摘要
目的建立一种特异、灵敏、简便的交叉引物恒温扩增(cross priming amplification,CPA)技术,快速检测副溶血性弧菌(Vibrio parahaemolyticus,VP)耐热直接溶血素基因(tdh)。方法根据Gen Bank上公布的VP tdh基因序列,在其保守区域设计CPA引物和探针,并对反应体系和反应条件进行优化,同时验证方法的灵敏度和特异性。结果本方法对tdh基因检测具有高度特异性;对tdh阳性质粒DNA和菌液检测的灵敏度分别达到了1.8×101copy/μl和6.2×103cfu/ml;对82份临床标本的检测阳性率与荧光PCR法相同,大大高于传统培养法。结论本研究建立的CPA法快速、简便、灵敏、特异,适用于VP腹泻患者的床边诊断以及食物中毒的现场快速检验中。
Objective To develop cross priming amplification（ CPA） technology for specific,sensitive,and simple detection of the thermostable direce hemolysin gene（ tdh） of Vibrio parahaemolyticus（ VP）. Methods According to the VP tdh gene from the Gen Bank,the CPA primers and probes were designed in the conservative region,and the reaction system and reaction conditions were optimized,and the sensitivity and specificity of the method were verified. Results The method was highly specific for the detection of the tdh gene; the detection limit of the positive plasmid and bacteria liquid was up to 1. 8 × 101 copy / μl,and6. 2 × 103 cfu / ml,respectively. The positive rate of CPA method in 82 clinical specimens was the same as the fluorescent PCR method,which was significantly higher than that of the traditional culture method. Conclusion The CPA method established in this study is rapid,simple,sensitive and specific,which is suitable for the diagnosis of VP patients with diarrhea and the rapid detection of food poisoning in the field.
出处
《中国卫生检验杂志》
CAS
2016年第13期1906-1908,1953,共4页
Chinese Journal of Health Laboratory Technology
基金
杭州市社会发展科研攻关项目(20130533B46)
浙江省医药卫生科技计划(2014ZHA003)
