猴D型反转录病毒巢式PCR检测方法的建立  

Establishment of Nested PCR Assay for SRV-1 Detection

作　　者：孙敏[1] 涂新明[1] 丛喆[1] 蒋虹[1] 佟巍[1] 于浩[1] 魏强[1] 

机构地区：[1]中国医学科学院实验动物研究所中国协和医科大学实验动物学部,北京100021

出　　处：《中国实验动物学报》2006年第4期287-289,共3页Acta Laboratorium Animalis Scientia Sinica

摘　　要：目的建立SRV-1巢式PCR检测方法并进行初步应用。方法针对SRV-1env基因的保守区序列,设计特异性引物,以感染SRV-1 Raji细胞提取出的含有前病毒DNA的基因组DNA为模板,进行巢式PCR反应。扩增产物测序后与GenBank报道的序列进行同源比对。将DNA样本进行10倍梯度稀释,以检测巢式PCR反应的灵敏度。使用该方法对正常Raji细胞以及感染SIV、STLV的外周血淋巴细胞DNA样本进行扩增,检测该方法的特异性。用建立的巢式PCR方法检测40份储存猴血标本。结果使用巢式PCR扩增出的特异片段经测序分析,结果证实与GenBank报道的序列一致。所建立的巢式PCR检测法检测限度可达1.5×10-3ng/μL,而且方法特异。用此方法检测40份猴血标本,未检测到阳性标本。结论初步建立SRV-1的巢式PCR检测方法,该方法灵敏、特异,为SRV-1的检测提供了一个快速、有效的手段。Objective To establish a nested PCR assay for SRV-1 detection ,and using this method to detect SRV-1 in 40 blood samples from rhesus macaques. Methods Two pairs of primers were designed according to the conserved env of SRV-1 sequence. DNA extracted from the Raji cells infected with SRV/D serotype 1 was used as amplification template and the PCR product was sequenced. SRV-1DNA samples were serially tenfold diluted to determine the sensitivity of the method, and DNAs extracted from normal Raji cells and PBMCs infected with SIV and STLV were used as template to determine the specificity of the method. 40 archived blood samples from macaques were assayed using this nested PCR method. Results The PCR product was proved to be identical with the SRV-1 sequence reported. This method was sensitive and specific, and the sensitivity was 1.5×10^-3 ng/μL. No positive result was obtained from the 40 blood samples using this method. Conclusion A nested PCR assay to detect SRV-1 has been established. This is a sensitive, specific, rapid method and can be used as an useful tool for SRV-1 detection.

关 键 词：β逆转录病毒 聚合酶链反应 检测 

分 类 号：R373.5[医药卫生—病原生物学]