Abstract
Escherichia coli and Staphylococcus aureus are the most important food borne pathogen transmitting from animal meat and meat products. Therefore, it is vital to design an accurate and specific diagnostic tool for identifying those food-borne pathogens in animal meat and meat products. In the current study, E. coli, methicillin-resistant and sensitive S. aureus (MRSA and MSSA) were simultaneously detected using a developed triplex PCR-based technique. To obtain an optimal reaction parameter, the multiplex assay was optimised by changing just one parameter while holding the others constant. Specificity of the assay was assessed using several porcine bacterial template DNA. The plasmid DNA was used to test the multiplex PCR assay’s sensitivity and interference in spiked pork samples. E. coli, MRSA, and MSSA each have PCR amplified products with sizes of 335, 533, and 209 bp, respectively. The assay detects a minimum microbial load of 102 CFU/μl for all the three pathogens and can identify bacterial DNA as low as 10−2 ng/µl. The assay was validated employing 210 pork samples obtained from retail meat shops and slaughter houses, with MRSA, E. coli, and MSSA with the occurrence rate of 1.9%, 42.38%, and 18.1%, respectively. The rate of mixed bacterial contamination in pork meat samples examined with the developed method was 6.19%, 1.43%, 1.90%, and 1.43% for MSSA & E. coli, MRSA & E. coli, MSSA & MRSA, and E. coli, MSSA & MRSA, respectively. The developed multiplex PCR assay is quick and efficient, and it can distinguish between different bacterial pathogens in a single reaction tube.
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Acknowledgements
Authors acknowledge the financial support received from Indian Council of Agricultural Research, New Delhi, India under the project of Indian Network for Fisheries and Animal Antimicrobial Resistance (INFAAR). Authors are thankful to Director, ICAR-National Research Centre on Pig, Guwahati, Assam, India for providing necessary facilities to conduct the experiment. It is to also acknowledge the All India Institute of Hygiene and Public Health, Government of India, Kolkata, West Bengal, India for providing scholarship to Dr Ranjeet Parihar, MVPH Scholar (first author) during the research period.
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Authors acknowledge the financial support received from Indian Council of Agricultural Research, New Delhi, India under the project of Indian Network for Fisheries and Animal Antimicrobial Resistance (INFAAR).
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RP: Conceptualized the idea, performing laboratory bench works, RD: Sensitivity of the assay, mentoring the research proposal, manuscript writing; JN, PT, GSS, JS: bacterial culturing, antibiotic sensitivity assay, assay optimization; SRP: sample collection; PJD: Insilico works; SR: Specificity of the assay with different bacterial isolates; AR: MRSA and MSSA sampling; VKG: Overall supervision of the study.
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Parihar, R., Deb, R., Niharika, J. et al. Development of triplex assay for simultaneous detection of Escherichia coli, methicillin resistant and sensitive Staphylococcus aureus in raw pork samples of retail markets. J Food Sci Technol (2024). https://doi.org/10.1007/s13197-023-05917-7
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DOI: https://doi.org/10.1007/s13197-023-05917-7