Nested-PCR检测恒河猴泡沫病毒

Detection of Simian Foamy Virus(SFV)by Nested-PCR

目的用nested-PCR检测恒河猴泡沫病毒SFV的前病毒形式。方法从SFV-1的pol区域选择两对引物分别对原代猴肾细胞(rhesus monkey kidney,RMK)及猴外周血淋巴细胞(peripheral blood lymphocytes,PBLs)进行体外扩增,扩增产物经1.0%的琼脂糖凝胶电泳,证实其特异性。阳性对照使用具有典型泡沫样病变的RMK377细胞株的前病毒DNA,阳性对照的PCR扩增产物经测序证实,阴性对照为恒河猴的SRV-1cDNA。结果nested-PCR能快速灵敏的直接从猴外周血淋巴细胞检出SFV的前病毒形式,与RMK细胞培养结果基本相一致。结论本实验所建立的检测SFV的nested-PCR法能快速准确的检测猴群中的SFV的带毒情况,对于提高实验猴的质量具有重要意义。

Objective To establishe a nested-PCR method for direct detection of SFV proviral DNA in peripheral blood lymphocytes（PBLs）of rhesus monkey.Methods Amplification products of two pairs of primer of SFV-1 pol region from PBLs and RMK cells of rhesus monkeys were separated by 1% agarose gel electrophoresis.The cultured rhesus monkey kidney RMK cell line served as a positive control showing foamy cell pathology.The amplification products of positive control was confirmed by sequencing analysis.The cDNA of SRV-1 served as negative control.Result DNA of SFV provirus was detected directly from PBLs and cultured monkey kidney cells.The results of nested-PCR detection of PBLs and cytological changes of cultured kidney cells were identical with each other.Conclusion This method is a rapid,safe,efficient to detect SFV in rhesus monkeys.It can be an important molecular biological technique in detection of virus infection in rhesus monkeys,and may significantly improve the quality of laboratory monkeys for both researches and vaccine production.