Quantification of HIV-1 RNA load by one-tube-one-step RT PCR and real time PCR assay with TaqMan probe.
OBJECTIVE
To develop a less expensive assay to calculate HIV-1 viral load for use in resource-limited countries.


METHODS
An In-house One-tube-one-step Viral Load Assay (IOVA) was developed by using real-time PCR-based with TaqMan probe. Primers and probe were designed from the conserved region of sequences from all HIV subtypes. A standard curve was generated from reference virus in various dilutions. IOVA was applied on 105 HIV-positive and 25 HIV-negative samples and compared with the results from ROCHE AMPLICLOR.


RESULTS
IOVA measured HIV RNA in the samples ranging from 125 to 2 x 10(6) copies/mL. The coefficient of variation of intra- and inter-assay ranged from 0.68% to 7.89%. The sensitivity, specificity, positive and negative predictive values were 92%, 100%, 100% and 79.5% respectively. The parallel quantitative analysis showed high correlation (r=0.95) between IOVA and AMPLICOR.


CONCLUSIONS
A new HIV-1 viral load assay was developed and validated. It was reliable and less expensive.
Watanaveeradej
Veerachai
V
Deparment of Pediatrics, Phramongkutklao Hospital, Bangkok, Thailand. veerachaiw@yahoo.com
Sirirattanaphoomee
Sudalak
S
Chantratita
Wasun
W
Nitayaphan
Sorachai
S
Viputtikul
Kwanjai
K
Kerdpanich
Angkool
A
Samakoses
Rudiwilai
R
Simasathien
Sriluk
S
D000328:Adult; D002648:Child; D015658:HIV Infections / Q000097:blood*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D015347:RNA Probes; D012367:RNA, Viral / Q000097:blood*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
D015347:RNA Probes; D012367:RNA, Viral