Application of PCR Techniques in Detection of Simian Immunodeficiency Virus (SIV) in Monkey Infection Models
  Revised:January 04, 2005
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KeyWord:Simian Immunodeficiency Virus (SIV),RT PCR, DNA PCR, Nested PCR, Peripheral blood mononuclear cells (PBMCs),Virus isolation
CONG Zhe,TU Xin ming,JIANG Hong,TONG Wei,WEI Qiang,SUN Min,YU Hao
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Abstract:
      Objective (1) To develop a new RT PCR method to assess the viral RNA in the plasma of SIV infected monkeys and to qualitatively compare the sensitivity of RT PCR and traditional methods in detection of virus isolated from plasma. (2) To develop a DNA PCR technique for measurement of proviral DNA load in peripheral blood mononuclear cells (PBMCs). (3) To verify the practicability and maneuverability of DNA PCR and RNA PCR, techniques used in SAIDS monkey models. Methods\ 9 rhesus monkeys (Macaca mulatta) were inoculated with SIVmac251 intravenously. Blood samples were colleted and the virus RNA was extracted from the plasma and the RNA was amplified by RT PCR. High molecular weight genomic DNA was extracted from PBMCs and amplified with specific nested DNA PCR. The PCR products were assessed by electrophoresis in 1 5% agarose gel. Results\ A 477bp specific fragment was amplified with nested DNA PCR and RNA PCR respectively. It was verified by sequencing. 7 days after challenging, among the 9 monkeys, the positive rate was 7/9 assessed by RNA PCR and 100% (9/9) by DNA PCR. At the same time the positive rate was only 5/9 assayed by virus isolation from the plasma. From then until 42 days after inoculation, the results of RNA PCR and DNA PCR were always 100% positive, whereas the positive rate of plasma virus isolation method went down to 4/9 at 35 days and 0 at 42 days postinoculation. Conclusions\ PCR method is more sensitive than virus isolation. Especially, it is true for DNA PCR. It can be used not only to detect infected cells with active virus replication, but also infected cells at transcription latent period. So it may become an important and necessary technique in SAIDS models, especially in the early stage of infection or in the middle and late stages but the viral load in the plasma is rather low or the virus is in a latent period. It should be more sensitive than virus RNA detection in plasma. scriptionlatentperi finfectionorinthe dbemoresensitive
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