不同核酸抽提方法对HCV-RNA检测效果的影响

Influences on the detection of HCV-RNA with different methods of extraction of nucleosides

采用TEK（0．2mol／LTris－HCLpH7．6．250mmol／LEDTA，200mg／L蛋白酶K）作为裂解剂处理样本，再用无水乙醇沉淀HCVRNA作为模板，用于多聚酶链反应检测HCVRNA，该法与AGPC法及TENS法比较，由于不使用SDS，避免TaqDNA多聚酶的强抑制原，省略了酚-氯抽提这一步骤，而又达到分高纯化RNA模板的目的。经对215例抗HCV阳性血清用上述三种方法进行比较分析，结果用三种方法制备的RNA模板进行多聚酶链反应，其HCVRNA阳性率无显著性差异．但TEK法较AGPC法及TENS法操作简单、稳定、不易污染，特异性好等优点，经用加挂生物素的合成寡核苷酸探针对HCVRNA逆转录PCR扩增产物作斑点杂交，显示有很好的特异性，具有明显的实用价值，批量测定在一个工作日可完成，此法的建立为制备RNA模板提供了方法及经验，适合标本量较大的临床单位使用，值得推广应用。

The samples were treated by splitting agent composed of TEK (0. 2mol/L Tris-HCl pH7. 6,250mmol/L EDTA.200mg/L proteinase K),RNA was precipitated with anhydrous ethanol and regarded as template to detect HCV RNA by polymerase chain reaction (PCR). Because sodium dodecyl sulfate (SDS) was not used to avoid a strong inhibitor of Tag DNA,the step of phenol-chlorine extraction was omitted,in comparison with AGPC and TENS. There Were no differences in positive rates of HCV RNA detected by PCR using three kinds of RNA templates prepared with TEK,AGPC and TENS in 215 serum samples of anti-HCV positive. The TEK was stable.specific,simple and less contamination than AGPC and TENS. A good specificity was presented by spot hybridizition of extended reverse transcription PCR products detected by synthesized oligonucleotide probe connected with biotin. The TEK can be accomplished for a batch of serum samples in one day and it is applicable for large scale determination.