细胞培养中逆转录酶活性检测方法的建立与应用

Establishment and application of reverse transcriptase activity detection method in cultured histiocytes

目的采用RT-PCR原理,建立有良好敏感性与可重复性的逆转录酶活性检测方法,用于组织细胞中逆转录酶活性的检测。方法以MS2噬菌体RNA为模板,设计一对引物,采用逆转录酶催化合成cDNA后再用Taq DNA聚合酶进行PCR反应,琼脂糖凝胶电泳检测有无特异大小的目的条带出现,从而确定样品是否具有逆转录酶活性。结果所建立的逆转录酶活性检测方法检出限为2×10-4U,目的条带为308 bp。经琼脂糖电泳能较好的观察判断,送检组织细胞培养液与裂解上清液样品均未检出逆转录酶活性阳性。结论所建立的方法能有效地用于组织细胞培养中逆转录酶活性的检测。

Objective To establish a method to detect the activity of reverse transcriptase in cell culture based on transcription and PCR amplification. Methods A pair of primers were designed with MS2 bacteriophage RNA as template to synthesize cD- NA by reverse transcriptase catalysis. Then Taq DNA polymerase was amplified by PCR, and agarose gel electrophoresis was employed to detect the target band to identify the activity of reverse transcriptase in samples. Results The detection limit of the reverse transcriptase activity detection method was 2 × 10-4 U, and the size of targeted fragments was 308 bp. The agarose gel e- lectrophoresis showed that normal cell culture medium MEM and mrc -5 cell lysates were both not found reverse transcriptase activity. Conclusion The developed method could be applied to detect the activity of reverse transcriptase in histiocyte cul- ture.