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

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/μl and 1.0 ng/μl of DNA in a reaction mixture (25 μl). The developed multiplex PCR technique could detect Campylobacter jejuni and Listeria monocytogenes upto 3 × 10 5 and 3 × 10 4 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.

Molecular Detection, Typing, and Quantification of Campylobacter spp. in Foods of Animal Origin

“…This method has an irreplaceable role as one of the basic approaches for DNA analysis (Stanek ). This method is both rapid and specific and can be applied for detection of pathogens in food, including Campylobacter in products of animal origin (Giesendorf and others ; Wegmüller and others ; Docherty and others ; Jackson and others ; Ng and others ; Waage and others ; O'Sullivan and others ; Sails and others ; Frasao and others , b; Raja and others ).…”

“…To obtain a pure and ideal concentration of DNA, appropriate techniques for extraction should be used. Raja and others () tested the efficiency of 3 types of extraction to obtain Campylobacter DNA from poultry meat: boiling, a bacterial DNA extraction kit (Qiagen), and phenol‐chloroform‐isoamyl alcohol (25: 24: 1) extraction. Of the methods tested, the Qiagen kit resulted in the highest purity and DNA concentration, whereas the boiling method presented low purity.…”

“…The choice of method will depend on the relevant conditions. Some authors use the boiling method (Shams and others ; Zang and others ), others prefer the kit (Frasao and others , b; Ayaz and others ; Gosselin‐Theberge and others ; Rodgers and others ), whereas others employ phenol‐chloroform‐isoamyl alcohol (25: 24: 1) extraction (Raja and others ).…”

“…In the 1st stage, the DNA is denatured, followed by cooling for annealing of the primer to the DNA strand; the sample is then subjected to a high temperature for DNA synthesis and extension (Hue‐Roye and Vege ). For Campylobacter , a temperature of approximately 94 °C is applied for denaturation, with annealing at 52 to 54 °C and extension at 72 °C (Frasao and others , b; Raja and others ; Zang and others ).…”

“…However, quantification is excluded (Postollec and others ). With PCR, specificity is enhanced by using primers specific for Campylobacter (von Altrock and others ; Lemos and others ; Han and others ), C. coli (Gonzalez and others ; von Altrock and others ; Fontanot and others ; Frasao and others , b; Lemos and others ; Han and others ), C. jejuni (von Altrock and others ; Fontanot and others ; Lemos and others ; Ayaz and others ; Han and others ; Raja and others ), C. lari (Fontanot and others ), and C. upsaliensis (Fontanot and others ) (Table ). In addition to these specific primers, it is possible to amplify to different sizes (bp) by using a primer linked to the 16S‐23S ITS sequence, as this region has a variable size and is interspersed within well‐conserved regions, allowing differentiation between C. jejuni and C. coli (Fontanot and others ).…”