恒温核酸扩增方法的建立及其扩增产物的鉴定

Nucleic acid isothermal amplification and its product

目的建立一种快速、简便的恒温核酸扩增（ＮＡＳＢＡ）方法。方法用由ＡＭＶＲＴａｓｅ、ＲＮａｓｅＨ、Ｔ７ＲＮＡ聚合酶以及相应的缓冲成分组成的恒温扩增系统，对４例丙型肝炎患者血清中丙型肝炎病毒（ＨＣＶ）Ｅ区ＲＮＡ序列进行扩增，并通过Ｎｏｔｈｅｒｎｂｌｏｔ杂交及限制性内切酶对扩增产物进行鉴定。结果经９０分钟或１８０分钟反应后均可扩增出预期大小的产物，放射自显影在预期位置出现阳性杂交信号。扩增产物经聚合酶链反应（ＰＣＲ）及内切酶ＳｍａⅠ消化后产生预期大小的１４４ｂｐ和３５９ｂｐ的片段，第１次ＰＣＲ产物经转录实验后与ＮＡＳＢＡ直接扩增的产物具有较好的同源性。结论ＮＡＳＢＡ法是一种快速简便的核酸扩增方法，此法扩增出的产物具有良好的特异性。

Objective To establish NASBA method which amplified DNA and RNA by isothermal way.Methods HCV RNA was extracted from the patient's serum by conventional method.HCV E RNA was amplified by standard NASBA system with two primers deduced from HCV E gene. The product of amplification was analyzed by Northern Blot and restriction enzyme. The NASBA product was converted to DNA by PCR assay using two primers inside this amplification area.Results The HCV E RNA was amplified after 90min NASBA reaction,and the product was positive in the Northern Blot assay,using an 32 Plabeled oligonucleic acid as aprobe.After the digestion of PCR product of NASBA with restriction enzyme SmaⅠ,two DNA bands were observed: 144bp and 359bp. Conclusion The product of NASBA method was specific and it can be auseful tool in the investigati on of HCV RNA and the diagnosis of HCV infection.