空肠弯曲菌多重聚合酶链反应基因鉴定及其毒力相关基因分析

张茂俊; 顾一心; 冉陆; 张建中

Abstract

张茂俊,顾一心,冉陆,张建中.空肠弯曲菌多重聚合酶链反应基因鉴定及其毒力相关基因分析[J].Chinese journal of Epidemiology,2007,28(4):377-380

空肠弯曲菌多重聚合酶链反应基因鉴定及其毒力相关基因分析

Multi-PCR identification and virulence genes detection of Campylobacter jejuni isolated from China

Received:October 10, 2006

DOI：

KeyWord: 空肠弯曲菌结肠弯曲菌多重聚合酶链反应毒力、毒素相关基因

English Key Word: Campylobacter jejuni Campylobacter coli Multiplex polymerase chain reaction Virulence and toxin genes

FundProject:

Author Name	Affiliation	E-mail
ZHANG Maojun	National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
GU Yixin	National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
RAN Lu	中国疾病预防控制中心传染病应急办公室肠道传染病组
ZHANG Jianzhong	National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China	Helic099@sina．com

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Abstract:

目的<\b> 建立空肠弯曲菌、结肠弯曲菌多重聚合酶链反应(m-PCR)鉴定方法<\b>,并对菌株毒力相关基因cadF、virB11、flaA、cdtA、cdtB、cdtC的分布进行初步分析.方法<\b> 利用m-PCR的方法<\b>对经过传统生物化学方法<\b>鉴定的65株弯曲菌进行鉴定,并通过空肠弯曲菌特异基因hipO、结肠弯曲菌glyA基因特异片段的PCR扩增对鉴定的结果<\b>进一步验证;利用特异引物PCR方法<\b>初步分析其毒力、毒素相关基因cadF、virB11、flaA、cdtA、cdtB和cdtC的分布.结果<\b> m-PCR扩增结果<\b>发现65株不同来源的弯曲菌,42株为空肠弯曲菌,23株为结肠弯曲菌.hipO、glyA基因PCR扩增结果<\b>与m-PCR扩增结果<\b>一致.16.9%菌株PCR鉴定结果<\b>与传统的生物化学鉴定结果<\b>不一致.100%(65/65)菌株cadF、flaA基因阳性,并且PCR扩增片段大小一致.virB的阳性率为10.8%(7/65),其中空肠弯曲菌阳性率11.9%(5/42),结肠弯曲菌阳性率为8.7%(2/23).空肠弯曲菌、结肠弯曲菌cdtA的阳性率分别为100%(42/42)和78%(18/23);空肠弯曲菌cdtB扩增的的阳性率为97.6%(41/42),而23株结肠弯曲菌扩增结果<\b>均为阴性;空肠弯曲菌、结肠弯曲菌cdtC的阳性率皆为100%,但PCR扩增产物大小不一致,空肠弯曲菌为555 bp,结肠弯曲菌约为465 bp.结论<\b> m-PCR的方法<\b>可以有效的对空肠弯曲菌和结肠弯曲菌进行属内分型.中国菌株细胞溶涨毒素基因簇cdt基因的特征与文献报道国外菌株存在差异.

English Abstract:

Objective<\b> This study was to simultaneously identify Campylobacter jejuni and Campylobacter coli isolates in China by Multi.PCR assav and to studv the prevalence of six virulence and toxin genes on them.Methods<\b> A multi-PCR method with three sets of primers specifically designed for application of a 16S rRNA as a universal control，mapA，ceuE based on the specific sequence of C.jejuni and C.coli，was applied to detect 65 Campylobacter isolates from China.Another two separately PCR Primers were directed towards the hippuricase gene(hipO) characteristic of C.jejuni and glyA gene characteristic of C.coli were performed for further confirmation.The presence of the cadF，virB11，flaA， cdtA，cdtB，cdtC genes among these 65 strains were investigated by PCR.Results<\b> From multi-PCR detection，42 is01ates belonged to C.jejuni，other 23 isolates belong to C.coli.Data showing the identification were 100％in concordance with the separated PCR for hipO and glyA amplification.The efficiencv(1 00％)of identification by these three primers multi-PCR method was higher than the biochemical test(83.1％).The cndF and flaA genes were detected from 100％(65／65)of the isolates and the PCR product of each gene were identical with each isolate.0nly 10.8％(7／65)of the isolates were positive for virB11.The cdtA gene was found in 92％(60／65)of the isolates.97.6％(41／42)of C.jejuni had cdtB gene，whereas no PCR product with this primers for all the C.coZi is01ates.cd￡C was presented in all the isolates but the lengths of PCR products were different.For C.jejuni，it was 555 bp， for C.coli，it was about 465 bp.Conclusion<\b> This three primers simultaneous multi-PCR method seemed to be useful for the identification of C.i8mHi and C.coZi isOlates from China since cadF and flaA genes were widelv spread in Campylobacter isolates in this countrv.The present report on virB11 was similar to previous reports fromother countries，but the distribution of cd￡gene cluster in Campylobacter species isolated from China might be different.

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