Detection of Emerging Food Pathogens in Chicken Meat Using Multiplex Polymerase Chain Reaction

Satheesh Raja, Appa Rao, Narendra Babu, Thamizhannal Thamizhannal

Abstract


A study was undertaken to develop a multiplex PCR (m-PCR) protocol for simultaneous detection of Campylobacter jejuni and Listeria monocytogenes in chicken meat. The extraction of DNA was carried out using commercial DNA extraction kit, Phenol Chloroform and boiling method. Samples with OD ratio (260:280) between 1.7 and 1.9 were considered good in terms of concentration and purity and were used for PCR amplification. DNA extraction kit and Phenol Chloroform method revealed good OD value were used for sample extraction process. PCR and m-PCR amplification was carried out using genus specific primers were designed by targeting its Hyp (500 bp) and prfA (290 bp) gene for Campylobacter jejuni and Listeria monocytogenes respectively. Electrophoresis of amplified PCR products and gel documentation revealed 500 bp and 290 bp in 2% Agarose. The multiplex PCR technique was standardized using the reference strains with the similar amplification procedure. The minimum detection level (sensitivity) by mPCR for Campylobacter jejuni and Listeria monocytogenes was found to be 0.2 ng/ul and 1.0 ng/ul of DNA in a reaction mixture (25 ul). The developed multiplex PCR technique could detect Campylobacter jejuni and Listeria monocytogenes upto 3 × 105 and 3 × 104 CFU/ml of artificially inoculated meat homogenate. Around 60 chicken meat samples were collected from different regions of chennai and were screened for the presence of Campylobacter jejuni and Listeria monocytogenes. All the samples screened were not positive either for Campylobacter jejuni and Listeria monocytogenes. The negative samples were further checked by culture methods and good correlation between these two methods was observed.  Hence, the m-PCR technique developed in this study can be used as a rapid screening test for detection of Campylobacter jejuni and Listeria monocytogenes from chicken meat within 24 hours.


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DOI: http://dx.doi.org/10.5539/jas.v8n9p217

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Journal of Agricultural Science   ISSN 1916-9752 (Print)   ISSN 1916-9760 (Online)  E-mail: jas@ccsenet.org

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