天津沿岸海水中创伤弧菌的PCR检测和定量

采用普通PCR方法和SYBR Green实时定量PCR方法,根据创伤弧菌16S rDNA和23S rDNA基因间隔序列(internal transcribed spacer,ITS)设计引物,对渤海湾天津沿岸海水中的创伤弧菌进行了定性和定量检测.8个样品采集自天津市汉沽区沿岸海水,采集时间分别为2008年7月、10月和2009年3月、5月.PCR检测结果表明,这些样品都能扩增出276 bp的ITS序列,这些序列和GenBank上的同源序列(DQ462478)的相似性为96%～98%.用SYBR Green实时定量PCR方法测定了8个海水样品,样品中创伤弧菌的浓度为7.12×10~3～8.40×10~5 个/L,表明天津沿岸海水存在严重的创伤弧菌污染.

PCR detection and quantification of Vibrio vulnificus in the coastal seawater of Tianjin

The paper is aimed at introducing our research on the way of how to detect and quantify Vibrio vulnificus in Tianjin near-coast seawater of Bohai Bay by means of conventional PCR and SYBR green real-time quantitative PCR with the primer pair based on 16S rDNA-23S rDNA internal transcribed spacer (ITS) of Vibrio vulnificus. For our research purpose, we have chosen eight samples from Hangu, one of the above said seawater areas in Tianjin, in July and Oct., 2008 and Mar. And May, 2009. PCR analysis shows that a 276 bp ITS sequence was amplified from all the eight seawater samples and the sequential results show that identity of these DNAs to the homologous sequences in the GenBank (DQ462478) were 96%-98%. In addition, the SYBR Green real-time quantitative PCR analysis also indicates that Vibrio vulnificus concentration in the above-mentioned samples range from 7.12×10~3 cells/L to 8.40×10~5 cells/L, suggesting that there is a serious water contamination problem in Tianjin coastal seawater. The entire work of detection and qualitative analysis of Vibrio vulnificus, including sample processing, extraction of bacterial DNA, conventional and real-time PCR amplifications, proves able to get finished in 12 hours, making it a rapid single-day assay.