Methodology Development of TaqMan qPCR for E.necatrix Vaccine ENYS Strain
In order to control the quality of E.necatrix ENYS in production and monitor its clinical application,this study performed whole genome resequencing on ENYS and ENGD strains,and established a TaqMan qPCR detection technology combined with bioinformatics analysis.The results showed that the En marker5 sequencing results were consistent with expectations,with the optimal primer and probe concentrations of 500 nM and 250 nM,respectively.The slope of the standard curve was-3.249,and the correlation coefficient was 0.999.The template concentration can be quantified by the Ct value.This method detected 290 copies/reactions and 50 follicles/reactions,with a sensitivity 10 times higher than traditional PCR;This method can specifically detect ENYS strains,but cannot amplify other vaccine strains,wild-type strains,and intestinal pathogens;The coefficient of variation for repeatability testing is less than 0.6%,indicating good repeatability.This study developed a highly sensitive,specific,and stable TaqMan qPCR detection method for the E.necatrix ENYS strain,providing technical support for the safety evaluation and clinical application of the E.necatrix ENYS strain.
E.necatrix.Attenuated live vaccineWhole genome resequencingMolecular markerTaqMan qPCR
万方数据
维普
