Establishment of Cheap and Reliable Real-Time PCR for Quantitation of HIV-1 Viral Load in Plasma

Kanittaporn Supadej MSc*, Sorasak Intorasoot PhD*

Affiliation : * Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand

Objective : To establish an inexpensive and reliable real-time PCR for quantitation of HIV-1 RNA from plasma samples. Material and Method: Previously analyzed 145 HIV-1 positive plasma samples with viral load ranging from less than 40 to approximately 1,000,000 copies/ml were included in the present study. HIV-1 gag gene was amplified and cloned into TA cloning vector. External standard curve was plotted using in vitro transcribed HIV-1 RNA and utilized for viral quantitation in the samples. Scramble nucleotides located in HIV-1 specific probe was subsequently constructed and used for individual systemic control. The correlation coefficient and Bland-Altman plot were applied for statistical analysis of the two methods.
Results : The limit of quantitation of the validated assay was 31 copies/ml and the linear range was approximate 31-1 x 107 copies/ml. After reproducibility determination using intra- and inter-run assay, it was implied that the coefficient of variation (%CV) was significantly increased while the low copy number of RNA was examined. A highly correlation (r2 = 0.8099) and good agreement were obtained when the two assays were compared.
Conclusion : Developed real-time PCR was inexpensive and reliable for quantitation of HIV-1 viral load in plasma.

Keywords : HIV-1 RNA, viral load, real-time PCR

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