Development And Evaluation Of A Real-Time PCR System For Enterococcus Spp. Detection In Food Samples

Enterococcus spp. is a well-recognized opportunistic pathogen, which is widely distributed in dairy and meat products. Due to its resistance to heat, Enterococcus spp. is likely to cause gastroenteritis at dinner party by ingestion of food containing vast bacteria. Furthermore, Enterococcus spp. has resistance to plenty of antibiotics,especially to vancomycin, which take greater risks to food security. Therefore, it is meaningful to develop a specific, sensitive and effective detection method for monitoring and control of Enterococcus spp.Real-time PCR technology had been applied widely in the detection of food borne pathogen since it is highly sensitive, specific and rapid. Nowadays, two common detective targets, i.e. 16S rRNA and 23S rRNA, are the common detective targets for real-time PCR detection of Enterococcus spp. However, both of them are highly homologous to other bacteria, which might result in false positive results. Moreover, PCR system was lack of valid effective evaluation when applied in food detection. Thereby, exploring new targets is important to increase the specificity of PCR detection for Enterococcus spp. as well as to effectively evaluate this method when used in food sample detection.In this study, genomic comparative analysis among Enterococcus and non-Entercoccus strains was conducted to screen the potential specific targets for enterococci. Thirty-six target fragments were selected, and fifty primer pairs were designed, while their specificity and sensitivity were evaluated by regular PCR and real-time PCR, respectively. Analysis results showed that primer EF1902 was confirmed as the best primer due to its specificity and sensitivity. Specificity of this real-time PCR system was tested with 44 Enterococcus strains and 23 non-Enterococcus strains. The results showed a specific amplification from Enterococcus strains, whereas no amplification from non-Enterococcus strains. After PCR parameter was optimized, an annealing temperature of 60℃, an optimal dye volume of 1μL and Mg2+ concentration of 0.2μmol/L were chosen to develop a stabile PCR system. A detection limit of 13.78 copies/PCR for purified genomic DNA and 38.4 cfu/PCR for bacteria cultures could be achieved after PCR parameter optimization. Artificial contamination assays showed that Enterococcus could be detected by real-time PCR after 6 h enrichment, with an initial spike of 2.63 cfu/mL bacterium in milk samples. The real-time PCR method developed in this study was also applied to detect 52 food samples, and the positive rate of detection was 57.69%. The accuracy was 94.23% compared with the conventional standard method, which demonstrates its potential for practical application.In conclusion, a specific molecular targets for Enterococcus spp. was explored through genomic comparative analysis and PCR methods, based on which a real-time PCR system was developed. This method was suitable for the rapid detection of Enterococcus spp. for its advantages in specificity, sensitivity, rapidness and accuracy.