A simple competitive RT-PCR assay for quantitation of HIV-1 subtype B and non-B RNA in plasma.
An easy, inexpensive competitive RT-PCR assay for HIV-1 RNA quantitation was constructed. A 138-bp sequence in the HIV-1 gag p24 region was selected as the target and co-amplified with competitor RNA containing an internal 44-bp deletion. Quantitation of serial dilutions of control RNA samples prepared from the LAI isolate demonstrated a good linearity (R(2)=0.991) within the range between 10 and 250 copies/sample. The detection limit of the assay was determined to be 3.8 copies/sample by Probit analysis and corresponded to 110 copies/ml in plasma. The intra-assay CV value was 9.1%, and the inter-assay value was 25.9%. Both were comparable to those obtained with commercially available HIV-1 RNA quantitation kits. The correlation efficient for the results obtained in 47 plasma samples from HIV-1-infected individuals (subtype A in 1, subtype B in 25, subtype C in 4, subtype F in 1, and CRF01 AE in 16) with the competitive RT-PCR and Cobas Amplicor HIV-1 Monitor test v1.5 was 0.956 for subtype B and 0.947 for subtype non-B. The assay devised is a good alternative for monitoring antiretroviral therapy in resource-poor countries.
Hamatake
Makiko
M
Department of Molecular Virology, Tokyo Medical Dental University, Bunkyo, Tokyo 1138510, Japan.
Nishizawa
Masako
M
Yamamoto
Naoki
N
Kato
Shingo
S
Sugiura
Wataru
W
D016655:HIV Core Protein p24 / Q000235:genetics; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D012367:RNA, Viral / Q000097:blood*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D019562:Viral Load
D016655:HIV Core Protein p24; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic