[Establishment of a real-time RT-PCR to detect plasma viral load of simian/human immunodeficiency virus CN97001 during its in vivo passage in rhesus monkeys].
OBJECTIVE
To establish a real-time RT-PCR based plasma virus quantification method and monitor the plasma viral load of SHIV-CN97001 during its in vivo passages in rhesus macaques.


METHODS
Viral RNA standards were prepared by in vitro transcription and one-tube real-time RT-PCR were established and optimized using TaqMan EZ RT-PCR CORE REAGENTS and TaqMan probes and primers directed to the 91 bases within the conserved gag region of SHIV. Plasma viral RNA of 126 plasma samples from rhesus macaques of different viral passages was quantified.


RESULTS
The PCR system was optimized by using serial dilution of standards, and the viral RNA load was detected. The lowest limit of the standard curve reached 2x10(-2) copies/ml. The correlation (r>0.99) and the repetition (CV=4.14 percent) also met the requirement. It was revealed that the viral RNA load of third passage was the highest. Generally, the viral load peaks (10(5)-10(6) copies/ml) appeared at the fourteenth day after the infection or inoculation.


CONCLUSIONS
The method of one-tube real-time RT-PCR was established successfully, which may provide a sensitive way to qualify SHIV viral load. This will contribute to the establishment and application of SHIV/rhesus macaque models. It was also found that the replicative ability of SHIV-CN97001 was enhanced during the first 2 in vivo passages.
Liu
Qiang
Q
National Center for AIDS/STD Prevention and Control, Chinese Center for Disease Control and Prevention, Beijing 100050, China.
Li
Ju
J
Yang
Gui-bo
GB
Xing
Hui
H
Dai
Jie-jie
JJ
Shao
Yi-ming
YM
D000818:Animals; D006678:HIV / Q000235:genetics / Q000302:isolation & purification*; D015658:HIV Infections / Q000821:virology*; D006801:Humans; D008253:Macaca mulatta; D012367:RNA, Viral / Q000097:blood* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012692:Serial Passage; D016097:Simian Acquired Immunodeficiency Syndrome / Q000821:virology*; D015302:Simian Immunodeficiency Virus / Q000235:genetics / Q000302:isolation & purification*; D019562:Viral Load
D012367:RNA, Viral