多重聚合酶链反应检测超广谱β内酰胺酶方法的研究

目的 建立超广谱β内酰胺酶(ESBLs)多重聚合酶链反应(multiplex PCR)的检测方法并对其进行应用评价。方法 通过排序比较选择TEM、SHV和CTX—M型基因编码区保守序列设计型特异性通用引物，建立多重PCR体系，对产ESBLs标准株及54个疑产ESBLs临床分离株进行ESBLs的检测，并通过PCR产物克隆测序及美国临床实验室标准化委员会表型确认试验的平行检测，对该法进行应用评价。结果 多重PCR法对纸片法阳性株及阴性株ESBLs检出率分别为94．3％(33／35)和26．3％(5／19)；测序结果证实了该法的特异性，且敏感性较高。84．2％(32／38)ESBLs基因型为CTX—M型，15．8％(6／38)为SHV型。结论 该法的建立为临床检测ESBLs及其分子流行病学研究提供了一种特异和敏感的工具。

Study on multiplex polymerase chain reaction for detecting extended-spectrum beta-lactamases in Southern China

Objective To establish and evaluate practicable method of Multiplex polymerase chain reaction(PCR)for detecting Extended-spectrum beta-lactamases(ESBLs).Methods Universal primers for TEM,SHV and CTX-M ESBLs genes were designed by aligning their conserved encoding sequences to set up Multiplex PCR systems. This method was then evaluated by testing ESBLs-producing standard and suspicious strains as compared with NCCLS phenotypic confirmatory test and the cloning and sequencing results of its PCR production.Results 94.3%(33/35) ESBLs positive strains and 26.3%(5/19) ESBLs negative strains for NCCLS disk susceptibility test were positive for Multiplex PCR.Of them,84.2%(32/38)are CTX-M-type and 15.8%(6/38) are SHV-type. The results of gene sequencing confirmed the specificity of this method. Conclusion CTX-M-type is the most common genotype in ESBLs-producing clinical isolates in southern China.This Multiplex PCR method for detecting ESBLs can be served as an useful tool in clinical laboratories and molecular epidemiological research.