关键词: 网状分枝扩增方法, 环形探针, 聚合酶链反应, 方法

Abstract: 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.Amo ng 32 isolates, 28 STEC isolates containing stx2 gene were positive by RAM assa y, 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.

Key words: ramification amplification method, circular probe, polymerase chain reaction, methods