Quantification of HIV-1 RNA Load by One-Tube-One-Step RT PCR and Real Time PCR Assay with TaqMan Probe
Veerachai Watanaveeradej MD*, Sudalak Sirirattanaphoomee BSc**, Wasun Chantratita PhD***, Sorachai Nitayaphan MD, PhD**, Kwanjai Viputtikul MSc**, Angkool Kerdpanich MD*, Rudiwilai Samakoses MD*, Sriluck Simasathien MD*
Affiliation : *Deparment of Pediatrics, Phramongkutklao Hospital, Bangkok, Thailand **Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand ***Mahidol University, Bangkok, Thailand
Objectives : To develop a less expensive assay to calculate HIV-1 viral load for use in resource-limited
countries.
Materials and 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 106 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.
Conclusion : A new HIV-1 viral load assay was developed and validated. It was reliable and less
expensive.
Keywords : HIV, Viral load, Real time PCR, TaqMan
