城市生活污水中志贺氏菌ipaH毒力基因的定量PCR检测

Quantitative detection of ipaH gene of Shigella in municipal wastewater by real-time PCR

基于ipaH毒力基因的实时荧光定量PCR检测,建立适合城市污水中志贺氏菌的定量检测方法。使用从临床分离出的志贺氏菌构建重组质粒作为实时荧光定量PCR的标准品。在ipaH基因模板量2.58×100～2.58×106copy范围内具有良好的线性关系,每100 mL水样中含有2.58×101copy以上的ipaH基因即可被检出。从西安市生活污水分离得到2株野生型志贺氏菌,分析ipaH基因数量和菌体数量的关系,从而确定ipaH基因定量检测志贺氏菌的可行性。该方法灵敏、快速、特异性好,适用于城市生活污水中志贺氏菌的检测。

Based on the invasion plasmid antigen gene （ipaH） of virulent Shigella spp. , a real-time fluo-rescent quantitative PCR detection method was established to detect Shigella spp. in municipal wastewater. The Shigella strain separated from clinical patients was used to obtain recombinant plasmid as real-time PCR standard product. The standard curve presented good linear relationship in 2.58 × 10^0 and 2.58 × 10^6 copy. According to the method, more than 2. 58 × 10^1 copy gene in 100 mL water samples can be detected. Two Shigella strains were separated from the municipal sewage, then were used to make certain the relation between ipaH gene quan-tity and bacteria number, which confirmed the feasibility of this method to test Shigella spp. The method is sensi-tive, rapid and specific, and can be used to detect Shigella in municipal wastewater.