摘要
建立套式引物聚合酶链式反应检测全血ＤＮＡ滤纸片中的ＨＩＶ１前病毒基因ｐｏ１ＤＮＡ片段。分别采集１２例ＨＩＶ１感染者的外周血约５０μｌ，经与ＥＤＴＡ－蛋白酶ｋ孵育后滴在无菌滤纸片上，３７℃下干燥，将滤纸片密封于塑料袋中，在４℃及室温下保存１周～６０周后，分别将滤纸片置０．５ｍｌ试管中直接进行ＨＩＶ１ｐｏ１基因的外侧引物ＰＣＲ检测，然后进行内侧引物的ＰＣＲ检测。结果表明，全血ＤＮＡ滤纸片在室温下保存２８周，在４℃下保存３４周仍可检出ＨＩＶ１目的基因。根据ＰＣＲ产物的琼脂糖凝胶电泳溴化乙啶染色带形并参比实验设立的标准对照可直接判断结果。该方法具有快速、特异、敏感的特点，敏感性可以达到检出１０个靶ＤＮＡ分子。
A method for detection of proviral human immunodeficiency virus type 1 DNA fragment encoded pol gene in whole blood DNA on filter paper by nested primers polymerase chain reaction (nPCR) has been developed.Aliquots of 50 μl whole blood from each case of 12 HIV1 infected persons were collected and dripped onto the pieces of filter paper after incubated with EDTA proteinase k,and the paper was air dried,sealed in a plastic bag at 4℃ or room temperature for 1 to 60 weeks.The PCR with outer primers was performed in 0.5 ml tube with the piece of filter paper,and then the PCR with inner primers was finished.The results showed that template DNA could be detected in pieces of filter paper which stored at 4℃ for 34 weeks as well as stored at room temperature for 28 weeks.The results could be directly determined by ethidium bromide staining of the PCR products,as compared with the PCR products of standard position control and DNA molecular weight marker in electrophoresis analysis on agarose gel.The method is rapid,specific,and sensitive,and has the ability to detect as little as 10 proviral template DNA.It could be used as an alternative method of antibody confirming detection in HIV1 infection.
出处
《山西医药杂志》
CAS
1998年第5期393-396,共4页
Shanxi Medical Journal
基金
山东省自然科学基金
