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
陈曦 1闫梅英 1文远汐 2阚飙 3樊粉霞1
作者信息
- 1. 中国疾病预防控制中心传染病预防控制所 北京 102206
- 2. 山东大学公共卫生学院 济南 250012
- 3. 中国疾病预防控制中心传染病预防控制所 北京 102206;山东大学公共卫生学院 济南 250012
- 折叠
摘要
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.
Abstract
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.
关键词
噬菌体/TaqMan-qPCR/DNA检测/灵敏度/霍乱弧菌Key words
Phage/TaqMan-qPCR/DNA detection/Sensitivity/Vibrio cholerae引用本文复制引用
基金项目
国家重点研发计划(2021YFC2300304)
中国疾病预防控制中心传染病预防控制所传染病监测防治技术能力提升项目(102393240020020000003)
出版年
2024
NETL
NSTL
万方数据