多重PCR-DHPLC快速检测食品中产志贺毒素大肠杆菌

Rapid detection of Shiga toxin-producing Escherichia coli by multiplex PCR and DHPLC

应用多重PCR(motiplex PCR)结合变性高效液相色谱技术(denaturing high-performance liquid chromatography,DHPLC)建立了快速检测食品中产志贺毒素大肠杆菌O111和O157的方法。以基因wzx O111、rfb EO157为靶基因,建立多重PCR-DHPLC方法,进行特异性和灵敏度测试,同时进行RT-PCR检测比较灵敏度。该方法具有良好特异性,可以一次PCR扩增同时检测O111、O157;灵敏度达到25 CFU/m L。129份牛肉样品中检出1例O111,3例O157阳性;74份鸡肉样品中检测出O111、O157阳性各1例,67份蔬菜样品中未检测到O111、O157。本文建立O111、O157多重PCR-DHPLC检测方法,操作简便,特异性强,适用于产志贺毒素大肠杆菌筛选检测。

The method of multiplex PCR combined with denaturing high-performance liquid chromatography was established for rapid detection of Shiga toxin-producing Escherichia coli O111 and O157 in food. The multiplex PCR assay for detecting Shiga toxin-producing Escherichia coli O111 and O157 was developed using primers with specifically amplifing segments of wzxOlll and rfbEO157 genes. The method was carried out for specificity and sensitivity testing and its sensitivity was compared with RT-PCR method at the same time. Results indicated that the multiplex PCR-DHPLC had good specificity; a PCR amplification could detect both O111 and O157 simultaneously. The detection limit of the mPCR was 25 CFU/mL for sensitivity. One of Olll and three of O157 were detected in 129 beef meat samples. In 74 chicken meat samples contained one of O111 and one of O157. Shiga toxin-producing Escherichia coli O111 and O157 were not detected in 67 vegetables. As a result, the multiplex PCR-DHPLC detection method for O111, 0157 was simple, specific and suitable for Shiga toxin Escherichia coli screening test.