The Siv Plasma Viral Load Assay For Detection And Optimization

The simian immunodeficiency syndrome model is believed to be the animal model that most closed to human immunodeficiency syndrome.The viral load testing is very important in the research related to simian immunodeficiency syndrome model.Although we had engaged in simian immunodeficiency syndrome model related research for a decade years,the method for testing SIV viral load hadn't been established in our lab.With the progress of technology and equipment,we established the SIV viral load testing method.According to the 83 SIV complete genome sequences searched in the Genbank, The conservative fragment was identified and emlpoyed as the PCR target sequence.The primer and probe for quantitative PCR were designed and another pair of primer was designed for amplifing a little longer target than quantitative PCR target which include the quantitative PCR target sequence, and the sense primer include a T7 promotor sequence.The SIVRNA was.amplied with the primer which include T7 promotor sequence and the product was retrieved for SIV standard RNA synthesis use T7 RNA polymerase.SIVmac251 was amplified in the CEM×174 cell line to be the viral particle standard and compared with the synthesised SIV RNA standard.The results showed that both the viral particle standard and synthesised SIV RNA standard are fitful to be a SIV standard for Quantitative PCR assay.However the viral particle standard is superior to the synthesised SIV RNA standard in practical use.The lower limit of the SIV quantitative PCR assay was explored.The comperison of TaqMan and allglo probe in the quantitative PCR method was conducted.The results showed that the lower limit of the SIV quantitative PCR assay is 50 copies/ml.The reproducibility of Allglo probe is significantly superior to the TaqMan probe in the lowest limit of 50copies/ml.The TaqMan probe method and allglo probe method was respectively used to test the plasma SIV load of samples retrieved from monkey experiments for Chinese herb anti-AIDS effect evaluation.The comperison of these two methods were conducted.Theω,namely ratio of the distance and median of duplication must smaller than 1 was introduced to judge if the duplication could be accepted. Theωwas also used in the comperison of TaqMan probe method and allglo probe method.The results showed that theωof Allglo probe method was smaller than TaqMan probe method overall.This research established a sensitive quantitative PCR method for SIV load testing.The creteria for accuracy judgement of duplication in quantitative PCR method was introduced.This research settle a base for the common practice of SIV load.