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Volume 14, Issue 4 (9-2011)                   J Arak Uni Med Sci 2011, 14(4): 60-68 | Back to browse issues page

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Falahi S, Ravashad M, Kenarkoohi A. Application of new degenerate primer set for simultaneous isolation of hyper variable subunit of ENV gene from distinct HIV-1 genotypes. J Arak Uni Med Sci. 2011; 14 (4) :60-68
URL: http://jams.arakmu.ac.ir/article-1-541-en.html
1- , ravanshad@modares.ac.ir
Abstract:   (12255 Views)
Background: Polymerase chain reaction is the most common technique in the field of molecular biology that use for amplification of a specific nucleic acid sequence. Degenerate primers have ability to amplify related but distinct sequences. The aim of current study was to use, two sets of degenerate primers in combination with Hemi Nested PCR for detection V3-Loop sequence of envelope gene from wide spectrum of Human Immunodeficiency Virus (HIV) subtypes. Material and Methods: In this experimental study we designed and optimized, a degenerate primer pair in combination with Hemi Nested PCR, to detect HIV-1 V3 loop from Envelop gene that has wide variations among genotypes. The developed assay was used to check, 40 HIV infected, 10 negative controls as well as 5 samples from each HCV, HBV, HGV, and TTV were analyzed using developed method. Results: after optimization, 35 out of 40 positive controls were positive using our test. None of the negative human and viral control samples showed specific band. Also, in positive samples, non-specific bands were not detected. Conclusion: In this study moreover than standard PCR, we used two degenerate primers that could detect specific region of genome. In fact, first round of PCR product act as a template for second round inner primers and can produce smaller sequence with high sensitivity due to degeneracy. Based on the current investigation, the developed assay had advantages including product confirmation and hence more sensitivity.
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Type of Study: Original Atricle | Subject: Infection
Received: 2009/11/29

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