Determination of HIV-1 subtypes (A-D, F, G, CRF01_AE) by PCR in the transmembrane region (gp41) with novel primers

Abstract

HIV-1 has a huge genetic diversity. So far, nine subtypes have been isolated, namely, subtypes A, B, C, D, F, G, H, J, and K. Epidemiological study provides information which may help in the development of HIV-1 prevention programs or health policies. In the future, subtyping may also be critical for vaccine development, and an effective anti-viral drug will need to be effective for different subtypes of HIV virus. The analysis of the nucleotide sequence of the v3 region is considered the most reliable method for determining the HIV-1 subtype. However, the procedures for determining the v3 sequences are complicated and time consuming, requiring expensive reagents, equipment, and well-trained personnel. The polymerase chain reaction (PCR) method using subtype-specific primers for HIV-1 subtyping is easier and faster. The objective of this study was to develop subtype-specific primers for subtyping PCR. The specific primers were designed for subtypes A, B, C, D, F, G, and CRF01_AE, and these primers could be applied to assay for various HIV-1 subtypes in the clinical samples. The specific primers were designed for each subtypes in the gp41 region. The result of PCR was compared with the subtypes which was determined by the v3 sequence. The results of subtyping by PCR using the newly designed primers could detect 29 of 33 patients tested, and all matched those obtained by nucleotide sequencing of the env v3 region except for three subjects, which were differentiated as CRF02_AG. The newly designed primers functioned accurately and conclusively. In comparison with PCR as a method for the determination of subtypes, sequence analysis requires better-trained personnel, more expensive reagents, and more equipment and time.

Testing a Subtype-Specific Gp41 Amplification Method for Genotyping Individuals Infected by Human Immunodeficiency Virus Type-1 in the Brazilian Population of Itajaí, South Brazil

“…Similarities between the patient sequences that were obtained in this study to other sequences in the NCBI HIV database (National Center for Biotechnology Information) were determined using the BLAST online tool (http://ncbi.nlm.nih.gov//BLAST). To verify the subtypes that were classified, a maximum likelihood phylogenetic tree was constructed based on the Tamura-Nei model using env reference sequences from the HIV database to determine the phylogenetic locations of the sequences from the patients that were subtyped using the method of YAGYU et al 29 . Confidence of the branches was evaluated by bootstrapping 500 replications.…”

“…Viral load (highest recorded, last recorded and mean of the last six exams) for each patient (n = 98) was determined by the NASBA (Nuclisens, Bioméreux) method. The viral load for each patient was used for a comparison with the HIV-1 subtype that was detected by PCR amplification using the method of YAGYU et al 29 . Patients also completed a form indicating their age and the mode by which they believed they were infected.…”

“…The gp41 region from the proviral HIV-1 DNA was amplified by PCR using subtype-specific primers and methods as described by YAGYU et al 29 (Table 1). This method consists of sequential, nested PCR amplification steps that result in the amplification of different DNA fragments of specific sizes (Table 1) based on the HIV-1 subtype.…”

“…Phylogenetic studies demonstrated that the gp41 sequence and the C2-V3 nucleotide sequence of gp120 showed concordance in the classification of HIV-1 subtypes 1,17 . Additionally, conserved regions in the gp41 coding region were identified, allowing the design of primers for subtype-specific amplification and identification of HIV-1 29 .…”

“…YAGYU et al 28,29 described a rapid and simple method for the identification of HIV-1 subtypes by PCR amplification of a region of gp41 using subtype-specific primers. Therefore, our aim was to test whether the method of YAGYU et al 29 could classify HIV-1 subtypes in seropositive individuals from the population of Itajaí (Santa Catarina, South Brazil).…”