LAMP技术检测单纯疱疹病毒-2型的初步应用

Detection of Herpes Simple Virus Type 2 DNA With Loop-mediated Isothermal Amplification Assay

目的建立一种对单纯疱疹病毒-2型(HSV-2)进行快速、高效、简便的LAMP检测方法。方法用病毒DNA提取试剂盒提取HSV-2基因组DNA,设计4条扩增HSV-2糖蛋白gG基因的LAMP引物,以灭菌水为阴性对照,进行LAMP,LAMP产物经电泳、酶切鉴定。将HSV-2 DNA作10倍系列稀释后进行LAMP,检测其敏感性。结果HSV-2 LAMP产物电泳后呈LAMP特征性梯状条带,对照组无扩增条带。LAMP产物的特异性通过限制性内切酶得到了证实。LAMP检测HSV-2的敏感性为0.01 ng/μL。结论检测HSV-2的LAMP方法特异、敏感、简便。

Objective To detect herpes simple virus type 2 （ HSV - 2 ） DNA with loop - mediated isothermal amplification （ LAMP） assay. Methods HSV - 2 DNA was extracted by Virus DNA extraction kit. four primers which recognized six distinct regions on the glycoprotein G （gG） gene of HSV - 2 were needed and used for LAMP assay. To evaluate the specificity of the assay, sterilized water was used as negative control. LAMP results were judged by electrophoretic analysis and restriction digestion. To evaluate the sensitivity of the LAMP, the HSV- 2 DNA was 10 -fold serially diluted with sterilized water and was amplified by LAMP. Results The LAMP product in HSV - 2 tube were confirmed by gel electrophoresis and a characteristic DNA ladder was observed. In constract, no bands was observed for the negative control in the study. The specificity of LAMP product was confirmed by restriction enzymes. The detection limit of LAMP assay was 0.01 ng/μL. Conclusion The LAMP assay for detection of HSV - 2 is specific, sensitive and simple.