生物制品中逆转录酶活性检测

Reverse transcriptase assay of biological products

在以往报道的逆转录酶 (RT)检测方法的基础上 ,以噬菌体MS2RNA为模板 ,在有外源RT作用下 ,缩短逆转录的时间 ,产生特异性cDNA ,经过PCR扩增 ,增加了试验的灵敏度。在PCR过程中 ,加入了RnaseA酶消化步骤 ,降低了逆转录反应的 pH值到 5 3,并用高浓度的琼脂糖凝胶观察扩增产物 ,减低了由于细胞内DNA聚合酶造成的假阳性结果的产生 。

In this study an assay using the MS2 RNA template and primers was developed from the previously established method by Pyra et al.RNA of bacteriophage MS2 serves as the template for RT mediated cDNA synthesis.A specific region of the cDNA product is then amplified by the polymerase chain reaction to increase the sensitivity of cDNA detection.A simple high resolution agrose gel was used as the endpoint for the assay.In addition,the pH of the RT reaction was lowered to pH 5.3,the time of RT incubation was 1 h,These reduce false positives caused by cellular polymerases and lead to an assay which is more specific and sensitive than conventional RT assays.