Establishment Of A Real-time PCR Assay And Comparison Of Several Methods For The Quantitative Detection Of Campylobacter Spp

Campylobacter spp is one of the most commonly important food-borne pathogens worldwide. The incidence of Campylobacter spp has exponentially increased in recent years. Studies have shown that more than90%of the campylobacteriosis is caused by the consumption of contaminated chicken products and the cross-contaminations. So far, we have generally gained the epidemiology and pathogenicity of Campylobacters in diffrrent products in China. However, there are still difficulties to quantify the results of such kind of work. Therefore, it is quite necessary to develope a rapid, quantitative detection method in order to provide basic data for the quantitative risk assessment, hazard analysis, and prevention and control of Campylobacter spp contaminations.1、Development of real-time PCR method for the quantitative detection of Campylobacter sppA real-time PCR assay targeting16S rRNA gene was developed in this study. Species-specific product can be detected after amplifying the DNA templates of Campylobacter jejuni and Camylobacter coli while other bacteria strains showing no corresponding product. It was shown that the detection limits of this PCR was10CFU in1mL sample. The correlation coefficient of standard curve (R2) was0.999, indicating a preferable linear relation between Ct value and template concentration. The intra reusable coefficient of variation was0.12%-0.4%. Repeatable coefficient of variation was0.17%between batches, showing a good reproducibility. Samples were prepared by sample direct thermallysis, the broth concentrate thermallysis method and genomic kit extraction method to obtain template DNA, and the effects of different sample preparation methods of quantitative PCR was evaluated, and the results showed that the broth concentrate thermallysis method is better for chicken wipe samples and chicken feces samples to extract template DNA, the DNA recoveries were46%and19.6%, respectively. Besides, we selected the genomic extraction kit to deal with chicken rinsing samples with a DNA recovery of1.2%. 2、Modification of selective CCDA plating method and selective Karmali plating methodWith reference to the quantitative detection of Campylobacter in the poultry established by Zhai Weihua et al, this study adjusted the concentration of antibiotic in the selective CCDA plates, and compared the effects on the detection of Campylobacter by adding three plates with different antibiotic concentrations, the results showed that, it was more conducive to the growth of Campylobacter when the concentrations of the three antibiotics oxygen-sulfamethoxazole, rifampin and cycloheximide were adjusted to0.01g/L,0.01g/L and0.1g/L, respectively; moreover, the amount of bacteria in the plate did not affect the determination of suspicious Campylobacter colonies, the conformity between antibiotics with these concentrations and pure cultures of Campylobacter line was (95±5)%.The selective Karmali plating method, with reference to the method that "China’s retail chicken curved bacteria pollution effects on human health preliminary quantitative risk assessment project," was validated, and applied in the quantitative separation of Campylobacter in samples.3、Comparison of real-time PCR, CCDA plating method and Karmali plating method for the detection of Campylobacter spp in chicken rinsing samples50chicken rinsing samples were detected by real-time PCR, selective CCDA plating method and selective Karmali plating method. The results of real-time PCR showed a positive rate of92%, the average number of detected Campylobacter was log2.03CFU/g, and the conformity rate was28.0%corresponding to the national standard method(GB method); while it was36%, logl.44CFU/g,60.0%respectively by the selective CCDA plating method; and56%, log2.08CFU/g,56%respectively by selective Karmali plating method. Due to the existence of undetected phenomenon in GB method, the conformity rates between the three methods and GB method were relatively low. The results showed the positive detection rate of the real-time PCR method was higher than both of the selective CCDA plating method and the Karmali plating method, but it presented the lowest conformity rate to the national standard method; As for the chicken samples, both the positive detection rate and average number of detected Campylobacter by selective Karmali plating method were significantly higher than that by the CCDA plating method.4、Comparison of real-time PCR and CCDA plating method for the detection of Campylobacter spp in chicken wipe and feces samples68chicken wipe samples were detected by both real-time PCR and CCDA plating method. real-time PCR method detected65Campylobacter positive samples, the positive rate was95.6%(65/68), and the average number of detected Campylobacter was log2.36CFU/100cm2; while the selective CCDA plating method detected60Campylobacter positive samples, with a positive rate of88.2%(60/68), the average number of Campylobacter was log1.25CFU/100cm2. The results showed that the two methods displayed a compliance rate of89.7%and the real-time PCR method demonstrated a higher positive detection rate and positive average number of Campylobacter than that of the selective CCDA plating method.56chicken feces samples were detected by both real-time PCR and the CCDA plating method.54samples were detected as Campylobacter positive by real-time PCR, the positive rate was96.4%(54/56), the average number of Campylobacter detected was log6.02CFU/g; while the result was75.0%(42/56) and log3.50CFU/g respectively by selective CCDA plating method with42Campylobacter positive samples. The results showed that the two methods presented a compliance rate of78.8%and real-time PCR method also showed higher positive detection rate and positive average number of Campylobacter than that of selective CCDA plating method.5、Comparison of CCDA plating method and Karmali plating method for the detection of Campylobacter spp in chicken feces samples18chicken feces samples were detected using both Karmali and CCDA plating method. Selective Karmali plating method detected9samples were positive, the positive rate was50%, and the average number of detected Campylobacter was log6.02CFU/g; while the CCDA plating method detected13positive samples, with a positive rate and an average number of detected Campylobacter of72.2%and log6.06CFU/g, respectively. The results indicated that as for the chicken feces samples, although these two detection displayed no significant difference, the positive detection rate of CCDA plating method exceeded the selective Karmali plating method.