多重荧光定量PCR甄别霍乱弧菌方法的建立
张政;金大智;朱水荣;叶菊莲;罗芸
【期刊名称】《中华流行病学杂志》 【年(卷),期】2010(031)009
【摘要】Objective To develop a rapid, sensitive and specific assay method, based on multiplex real time PCR for identifying Vibrio cholerae and distinguishing Vibrio cholerae O139 serotype from Vibrio cholerae. Methods Cholera toxin A subunit gene (ctxA) and glycosyltransferase gene (LPSgt) were chosen as targets according to Vibrio cholerae and Vibrio cholerae O139 serotype,and then the primers and TaqMan-MGB probe were designed. The 5'end of probes was labeled with FAM and VIC fluoresceins respectively while the 3' end of probes was labeled with MGB. The PCR reaction was optimized systematically. Then the specificity, sensitivity and reproducibility of multiplex real time PCR were estimated. Finally, multiplex real time PCR was applied to detect the clinical specimens. Results Vibrio cholerae was identified by multiplex real time PCR accurately and quickly,which could distinguish Vibrio cholerae O139 serotype from Vibrio cholerae. Vibrio cholerae was identified positive for primer pairs and probes from ctxA gene, and Vibrio cholerae O139 serotype tested was positive for LPSgt gene. Meanwhile, none of other bacteria was identified. Sensitivity of the test was 2 × 102 cfu/ml. When this assay was applied directly to
多重荧光定量PCR甄别霍乱弧菌方法的建立
