Routine use of duplex real-time PCR assays including a commercial internal control for molecular diagnosis of opportunistic DNA virus infections

► Improving the validity of laboratory-developed real-time PCRs for the detection/quantitation of DNA viruses. ► Use of the commercial Simplexa™ extraction and amplification control (SEAC) set. ► The SEAC showed high reproducibility and high stability. ► Procedure for validation of PCR results in quality assurance programs. The aim of this work was to improve the validity of laboratory-developed real-time PCR protocols implemented in the laboratory for molecular diagnosis of opportunistic DNA virus infections using the Simplexa™ extraction and amplification control (SEAC) which allows the monitoring of the whole extraction and amplification process. Herpes simplex virus (HSV), varicella-zoster virus (VZV), human cytomegalovirus (CMV), Epstein–Barr virus (EBV), BK virus (BKV), and adenovirus (AdV) genomes were investigated in 152 different clinical specimens. The use of the SEAC did not influence the results of the different virus-specific PCRs. The SEAC results showed high reproducibility with a mean Cp value of 31.08±1.44, and were not influenced by the virus-specific PCR performed or the type of clinical specimen tested. The SEAC in the DNA extracts showed high stability during storage at both +4°C and −20°C. These data allowed establishing a new procedure for the validation of viral PCR results. In conclusion, the SEAC provides a reliable option for improving the diagnosis of opportunistic viral infections by laboratory-developed real-time PCR assays in quality assurance programs.