Quantification of Simian Immunodeficiency Virus(SIV) Viral Load Using Real-Time Quantitative RT-PCR with SYBR Green I

Objective To develop a real-time quantitative RT-PCR method with SYBR Green I to assess the viral load of SIV.Methods A 477 bp specific fragment was amplified with nested RT-PCR and ligated into a pGEM T vector.The recombinant pGEM-SIVgag477 was transformed into E.coli DH5α competent cells.Large amount of cells were collected and purified after cultured in LB medium.Plasmids DNA were digested by restriction enzyme Not I to linearize DNA after purified from the cells.The linearized plasmids DNA were transcripted into RNA using riboprobe in vitro transcription systems(Promega,P1420).10-fold serial dilutions of the RNA standards were quantified and the actual copy numbers were assessed using real-time quantitative RT-PCR with SYBR Green I by Roche LightCycler.Results From 1×10~8 copies/μL to 1×10~2 copies/μL of 10fold serial diluted RNA could be quantified with real-time quantitative RT-PCR.The standard curve showed that they had good linear correlation and could be served for the quantification of other samples.The specification of amplified products is checked by melting curve analysis.Conclusions The RNA standards could be used as an external standards of the real-time quantitative RT-PCR method with SYBR Green I.The method has high sensitivity,specificity and stability so that it could be used for the quantification of SIV RNA load.