Comparison of Multiplex PCR-ELISA and conventional Multiplex PCR for detection of HIV-1/HCV co-infection

Abstract

Background and Objectives: PCR is a sensitive assay and could be used as an accurate diagnostic method for detecting various types of microorganisms' genome in low concentration in biological specimens. The demand for sensitive, rapid, safe and easy detection of PCR products has led researchers to a combination of this method with ELISA. Materials and Methods: Conserved sequences were selected for design of primers. Samples were tested by ELISA (for detection of specific HIV and HCV proteins) and real- time PCR for detection of specific nucleic acid and viral genome respectively. Viral genome was extracted and reverse transcription was performed with M-Mulv and the cDNA kept at -80º C. The PCR products were labeled by DIG-dUTP. Diluted PCR products were analyzed with both electrophoresis and ELISA methods. Results: Thirty-five samples were tested with the PCR-ELISA method. False positive or negative reactions were not observed. ELISA results of diluted products were compared with results obtained by electrophoresis. In gel electrophoresis, dilution of 1/10 was positive, but in ELISA, optical density of 1/100 dilution was much more than the cut-off value. Conclusion: Detection limits for gel electrophoresis as well as ELISA have been evaluated. It was shown that the PCR-ELISA method is ten times more sensitive than conventional PCR.

Keywords

• Multiplex
• PCR- ELISA
• HIV
• HCV