Use of whole blood and dried blood spot for detection of HIV-1 nucleic acids using reverse transcription loop-mediated isothermal amplification

For monitoring viral load (VL) or Early Infant Diagnosis (EID) of HIV-1, real-time Polymerase Chain Reaction (qPCR) is used to perform on plasma or Dried Blood Spot (DBS) sample. The qPCR method is expensive and requires sophisticated equipment. Therefore, there is a requirement for newer and cheaper technology for VL measurement or EID. In this analytical study, a Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) assay was optimized and applied for amplification of HIV nucleic acids (NA) extracted from plasma, heat-treated plasma, heat-treated whole blood and lysis buffer-treated dried blood spot (DBS). The amplified product of RT-LAMP assay was detected by color change of Hydroxy naphthol blue (HNB) dye, step ladder pattern band on agarose gel after electrophoresis and sigmoid-shaped curve in the real-time thermal cycler. Comparing the results from RT-LAMP testing of all conditions with the results obtained by RT-qPCR results, viewed as the gold standard; a relative analytical sensitivity and specificity of RT-LAMP was calculated as 100 % and 90 % respectively. The corresponding positive predictive value (PPV) and negative predictive value (NPV) were 93.75 % and 100 %, respectively. The percentage of agreement between the RT-LAMP and RT-qPCR was 88.46% and Cohen's kappa value was 0.75 shows a substantial agreement between the two tests. This study suggests that whole blood or DBS may be useful specimens for analysis by HIV-1 specific RT-LAMP, to provide a cost effective alternative to RT-qPCR for the detection of HIV-1 nucleic acid at the point of care, or in early infant diagnoses.