Design and optimization of a multiplex real-time PCR assay for detection of Hepatitis C virus (HCV), Human immunodeficiency virus-1 (HIV-1) and Human hepegivirus-1 (HHpgV-1).

Aim: The objective of this study was to develop a real-time PCR method to detect hepatitis C virus (HCV), human immunodeficiency virus-1 (HIV-1), and also recently discovered human hepegivirus-1 (HHpgV-1) through melting curve analysis using SYBR Green dye in a single reaction. Background: It is estimated that chronic viral infections affect millions of people worldwide, and a significant number of these individuals are unaware of their infection. In addition, infection with newly discovered and emerging viruses may put public health at potential risks. Precise and early detection is a primary and important task for controlling infections and helping patients. Methods: A set of specific primers were designed for detection of HCV, HIV-1, and HHpgV-1 in a single tube. The real-time polymerase chain reaction was performed and melt curves were analyzed. Specificity was assessed by cross-reaction tests with other common blood-borne pathogens. The established assay was evaluated for the detection of viruses in clinical samples. Results: The three viruses were clearly distinguished by their respective melting temperature values. The detection limits of this assay were 102 copies/ml for each virus. The clinical evaluation of this assay was demonstrated by analyzing 134 patients' serum samples. The specificity of the developed assay considered as 100%. Conclusion: The developed multiplex real-time PCR based on SYBR Green dye is a well-suited detection assay of single or co-infections of HCV, HIV, and HHpgV-1 in clinical and research laboratories. It can be used as a cost-effective, rapid tool for routine diagnostic in-house tests.