Feasibility of detection of HTV-1 proviral DNA on dried blood spot samples on filter paper

Objective To assess the feasibility of detection of HIV-1 proviral DNA on Dried Blood Spot (DBS) samples by nested-PCR assay. Methods 59 HIV-1 positive samples of IDUs and 22 HIV-1 negative samples of healthy persons collected freshly EDTA anticoagulant whole blood were respectively dropped on the filter paper(903# SS) to prepare DBS samples, reserved the corresponding whole blood and centrifuged to obtain plasma. 200μl 10% chelex100 suspension was used to extract the DNA genome of DBS on filter paper. DNA extraction from 200μl whole blood was performed by the QIAamp mini kit. Roche Cobas Amplicor RT-PCR assay were used to test viral load in plasma samples. A total of 10μl of DNA solution extracted from whole blood or from DBS was used for in-house nested PCR to amplify parts of the HIV-1 env, pol and gag genome regions. To test for the presence of PCR inhibitors in elution of DBS, the humanβ-actin gene was amplified in PCR assay. At least two parts of the HIV-1 three genes were positive if theβ-actin gene was amplified, the sample were considered HIV-1 positive. The results obtained on DBS by the three nPCR assays were compared to the corresponding results of whole blood. Results Theβ-actin genome on all DBS samples was amplified by PCR. The sensitivity of HIV-1 nPCR tests to detect HIV-1 proviral DNA on 59 HIV-1 positive DBS samples was 93% (95% CI 89% -97% ), and that on 34 whole blood samples was 94% ( 95% CI 89% ~ 98% ), x2 analysis, P = 0. 899 0.01, no statistically difference between the two kinds of samples in detection of HIV-1 infection. The specificity of HIV-1 proviral DNA by nPCR tests on 22 HIV-1 negative DBS versus whole blood samples were both 100% (95% CI 95. 93% ~ 100% ). From the trend (no statistically analysis) , repeatability and efficiency of detection of HIV-1 on 8 DBS samples corresponding plasma Viral Load( VL) 4. 0 Log was precede 5 DBS samples whose plasma VL 4.0 Log. Concordance of HIV-1 proviral DNA detection by nPCR from 18 paired DBS and whole blood samples was 94% , for one whole blood sample nPCR test was positive, but DBS sample was negative. Conclusions DNA genome extracted from DBS specimen can be amplified by in-house nested-PCR to diagnosing HIV-1 infection. But there are much less HIV-1 pro-viral DNA in three 3-mm discs DBS ( absorbed 15μl whole blood) when VL 4. 0 Log, to enhance detecting probability of HIV-1 DNA on DBS, it is necessary to diploid and triplicate nPCR tests. DBS on filter paper facilitate blood collection and reduce the biohazard risk to health care workers, and provide a practical way for sample processing in remote area or difficulty in whole blood collection of HIV-1 positive patients, furthermore, give a reliable way early diagnosis of HIV-1 infection in children born to HIV-infection mothers.