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

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 and 3 × 10 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.


Introduction
Recently many pathogens such as Listeria monocytogenes, Campylobacter spp., Vibrio spp., etc. have been identified as emerging meat borne diseases.Campylobacter jejuni is present in the gastro intestinal tract of all animals.Due to improper slaughtering method, the carcass may get contaminated with intestinal contents.Contaminated raw or undercooked poultry meats and/or by-products are particularly important to cause food-borne Campylobacteriosis in humans (CDC, 2005).The prevalence of contamination with Campylobacter species were approximately 60% in any portions of raw poultry meats and by-products except for fillets and hearts (Suzuki & Shigeki, 2008).Listeria monocytogenes is widely distributed and found in many food commodities.It is very persistent microorganism that survives on surfaces and equipment of food processing units in conditions of insufficient cleaning.Post processing contamination is the major source and cross contamination may also occur at the retail shop and also in products due to improper hygienic practices.Ingestion of uncooked meat contaminated during processing can produce infection.Contaminated raw meat was also a potential source for cross contamination to heat-treated or ready-to-eat products during processing or in the kitchen (Meyer, 2011).L. monocytogenes was found prevalently high in poultry (24.5%), intermediate in beef (24.4%) and less prevalent in pork (21.4%).Prevalence of L. monocytogenes in the eight isolated strains from ham and sandwiches was 37.5% and 25.0% respectively in Italy (Pesavento et al., 2010).
In earlier days, it was customary to sell the freshly slaughtered and dressed hot chicken meat with neither refrigeration nor storage facilities.Currently, increase in population (number of working women), changing life style and food habits have propelled consumers to choose hygienic, packed, ready-to-cook meat and meat products.Hence, centralized meat processing units have been established to cater to this demand where there is possible risk of contamination of meat by many microorganisms.Therefore it is necessary to process and handle meat hygienically.Any discrepancy in maintaining the hygienic activities leads to increase in the microbial load in final product.Therefore all processors are opting for the implementation of HACCP in their plants to ensure food safety.It helps in identifying all biological hazards in various processing areas and monitors the critical points for which methods for rapid detection of pathogenic organism are required.
Therefore, there is a thrusting need to develop rapid and reliable method to detect the emerging food pathogens in meat while processing to deliver safe and quality meat and meat products to the consumers.Multiplex PCR is a type of PCR which enables simultaneous amplification of many target sites (template DNA) in one reaction by using more than one pair of primers for different organisms.It helps in minimizing the use of chemicals and also time needed for detecting single organism by normal PCR.Hence, the present study is undertaken for simultaneous detection of Campylobacter jejuni and Listeria monocytogenes from chicken meat by using multiplex polymerase chain reaction.

Multiplex PCR (m-PCR)
A 25 µl of reaction mixture was set up in 0.2 ml PCR tube with following components such as master mix -12.5 µl, forward primer (Listeria) -1 µl, reverse primer(Listeria) -1 µl, forward primer (Campylobacter) -1 µl, reverse primer(Campylobacter) -1 µl, template DNA -2 µl and nuclease free water -6.5 µl.The PCR amplification was carried out in Master Cycler Gradient Thermo cycler (M/s.Eppendorf, Germany) with the following cycling of initial denaturation at 94 ºC for 5 minutes, followed by 30 cycles of denaturation (94 ºC for 30 seconds), annealing (52 ºC for 30 seconds) and extension (72 ºC for 30 seconds) and subsequently a final extension at 72 ºC for 7 minutes.The PCR product was subjected to electrophoresis in 2% agarose gel with ethidium bromide added at a concentration of 1 μg/ml of agarose.Electrophoresis was carried out in 1 X TAE buffer at 100 volts for 30 minutes.The gel was viewed under UV transiluminator and photographed in gel documentation system.

Minimum Detection Limit or Sensitivity of Multiplex PCR
DNA from reference strain was extracted and quantified using Biophotometer plus (M/s Eppendorf, Germany).The quantified DNA was serially 10 fold diluted in sterile nuclease free water and for each dilution PCR amplification was carried out.Sensitivity of m-PCR in terms of DNA concentration was determined.The highest dilution of the DNA showing a visible band in gel was taken as the detection limit for Campylobacter jejuni and Listeria monocytogenes.

Artificial Inoculation of Campylobacter jejuni and Listeria monocytogenes in Chicken Meat
Pellets obtained from overnight incubated Bolton broth and Brain Heart Infusion broth containing pure cultures of Campylobacter jejuni and Listeria monocytogenes (MTTC 657) respectively were separately suspended in sterile saline solution.The cell numbers were adjusted from 3 × 10 8 CFU/ml to 3 × 10 2 CFU/ml by doing serial 10 fold dilutions.The cell numbers in each dilution were confirmed by plate count method using Blood free Campylobacter Selective agar and Listeria Identification agar (PALCAM).
The chicken meat samples which were negative for Campylobacter jejuni and Listeria monocytogenes were confirmed by culture methods was used for artificial inoculation study.Both of these bacteria in respective tenfold dilutions from 3 × 10 8 CFU/ml to 3 × 10 2 CFU/ml (0.2 ml of each dilution) were inoculated together in 1.8 ml of meat homogenate obtained by homogenizing 25 gram of chicken meat in 225 ml of BPW.DNA was extracted before pre-enrichment of meat homogenate, by using Bacterial DNA Extraction Kit.The minimum detection level (sensitivity) for m-PCR in terms of number of organisms was determined.The highest dilutions of the cultures showing visible bands in gel were taken as the detection limit for Campylobacter jejuni and Listeria monocytogenes.

Screening of Chicken Meat Samples
Around 60 chicken meat samples were collected from different regions of chennai.These samples were screened for the presence of C. jejuni and L. monocytogenes by the m-PCR technique developed in this study.The meat homogenate obtained was then subjected to DNA extraction using Phenol-Chloroform-Isoamylalcohol mixture (25:24:1) and m-PCR analysis for the presence of C. jejuni and L. monocytogenes.The chicken meat samples, in which either Campylobacter jejuni and Listeria monocytogenes or both were detected by m-PCR, were further confirmed by culture methods.

DNA Extraction
Three methods of DNA extraction were tried, out of which the Phenol Chloroform Isoamyl alcohol (25:24:1) extraction method was found to be cost effective and the purity and concentration of DNA (Table 1) obtained by this method was almost equal to that of Real Genomics DNA extraction kit method.The purity of DNA (OD ratio, 260 nm:280 nm) was considered good when the values of OD ratio were between 1.7 and 1.9 (Stephenson, 2010).For boiling method, the purity of DNA was not considered good, the reason for not getting good purity may be due to presence of other compounds than DNA and the other materials are not properly separated or removed during extraction procedure.Among the two extraction methods, Qiagen kit method was best because of higher concentration and purity of DNA which may be due to the presence of spin column which can easily remove or filter the other impurities leaving the DNA.Even though the kit method was considered as best, Phenol-Chloroform-Isoamyl alcohol (25:24:1) extraction method was cost effective.Hence Phenol-Chloroform-Isoamyl alcohol method was standardized and used for further screening of retail chicken meat.

Multiplex PCR (m-PCR) Standardization
Using the genus specific primers newly designed for Campylobacter jejuni and Listeria monocytogenes by targeting the Hyp gene and prfA gene, DNA were amplified which gave the product size of 500 bp and 290 bp respectively by PCR (Figure 1).The multiplex PCR technique was standardized to detect both Campylobacter jejuni and Listeria monocytogenes simultaneously in the same reaction tube (Figure 2).Electrophoresis performed in 2% agarose, consistently gave good results with 500 bp of Campylobacter jejuni and 290 bp of Listeria monocytogenes.The results of this study were congruent with that of Wang and Slavik (2005) who developed a multiplex PCR assay to detect C. jejuni, E. coli O157:H7, S. typhimurium and L. monocytogenes with amplified PCR products of sizes 159 bp, 252 bp, 360 bp and 450 bp respectively.Similar findings were also reported by Yamazaki-Matsune et al. (2007) who developed a multiplex PCR for different species of Campylobacter.

Table 1 .
Mean ± SE values of purity of DNA and concentration of DNA