柯萨奇病毒B3的TaqMan实时荧光定量RT-PCR检测方法的建立  

Development of a TaqMan fluorescent quantitative RT-PCR assay for detection of coxsackievirus B3 mRNA

作　　者：代海丽[1] 高铁磊[1] 朱琳[1] 曹威[2] 栾颖[2] 靳占峰[1] 

机构地区：[1]哈尔滨医科大学病理学教研室和法医教研室,哈尔滨150081 [2]哈尔滨医科大学附属第二医院心内科,哈尔滨150081

出　　处：《中国卫生检验杂志》2012年第1期8-10,共3页Chinese Journal of Health Laboratory Technology

基　　金：黑龙江省教育厅科学技术研究项目(11531079)

摘　　要：目的:建立一种检测柯萨奇病毒B3(Coxsackievirus B3)的实时荧光定量RT-PCR方法,为临床CVB3的快速、准确检测提供一种新的方法。方法:针对CVB3基因的VP4区和管家基因β-actin的核苷酸序列分别设计了特异性的引物和TaqMan荧光探针,以重组质粒pMD18-T-CVB3为标准品,进行实时荧光定量RT-PCR检测,并构建CVB3的荧光定量RT-PCR标准曲线。结果:建立的方法在1.0×101～1.0×108copies/μl模板范围内具有良好的线性关系(r2>0.999),扩增效率高于98.0%,至少可检测10 copies/μl的阳性标准品。用CVB3感染HeLa细胞来模拟实际样品,灵敏度可达到1.0×101copies。结论:建立的荧光定量RT-PCR具有较高的敏感性、特异性和重复性,可作为CVB3的快速诊断方法,为CVB3感染的临床治疗提供快速可靠的诊断依据。Objective：To establish a real -time fluorescent quantitative PCR assay for detection of coxsackievirus B3, and to provide a fast and precise testing way for CVB3. Methods： Specific primers and fluorescent probes based on VP4 gene of CVB3 and housekeeping gene β -actin were designed respectively, the plasmid of pMD18 -T- CVB3 was used as standard product, a real - time fluorescent quantitative PCR was used to construct the standard curve of CVB3. Results： The assay for the quantitation of CVB3 template was well from 1.0×10^1- 1.0 ×10^8 copies/μl with excellent linearity. The coefficient correlation r2 reached 0.999 and amplification efficiency was higher than 98. 0%. The sensitivity was sufficient to detect at least template of 10 copies/μl. Conclusion： The established RT- PCR assay was a highly sensitive, specific and reproducible approach for rapid detection of CVB3, which can provide rapid and reliable diagnosis evidence for clinical therapy of CVB3.

关 键 词：柯萨奇病毒B3 荧光定量RT—PCR TaqMan荧光探针 快速检测 

分 类 号：R446.5[医药卫生—诊断学]