Protocol for the use of a rapid real-time PCR method for the detection of HIV-1 proviral DNA using double-stranded primer.
This chapter describes a real-time PCR method for the detection of HIV-1 proviral DNA in whole blood samples using a novel double-stranded primer system. The assay utilizes a simple commercially available DNA extraction method and a rapid and easy-to-perform real-time PCR protocol to consistently detect a minimum of four copies of HIV-1 group M proviral DNA in as little as 90 min after sample (whole blood) collection. Co-amplification of the human RNase P gene serves as an internal control to monitor the efficiency of both the DNA extraction and amplification. Once the assay is validated properly, it may be suitable as an alternative confirmation test for HIV-1 infections in a variety of HIV testing venues including the mother-to-child transmission testing sites, clinics, and diagnostic testing centers.
Laboratory Branch, Division of HIV/AIDS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, GA, USA. chp3@cdc.gov
Chou-Pong
CP
Pau
Susan K
SK
Wells
Timothy C
TC
Granade
D001769:Blood / Q000821:virology; D017931:DNA Primers / Q000235:genetics*; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics* / Q000302:isolation & purification; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011533:Proviruses / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020079:Terminal Repeat Sequences / Q000235:genetics; D013997:Time Factors
D017931:DNA Primers; D004279:DNA, Viral