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