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. 2012 Sep;52(3):456-63.
doi: 10.1007/s12088-012-0271-1. Epub 2012 May 11.

Design and Development of a Multiplex Real-Time PCR Assay for Detection of HIV-1 and HCV Using Molecular Beacons

Mahdi Paryan  1 , Samira Mohammadi-Yeganeh, Siamak Mirab Samiee, Houri Rezvan
Affiliations

Design and Development of a Multiplex Real-Time PCR Assay for Detection of HIV-1 and HCV Using Molecular Beacons

Mahdi Paryan et al. Indian J Microbiol. 2012 Sep.

Abstract

At least 10 million individuals worldwide are co-infected with immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). These two viruses are transmitted most primarily by exposure to infected blood or blood products. Various nucleic acid assays have been developed for diagnostics and therapeutic monitoring of infections. In the present study, a multiplex real-time PCR assay for simultaneous detection of HCV and HIV-1 using molecular beacons were designed and validated. A well-conserved region in the HIV-1 pol gene and 5'NCR of HCV genome were used for primers and molecular beacon design. The analysis of scalar concentrations of the samples indicated that this multiplex procedure detects at least 1,000 copies/ml of HIV-1 and 100 copies/ml of HCV with linear reference curve (R (2) > 0.94). The results demonstrate that a specificity of 100 % and sensitivity of 96 % can be achieved. The analytical sensitivity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes only detected HIV-1 and all major variants of HCV. This assay may represent an alternative rapid and relatively inexpensive screening method for detection of HIV-1/HCV co-infection especially in blood screening.

Keywords: HCV; HIV-1; Molecular beacon; Multiplex real-time PCR.

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Figures

Fig. 1
Fig. 1
Schematic mechanism of molecular beacon
Fig. 2
Fig. 2
Conserved genomic sequences were applied for HCV primers and probe design
Fig. 3
Fig. 3
Conserved genomic sequences were applied for HIV-1 primers and probe design
Fig. 4
Fig. 4
Real-time detection of two different viral genomes in a multiplex format. Each reaction contained two sets of PCR primers specific for unique HIV-1 and HCV nucleotide sequences and two molecular beacons, each specific for one of the two amplicons and labeled with a differently colored fluorophore. HCV is plotted in solid black, multiplex format plotted in dotted–dashed black, HIV is plotted in solid gary and Non template control is plotted in dotted gary line. a FAM/Syber window for HCV detection b JOE/Tet window for HIV-1 detection
See this image and copyright information in PMC

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