Effective light-upon-extension real-time PCR primer systems for rapid detection of human viruses

Abstract

Background: Real-time polymerase chain reaction (PCR) assays for quantitative detection and typing of viruses are fast, simple, and provide valuable information about the progression of an infection. Identification of human papilloma and polyomaviruses is used to estimate the risk of cancer (papilloma), polyomavirus-associated-nephropathy (BKV), or progressive multifocal leukoencephalopathy (JCV) in individual patients. Thus, non-invasive and specific diagnostics for viral detection should be regarded. Methods: We describe the design, optimization, and application of effective Light-Upon Extension (LUX) real-time PCR systems for detection of papillomaviruses, BKV, and JCV. Viral and human controls along with clinical samples were examined during the optimization process. D-LUX designer software and the Opticon 2 Real-Time System (Bio-Rad, Hercules, CA) were used. Results: We confirm the specificity, rapidity, and non-invasiveness of the method for demonstration of ongoing replication. We believe LUX technology is comparable to probe based real-time PCR assays for the detection of viruses, but it has an advantage in terms of cost-effectiveness, convenience, and possibility for a dissociation analysis. Conclusions: The fluorogenic primer method (LUX) is rapid, uses few toxic chemicals, and is useful for the specific and accurate detection of viral DNA among different types of samples.