聚合酶链反应检测SEN病毒的方法学探讨

Optimal condition of polymerase chain reaction for detection SEN virus DNA

目的 探讨聚合酶链反应 (PCR)扩增SEN病毒 (SENV)核酸的优化条件。方法 将标本分别以AcupureDNA/RNA提取法(方法 1)、SDS 蛋白酶K方法 (方法 2 )及裂解液提取TTV核酸法(方法 3)抽提SENVDNA模板,并用 3组针对SENVDNA不同区的引物进行扩增,比较不同模板提取方法、不同扩增引物及不同体积标本对SENVDNA检出率的影响。结果 在 191份血清中,方法 1、方法 2和方法 3提取核酸后可分别检测出 15份、9份和 7份。采用方法 1提取SENVDNA的基础上,采用 2组套式引物分别扩增出 15份和 11份,一次PCR引物仅检出 2份SENVDNA阳性标本;应用引物 2扩增至少需要 50μl血清。结论 不同引物扩增方法和不同模板提取方法可能是影响SENVDNA检出率重要因素。

Objective To explore the optimal SENV DNA isolation and primers to develop a sensitive ,rapid and specific polymerase chain reaction for the detection of SEN virus Methods Acupure DNA/RNA, SDS proteinase K and distinctive TTV DNA isolation procedures were used for isolation the viral genome, and three groups primers were used to amplify SENV DNA, the influence of different sera isolation method, amplification primers and the sample volume for the positive detection rate of SEN virus were compared Results Among 191 samples, 15, 9 and 7 samples were positive in Acupure DNA/RNA isolation, proteinase K isolation and distinctive TTV DNA isolation respectively The positive PCR result of two nest PCR primers were 15 and 11, only 2 samples were detected with one time PCR and at least 50μl of sample was needed when used primers group 2 Conclusions Different sera isolation method and amplification primers influence the positive detection rate of SEN virus