Application of these molecular techniques as diagnostic tools has become increasingly important. In this respect, World Health Organization (WHO) has recommended that diagnostic devices should be “ASSURED”: affordable, sensitive, specific, user-friendly, rapid and robust, equipment free, and deliverable to end users, especially in case of developing countries. Typically NAATs 1 require a significant investment in equipment, training and infrastructures. In the past decade, molecular techniques and particularly PCR as a rapid and sensitive method has revolutionalized detection of a variety of infectious viruses. This assay may represent an alternative rapid and relatively inexpensive screening method for detection of HIV-1/HCV co-infection especially in blood screening. The primers and molecular beacon probes only detected HIV-1 and all major variants of HCV. 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 results demonstrate that a specificity of 100 % and sensitivity of 96 % can be achieved. 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). A well-conserved region in the HIV-1 pol gene and 5′NCR of HCV genome were used for primers and molecular beacon design. 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. Various nucleic acid assays have been developed for diagnostics and therapeutic monitoring of infections. These two viruses are transmitted most primarily by exposure to infected blood or blood products. At least 10 million individuals worldwide are co-infected with immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV).
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