改良二氧化硅吸附法检测丙型肝炎病毒核酸

Detection of HCV RNA by polymerasc chain reaction using modified silica adherence method

建立了改良二氧化硅吸附法检测丙型肝炎病毒（ＨＣＶ）ＲＮＡ的方法。将待检血清５０μｌ、二氧化硅悬液２０μｌ、裂解液３００μｌ（含硫氰酸胍、ＥＤＴＡ、ＴｒｉｔｏｎＸ—１００、Ｔｒｉｓ·Ｃｌ）混匀置室温６０分钟，病毒颗粒裂解释放的核酸吸附在二氧化硅颗粒上，经离心和双蒸水洗脱，所得ＨＣＶＲＮＡ模板以５＇ＮＣ区引物作套式聚合酶链反应扩增。使用该方法可检出１０μｌ血清中的ＨＣＶＲＮＡ。１１０份抗－ＨＣＶ阳性血清中ＨＣＶＲＮＡ的检出率为８２．７％。实验全过程约７小时，为丙型肝炎检测提供了快速、简便、灵敏的新方法。

simple,rapid and sensitive method of extracting HCV RNA from serum by using silica is re-ported.Fifty μl of serum sample, 20 μl of silica suspension and 300μl of splitting buffer(containingguanidine thiocyanate, EDTA, Triton X-100 and Tris· HCl) were mixed at room temperature for 60min and then spun for 1 min in a ultracentrifuge. After discarding the supernatant the precipitate waswashed with 800μl of 70% ethanol. HCV RNA adhered onto the cilica particles was eluted with doubledistilled water, and amplified by RT-nested PCR with two sets of primer derived from 5'NC region ofHCV. Using this technique HCV RNA could be detected from as small as 10μl of positive serum. HCVRNA was detected in 91 of 110(82.7%) anti-HCV positive patients.The whole course of the experimentrequired only 7 hours.