空肠弯曲菌套式PCR快速检测方法的建立与初步应用

目的建立套式PCR快速检测实验动物空肠弯曲菌的方法。方法根据GenBank数据库的空肠弯曲菌flaA基因设计2对引物，对空肠弯曲菌抽提DNA作PCR扩增及克隆测序。同时，对其他病原菌抽提核酸扩增，并对120份临床样品进行检测。结果空肠弯曲菌经2对引物分别扩增出1719bp和640bp片段，与预期大小一致。套式PCR检测的最低限度为10CFU。整个检测过程可在8h内完成。而其他病原菌未出现特异性扩增条带。采用套式PCR对121份临床样品进行检测，检出31份阳性，与传统的细菌分离方法完全一致。结论本研究建立的套式PCR方法具有很好的特异性和敏感性，可用于实验动物空肠弯曲菌的快速检测。

Establishment and Application of Nested Polymerase Chain Reaction for Rapid Detection of Campylobacter jejuni

Objective To establish a nested polymerase chain reaction(Nested-PCR) assay for rapid detection of Campylobacter jejuni(C.jejuni) in laboratory animals.Method Two pairs of primers were designed according to the flaA gene of C.jejuni.DNA was extracted from C.jejuni.The specific amplified fragment of flaA gene which encoding flagellin A protein of C.jejuni was cloned into the pGEM-T vector and transformed E.coli DH5.The recombinant plasmid was identified by restriction endonuclease analysis,and confirmed by sequencing.Non-C.jejuni and 121 clinical specimens were submitted to detect C.jejuni by nested-PCR.Results C.jejuni PCR amplification product of the external primer was 1 719 bp,and nested-PCR amplification product of the internal primer was 640 bp.The sensitivity of nested-PCR was 10 CFU.The whole detection could be finished within 8 h.Non-C.jejuni did not show specific amplified fragment.121 clinical samples taken randomly from laboratory animals were submitted for detection by nested-PCR and bacteria separation method,with a positive detection rate of 25.6%(31/121) and an agreement of 100%.Conclusion These data demonstrated that the nested-PCR assay established here is a specific and sensitive method for rapid detection of C.jejuni in laboratory animals.