Abstract:The aim of the study was to explore new detective targets for Enterococcus spp.,with better performance in specificity and sensitivity,and subsequently to develop a real-time PCR method which could be effectively applied to rapid quantitative detection of enterococci.Genomic comparative analysis among Enterococcus and non-Entercoccus strains was conducted to screen the specific targets for enterococci,and 42 target fragments were selected for the design of 50 primer pairs.Their specificity and sensitivity were then evaluated by regular PCR and real-time PCR,respectively.Finally,the method was effectively evaluated while it was used in the detection of food samples.Analysis results showed that primer EF1902 was confirmed as the best primer due to its high specificity and sensitivity in real-time PCR tests.The results showed a specific amplification from 44 Enterococcus strains,whereas no amplification from 23 non-Enterococcus strains.A detection limit of 13.78 copies/PCR for purified genomic DNA and 38.4 cfu/PCR for bacterial 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 various 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,the real-time PCR system developed in this study was suitable for the rapid detection of Enterococcus spp.in foods for its advantages in specificity,sensitivity,rapidness and accuracy.
