摘要
目的利用实时PCR技术,建立检测副溶血弧菌及其毒力株的方法。方法根据副溶血弧菌的跨膜转录激活蛋白toxR基因序列设计引物和改良分子信标(ROX标记),建立检测所有副溶血弧菌菌株的方法。通过与文献报道的检测直接耐热溶血素(TDH)基因的实时PCR体系合并,建立了同时检测副溶血弧菌及其毒力株的双重实时PCR方法。结果通过对9个属的101株菌株进行试验,所有的52株副溶血弧菌均产生ROX阳性信号(toxR^+),其余菌株均检测为阴性,其中46.2% (24/52)的副溶血弧菌产生HEX阳性信号(tdh^+)。检测toxR基因的实时PCR体系DNA灵敏度为10～100fg/PCR体系,菌液灵敏度为59.2～592 CFU/ml或2.96～29.6 CFU/PCR体系。另外成功构建了双重实时PCR体系,能同时对副溶血弧菌的toxR和tdh基因进行检测。结论建立的双重实时PCR体系能同时检测副溶血弧菌及其毒力株,从而为食品的安全检疫提供有效手段。
Objective To establish real-time PCR methods to detect total and toxigenic V. parahaemolyticus. Methods Primers and probe for total V. parahaemolyticus were designed based on the specific toxR gene sequence of V. parahaemolyticus. PCR detection of toxigenic V. parahaemolyticus was done as previously reported. The detection methods were validated through specificity test and sensitivity test. Through integrating the two reaction systems, a duplex real-time PCR for detection of total and toxigenic V. parahaemolyticus was established. Results One hundred and one isolates of 9 genera were tested with real-time PCR targeting to V. parahaemolyticus specific toxR gene and tdh gene. Only V. parahaemolyticus straius（52/52） gave ROX signals （ toxR＊ ）, and no cross-reaction was observed with other bacteria. Only 46.2% （24/52） of V. parahaemolyticus strains gave HEX signals （tdh＊ ）. The sensitivity of the assay for total V. parahaemolyticus was 10-100 fg DNA per PCR reaction, or 2.96-29.6 CFU per PCR reaction （59.2-592 CFU/ml in pure cultures）. Also established was a duplex realtime PCR assay that could detect total and toxigenic V. parahaemolyticus strains simultaneously. Conclusi The established duplex real-time PCR assay could be not only used in the detection of total V. parahaemolyticus, but also provide useful information about its potential pathogenicity.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2007年第7期664-668,共5页
Chinese Journal of Microbiology and Immunology
基金
深圳出入境检验检疫局资助项目(SZK19-2005)
关键词
副溶血弧菌
毒力株
实时PCR
Vibrio parahaemolyticus
Modified molecular beacon
Real-time PCR
