肺炎克雷伯菌Ⅲ型菌毛PCR检测方法的建立

Establishment of PCR assay for detection of type 3 fimbriae of Klebsiella pneumoniae

以肺炎克雷伯菌Ⅲ型菌毛主要结构亚单位基因为靶序列,设计合成了1对可扩增长609 bp目的片段的引物,建立检测肺炎克雷伯菌Ⅲ型菌毛结构基因的PCR方法。特异性试验表明,本室保存的表达Ⅲ型菌毛的鸡源肺炎克雷伯菌临床分离株Kpn7扩增出609 bp的特征性片段,而大肠埃希菌、副伤寒沙门菌、胸膜肺炎放线杆菌、猪链球菌和金黄色葡萄球菌扩增产物均未检出;敏感性试验表明,PCR方法检测该菌基因组DNA的灵敏度为16.8 ng/L,检测该菌菌液的灵敏度为3.7×102CFU/mL。用此方法对10份临床疑似病例分离物进行检测,结果有5株阳性。表明此方法特异性和敏感性都很高,可用于临床Ⅲ型菌毛肺炎克雷伯菌病的辅助诊断和流行病学调查。

A pair of primers was designed to specifically amplified a 609 bp fragment according to type 3 fimbriae structural gene sequences.The PCR method for the detection of type 3 fimbriae of Klebsiella pneumoniae was established successfully.The specificity assay showed that type 3 fimbriae of isolation strains avian Klebsiella pneumoniae which had been preserved in our laboratory were positive.No positive results appeared in E.coli,S.paratyphi,A.pleuropneumoniae,S.suis and S.aureus.The sensitivity result showed that as little as 16.8 ng/L of DNA and 3.7×102 CFU/mL could be detected by the established PCR.The detection results for clinical samples tissue showed that 5 strains were positive.This assay was proved to be specific and sensitive for diagnosis and epidemiological survey of the disease caused by type 3 fimbriae of Klebsiella pneumoniae.