne pathogens – Escherichia coli O157:H7, Campylobacter jejuni, and Listeria monocytogenes. Three previously reported species-specific primer pairs were modified and used to directly amplify samples without DNA extraction. The assay was also validated for its specificity, sensitivity, and applied to test several samples obtained from a local market and clinical samples. The results showed the expected PCR fragments of approximately 490, 343, and 209 bp for E. coli O157:H7, C. jejuni, and L. monocytogenes, respectively. The assay was specific to the targeted pathogens and was sufficiently sensitive and robust to effectively analyze market samples. The whole process took less than 1 h to complete indicating that the assay is suitable for reliable, rapid, and inexpensive identification of these three foodborne pathogens, which could be useful in microbial forensic investigation.
Pathogenic bacteria and their toxins can occasionally be used as a biological weapon in food poisoning as a terrorist act. Intentional or unintentional food poisoning due to pathogenic bacteria have results in illnesses and deaths [
], highlighting the concern to public health. Microbial forensic science can therefore play an important role in bacterial species identification from relevant evidence to investigate these bio-crimes for food safety standards improvement and legislation.
The gold standard for bacterial species identification depends on bacterial cultivation combined with biochemical tests. The method is time-consuming (typically takes four to seven days), labor-intensive, expensive, and requires specialist knowledge. A molecular approach is used as an alternative to overcome these obstacles; it is also more reliable and sensitive. According to World Health Organization (WHO), Escherichia coli O157:H7, Listeria monocytogenes, and Campylobacter jejuni, are the most common virulent foodborne pathogens [
]. Thus, in this study, we developed a rapid molecular-based method called ‘multiplex-direct PCR assay’ to simultaneously detect these three foodborne pathogens. The assay was validated for its reproducibility, specificity and sensitivity.
2. Materials and methods
2.1 Primer design/modification
Primers specific to the three target pathogens were modified from the previous reports [
] using an alignment of virulent gene sequences of target and other bacterial species obtained from Genbank using MEGA 4. The reported primer sequences were identified in the alignment and then modified in length at the 5′-end. Candidate primers were evaluated for their physical parameters, self-complementarity and secondary structure by Oligocalc and AutoDimer.
2.2 Multiplex PCR
To prepare dilution samples, the target strains were suspended in PBS buffer and subjected to 2 min of heating. The dilution samples were then directly added to PCR and amplified as a multiplex reaction using the Q5™ High Fidelity PCR kit (BioLabs, UK), according to the manufacturer's protocol. PCR products were then separated by 2% agarose gel electrophoresis and detected under an LED transilluminator.
Test reproducibility was conducted by amplifying each bacterium five times. Specificity test was performed with the three target strains and other 13 strains related to the target strains or commonly found in food. The assay was also used on market samples to validate its robustness.
3. Results and discussion
The multiplex-direct PCR assay was successfully developed to simultaneously identify the three foodborne pathogens and provided based on the generation of the expected PCR fragments of 490 bp, 343 bp and 209 bp for E. coli O157: H7, C. jejuni, and L. monocytogenes, respectively (Fig. 1). The assay specificity test was conducted with the three target strains and other 13 bacterial strains. Table 1 shows that the assay was specific to the target strains; all non-target strains failed to amplify. The assay detection limit for E. coli O157:H7, C. jejuni, and L. monocytogenes were as low as 100, 103, and 102 CFU/mL, respectively. The assay was comparable, or better than, previously reported DNA-based assays [
]. Raw meats separately spiked with the three pathogens gave positive results accordingly.
Fig. 1The multiplex-direct PCR of three species; lane M – DNA marker, lane 1 – E. coli O157:H7, lane 2 – C. jejuni, lane 3 – L. monocytogenes, lane 4 – all species multiplex PCR, and lane 5 – negative.
Table 1Specificity test for the multiplex-direct PCR assay; a minus (−) indicates the absence of a band and a plus (+) indicates the presence of a band.
The multiplex-direct PCR assay for simultaneous detection of the three foodborne pathogens; E. coli O157:H7, C. jejuni, and L. monocytogenes was successfully developed and validated. The assay is reliable, rapid, specific, and robust. Therefore, it can be another tool for the investigation of microbial contamination in raw food and food products for microbial forensic purposes.
Conflict of interest
None.
Role of funding
Graduate Studies Research Grant, Prince of Songkla University.
Acknowledgements
The Microbiological Laboratory of Prince of Songkla Hospital, Thailand.
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