基于噬菌体核酸检测霍乱弧菌的TaqMan-qPCR方法的建立

Establishment of a Direct Phage DNA Detection-based TaqMan-qPCR Method for Vibrio cholerae Identification

目的:建立一种基于噬菌体特异基因检测霍乱弧菌的TaqMan-qPCR方法。方法:基于VP1噬菌体的特异性基因gp46,建立TaqMan-qPCR方法,根据VP1潜伏期为50~60 min,爆发周期持续110~120 min,确定TaqMan-qPCR体系检测时间点。比对分析基于gp46与基于霍乱弧菌毒力基因ctxB建立的两种TaqMan-qPCR方法。结果:基于VP1噬菌体gp46成功建立TaqMan-qPCR反应,扩增效率接近93%,Ct值与样本中噬菌体浓度的相关系数高达0.998。TaqMan-qPCR和琼脂双层噬菌斑计数两种方法结果一致性很高,偏差低于2.45%。基于噬菌体gp46基因比基于毒力基因ctxB建立的TaqMan-qPCR方法的LB模拟样本灵敏度提高10倍,粪便及环境水体模拟样本灵敏度可提高100倍。结论:成功建立快速、高灵敏度和高特异性的TaqMan-qPCR方法,可实现在几小时内对含有霍乱弧菌疑似样本进行快速检测。

Objective:To establish a TaqMan-qPCR method for the detection of Vibrio cholerae based on phage specific genes.Methods:Based on the specific gene gp46 of V.cholerae virulent phage VP1,primers were designed and a TaqMan-qPCR method was established.The detection time of the TaqMan-qPCR system was determined based on the incubation time of VP1 phage under LB,fecal and environmental water substrate conditions,which was 50-60 min,and the outbreak time was 110-120 min.A comparative analysis of two TaqMan-qPCR detection methods was perofrmed based on the phage gene gp46 and the virulence gene ctxB of V.cholerae.Results:A TaqMan-qPCR reaction system based on VP1 phage gp46 was successfully established with an amplification efficiency of nearly 93%.The correlation coefficient between the Ct value and the phage concentration in the sample was as high as 0.998.The consistency of the results between TaqMan-qPCR and agarose double-layer plaque counting methods is high,with a deviation of less than 2.45%.We conducted two TaqMan-qPCR approaches,targeting on the phage-derived gp46 gene and the virulence-associated ctxB gene of V.cholerae respectively.The former has a sensitivity increase of 10 times compared to the latter in LB simulated samples,and the sensitivity of fecal and environmental water simulated samples can be increased by 100 times.Conclusions:The TaqMan-qPCR established in this study has the characteristics of high speed,high sensitivity,and high specificity.It can obtain positive test results within a few hours without the need for bacterial isolation and DNA extraction,and can quickly detect suspected samples containing V.cholerae.