网状分枝扩增技术快速检测大肠埃希菌O157∶H7及其他产志贺样毒素大肠埃希菌

目的：建立一种简便、快速、灵敏和特异的检测食品和临床标本中大肠埃希菌O157∶H7及其他产志贺样毒大肠埃希菌(STEC)的方法。方法：设计并合成了检测志贺毒素2(stx2)基因特异的环形探针和捕获探针，确定网状分枝扩增方法（RAM）的灵敏度；含有stx2基因的不同血清型的菌株包括O46∶H38、O22∶H8 、O111∶NM，用于RAM特异度的测定。用RAM检测所有分离的菌株，并进一步对瞬时RAM进行研究。结果：RAM最低能检测10个拷贝的stx2合成靶基因和临床分离的菌株，表明RAM与PCR具有一样的灵敏度。特异度的测定结果表明不同血清型的大肠埃希菌均为stx2阳性，而非致病性大肠埃希菌ATCC23716则为阴性。在32株菌株中，28株细菌含有stx2基因，其余则为阴性， 与PCR检测结果100%一致。瞬时RAM结果也表明最低能检测10个细菌，检测信号的出现依赖于样品的浓度。 结论：RAM的简便和等温扩增特点可替代PCR检测各种标本中大肠埃希菌O157∶H7和STEC。

Rapid detection of E. coli O157: H7 and other Shiga toxin-producing E. coli using ramification amplification method

Objective To develop a sensitive, specific, rapid and easy method for detecting E. coli O157 : H7 and other Shiga toxin-producing E. coli in food and clinical specimens. Methods A circular probe and capture probe specific for Shiga toxin-2 (stx2) gene had been synthesized and was used for determining the sensitivity of ramification amplification method (RAM). Different serotypes which contained stx2 gene, including an E. coli O157 : H7, an E. coli O46 : H38, an E. coli O111 : NM, an E. coli O22 : H8 and E. coli ATCC23716 (stx2 gene negative) were used for determining the specificity. All strains were detected by RAM to determine whether they contained stx2 gene. Real-time RAM was further studied to detect stx2 gene. Results The lowest number targets detected by RAM assay was 10 copies of stx2, indicating that RAM assay was as sensitive as conventional PCR. The result of specificity showed that different serotypes of strains were all positive for stx2 gene, while nonpathogenic E. coli ATCC23716 was negative. Among 32 isolates, 28 STEC isolates containing stx2 gene were positive by RAM assay, while others were negative. The RAM results were 100% in concordance with that of PCR. The real-time RAM results also showed that as many as 10 bacteria can be detected and the time of appearance of detectable signal was depended on the target concentration. Conclusion RAM assay can offer an alternative method for PCR to detect E. coli O157 : H7 and STEC in all types of specimens because of its simplicity and isothermal amplification.