用检测ipaH基因的PCR检测志贺菌属

Detection of ipaH gene of Shigella by PCR

目的建立一种检测志贺菌属快速敏感的PCR方法。方法根据志贺菌属侵袭性质粒抗原H(invasive p lasm id,ipaH)编码基因序列,设计1对PCR引物特异性扩增长度为204 bp的靶基因片段,通过检测3株志贺菌属菌株和6株非志贺菌属菌株来评价该方法的特异性;对福氏志贺菌菌液做一系列10倍稀释后进行PCR分析敏感性,PCR扩增产物经电泳和测序鉴定。结果3株志贺菌属菌株出现特定大小的目的条带,6株非志贺菌属菌株未出现目的条带;测序也证实了PCR产物的特异性;PCR检测福氏志贺菌ipaH基因的下限为10 CFU/m l。结论本研究建立的方法快速、特异、敏感。

Objective To develop a polymerase chain reaction （PCR） assay for rapid, specific and sensitive detection of ShigeUa ipaH gene. Methods According to the sequences of the ipaH gene, primers were designed to amplify ipaH gene. The length of amplicon was 204 bp. To evaluate the specificity of the assay, 3 strains of Shigella and 6 strains of non-Shigella were tested by PCR. One of ShigeUa flexneri was 10-fold serially diluted and amplified by PCR to verify the limit of detection. PCR results were judged by eleetrophoresis and DNA sequence analysis. Results For 3 strains of Shigella, the results of the PCR assay were observed. No amplified bands were observed in 6 strains of non-Shigella. The specificity of PCR assay was also confirmed by DNA sequence analysis. The detection limit of PCR assay was 10 CFU/ml of Shigella flexneri. Conclusions These results indicated that the PCR assay is a rapid, specific and sensitive detection method for Shigella ipaH gene.