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目的研究与解决ＰＣＲ检测ＨＣＶＲＮＡ过程中常见的产物污染问题．方法以稀释的含ｄＵＴＰ的ＰＣＲ产物加入ＲＴＰＣＲ反应的不同阶段以模拟ＰＣＲ产物污染，再采用尿苷酶以清除污染的ＰＣＲ产物分子．结果在检测ＨＣＶＲＮＡ的套式ＰＣＲ反应中，假阳性结果主要是由于ＰＣＲ终产物在实验过程中的任何阶段污染所引起，但污染的模板分子仅在第２次ＰＣＲ反应时扩增．将ＨＣＶＲＮＡ套式ＰＣＲ检测试剂盒中的第２次ＰＣＲ反应液中加入一定量的ｄＵＴＰ，可使反应终产物核酸分子上含有ｄＵＴＰ．此后，在每进行第２次ＰＣＲ反应前，均在每３０μｌ反应体系中加入尿苷酶１Ｕ，３７℃消化１ｈ，然后９４℃１０ｍｉｎ灭活尿苷酶，即可降解其中含ｄＵＴＰ的污染核酸分子．结论此法能消除同一实验室内套式ＰＣＲ终产物的污染，防止假阳性结果的出现．
Objective To study and solve the common problem of product contamination during PCR detection of HCV RNA. Methods The diluted PCR products containing dUTP were added to different stages of RT-PCR reaction to simulate the contamination of PCR products, and uridine enzyme was used to remove the contaminated PCR product molecules. Results In the nested PCR reaction for detecting HCV RNA, the false positive result was mainly due to the contamination of the PCR final product during any stage of the experiment, but the contaminated template molecule only amplified during the second PCR reaction. In the second PCR reaction in the HCV RNA nested PCR detection kit, a certain amount of dUTP is added to enable dUTP to be contained in the nucleic acid molecule of the final reaction product. Thereafter, before each second PCR reaction, 1 U of uridinease was added per 30 μl of the reaction system, digested at 37 ° C for 1 hour and then inactivated at 10 ° C at 94 ° C to degrade the contaminating nucleic acid molecule containing dUTP . Conclusion This method can eliminate the contamination of the end-products in the same laboratory and prevent the occurrence of false-positive results.