To establish a novel and high-sensitive droplet digital polymerase chain reaction PCR(ddPCR)method to detect Vibrio cholerae(Vc)and Vibrio parahaemolyticus(Vp).The methods of ddPCR reaction system were established and optimal reaction conditions were confirmed based on Real-time PCR Method(SN/T 2425-2010)(SN/T 2424-2010)were selected.The bacterial Vc and Vp genomic DNA and quantitative accuracy analysis was performed on the result by evaluation for linearity,limit of detection and specificity.The method of ddPCR is feasibility and accuracy.The methods were tested of the background and test sample which results VcandVp performed good specificity.VcandVp were performed good specificity in ddPCR method in this experiment.The results indicated that the method was approximately 1×105 copies/reaction.In the artificial contaminated experiment,the result of ddPCR for quantification of VcandVp were closer to theoretical value,The cross-reactions was performed with other pathogens,and negative amplification of the cross-reactions methods demonstrated the high specificity of this assay.and the identification results of five testing centers are consistent.The result showed that this high sensitivity and good specificity method accurate quantitative detection of Vc and Vp by ddPCR efficient molecular biology tool to diagnose,which had accurate technique and practical application valueand it promotes the development of ddPCR in China.
关键词
微滴数字PCR/霍乱弧菌/副溶血性弧菌/定量检测
Key words
Droplet digital polymerase chain reaction/Vibrio cholerae/Vibrio parahaemolyticus/quantitative detection
维普
万方数据