Molecular Assay System Validated for Opportunistic Virus Infections

Laboratory-developed real-time polymerase chain reaction (PCR) protocols have been implemented for the molecular diagnosis of opportunistic DNA virus infections.

The validity of a duplex real-time PCR protocols for viral DNA have been improved by using an extraction and amplification control that allows for the monitoring of the molecular diagnostic process.

Virologists at the Pierre and Marie Curie University (Paris, France) tested 152 clinical samples including 77 whole bloods, 30 bronchoalveolar lavages, 28 viral transport medium containing mucocutaneous swabs, 12 urine, and 5 stool samples. The genomes of herpes simplex virus (HSV), varicella-zoster virus (VZV), human cytomegalovirus (CMV), Epstein–Barr virus (EBV), BK virus (BKV), and adenovirus (AdV) were investigated.

The Simplexa extraction and amplification control (SEAC) set (Eurobio; Courtaboeuf, France) was evaluated in this study. This internal control corresponds to a 577-base pair DNA fragment derived from the gene encoding ribulose-1,5-bisphosphate carboxylase oxygenase large unit N-methyltransferase of the plant Arabidopsis thaliana. This is a noncompetitive internal control with its own mix, containing primers and a Quasar 670 labeled-probe specifically designed for its amplification (Biosearch Technologies; Novato, CA, USA). Viral DNA amplifications were performed on the Light cycler LC480 system (Roche Diagnostics; Meylan, France) using laboratory-developed real-time PCR assays based on hydrolysis probe technology implemented in the laboratory for virological diagnosis activity.

The SEAC results showed high reproducibility with a mean crossing point (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 use of the SEAC did not influence the results of the different virus-specific PCRs compared to other systems. The SEAC in the DNA extracts showed high stability during storage at both +4 °C and -20 °C.

The authors concluded that the use of the commercial SEAC is simple, straightforward, and beneficial. It makes not only the detection of opportunistic viruses in clinical samples more convenient and cost-effective, but it ensures also the effectiveness of the whole molecular process implemented in the laboratory to perform the diagnosis of viral infections. The SEAC provides a reliable option to improve the diagnosis of opportunistic viral infections in laboratories using in-house real-time PCR assays. The study was published in the October 2012 edition of the Journal of Virological Methods.

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Pierre and Marie Curie University
Eurobio
Biosearch Technologies