Establishment of a real-time fluorescent quantitative PCR for the rapid detection of viral reservoir in the peripheral blood of SIV-infected rhesus monkeys / 中华微生物学和免疫学杂志

ABSTRACT

Objective To establish a TaqMan probe-based real-time fluorescent quantitative PCR ( real-time PCR) for the quantitative and rapid detection of viral reservoir in peripheral blood mononuclear cells (PBMCs) isolated from rhesus monkeys with simian immunodeficiency virus (SIV) infection and to evaluate its preliminary application. Methods A pair of primers and one TaqMan probe were designed ac-cording to the conserved sequence of SIVmac239 strain for real-time PCR amplification. A length of 2 090 bp of nucleotide fragment was digested from the plasmid p239SpSp5 containing 5′-end long segments of SIV-mac239 strain by restriction enzymes EcoRⅠand SpeⅠ. The standards used for quantitative detection of SIV DNA in peripheral blood samples were prepared by a 10-fold serial dilution and used for graphing the stand-ard curve. The numbers of SIV DNA ( copies per 106 PBMCs) in rhesus monkeys during acute and chronic phases of SIVmac239 infection were determined and the virological characteristics of SIV DNA at different phages of infection were analyzed. Results A linear positive correlation between cycle threshold ( Ct) val-ues and concentrations (10 copies/μl to 109 copies/μl) of the standards was found. High levels of SIV DNA were monitored in SIV-infected monkeys 14 to 22 days after acute infection. The levels of SIV DNA in the acute phase of infection were about 1 to 2 logs higher than those in the chronic phase of infection. The num-bers of SIV DNA ( copies per 106 PBMCs) were 1 log lower than the SIV viral load in peripheral blood of the same monkey. The ratios of SIV DNA load to SIV RNA load ( DNA/RNA) in chronic phase of infection were higher than those in the acute phase. Conclusion The established TaqMan probe-based real-time fluorescent quantitative PCR was a highly sensitive and specific assay for the detection of SIV DNA with an advantage of wide linear range. It could be used for the quantitative evaluation of latent reservoirs of SIV.