兔多杀性巴氏杆菌16S rRNA PCR检测方法的建立

Establishment of PCR assay based on 16S rRNA for the diagnosis of Pasteurella multocida infection in rabbits

为建立一种快速准确检测兔多杀性巴氏杆菌(P.multocida)的PCR方法,本研究以P.multocida的高度保守的16S rRNA为靶基因,参考已公布的P.multocida的16S rRNA基因设计1对特异性引物,优化PCR反应条件,建立了P.multocida PCR快速检测方法。该PCR方法的敏感性达到60 cfu/mL,采用该PCR方法扩增P.multocida标准株和分离株均能扩增出643 bp的目的片段,扩增兔大肠杆菌、支气管败血波氏杆菌结果为阴性,证明本实验所建立的P.multocida PCR检测方法快速、敏感、特异、可靠,可用于P.multocida的快速鉴定与诊断。同时用建立的PCR方法对临床疑似病兔的脏器分离菌进行扩增,可扩增出目的条带,与细菌的分离结果相一致。

To establish rapid method for detection of Pasteurella multocida, a PCR detection method was developed with a pair of primers designed according to a conservative seqence of 16S rRNA gene of P. multocida available in GenBank. Under the optimized conditions of the PCR reaction, the specific fragment of 643 bp was amplified from either standard strain or clinically isolated P. multocida with a limit detection of 60 cfu/mL, but no cross-reaction amplification were fotmd in E. coli and Bordetella bronehiseptica. The results demonstrated that the method was sensitive, specific and could be deserved for further clinical application.