Design and development of TaqMan Real-time PCR assay for detection and viral load determination of HIV-1

Human Immunodeficiency Virus Type 1 (HIV-1) is one of the most important blood-borne infectious viruses that are considered a global problem, thus it is important to diagnose it with high accuracy and sensitivity. Serologic methods do not adequately detect this infection. Therefore, the purpose of the present study was to design a sensitive method based on TaqMan Real-time PCR method for diagnosis of HIV-1. Primers and probes were designed using bioinformatics softwares for a region of 200 pairs of HIV-1 INT gene. The sequence was cloned into T/A cloning vector and in-vitro RNA transcription was performed to prepare standards for analytical sensitivity assay. To determine the analytical specificity, NCBI BLAST and different viral and bacterial samples were used. Clinical specificity was determined using negative plasma samples. The method introduced was able to detect as low as 10 copies of HIV-1 RNA/ml. Furthermore, it was linear in the range of 10-109 copies/ml. By examining the negative samples, the specificity of this method was determined to be 100%. Intra- and Inter-assay results ranged from 0.3% to 2.5% and 0.7% to 4.5%, respectively, that showed high reproducibility of the assay. Due to proper sensitivity and specificity, rapid analysis, being user-friendly, and relatively low cost, as compared with commercial kits, the method introduced in the present study can be suitable to accurately diagnose HIV-1 virus. Applying this in-house Real-time PCR assay, viral infection can also be detected before seroconversion and appearance of bloodstream antibodies, which can reduce window period of this infection.