RAPID, SENSITIVE, SPECIFIC DETECTION OF ENTEROVIRUSES AND RHINOVIRUSES USING A REAL-TIME PCR FOR ENTEROVIRUSES THAT DETECTS ALL RHINOVIRUSES BY GEL ELECTROPHORESIS

Introduction:  Entero (EV) and rhinoviruses (RV) are important causes of disease in humans.  Rhinoviruses have been reported to cause over 50% of the “common cold” as well as asthma exacerbations and lower tract respiratory infections, while enteroviruses are associated with a wide range of syndromes, including encephalitis and myocarditis.  Traditional cell culture methods for detection of these agents require days to weeks and are less sensitive than nucleic acid detection. For rapid, sensitive identification of EV and RV we evaluated a PCR method that amplifies both RV and EV with the same primers and distinguishes between them by using an EV specific probe. 

Method:  A Real-time PCR assay that can detect EV and RV by SYBRGreen or EV by probe technology (Kares, S., Lonnrot, M., Vuorinen, P., Oikarinen, S., Taurianen, S. and H Hyoty.  Real-time PCR for rapid diagnosis of entero-and rhinovirus infections using LightCycler. 2004. J clin virol. 29:99-104) was adapted by the California Department of Public Health, Viral and Rickettsial Disease Laboratory.  The assays were evaluated for detection and differentiation of EVs from RVs by testing 59 prototype EVs and 87 prototype RVs.  Sensitivity was determined by comparing TCID₅₀ titer of Coxsackie B-3 virus to sensitivity of detection by real-time PCR.   The Viral and Rickettsial Disease Laboratory currently uses this assay for testing clinical specimens for EVs and RVs, and for confirmation of cell culture isolates from respiratory specimens as RVs. 

Results:  All 59/59 prototype EV serotypes tested reacted with the probe giving a positive Real- time PCR reaction.   87 of 87 prototype RVs tested produced a band at the MW constituent with that expected from a RV.  Three of the prototype rhinoviruses, RV 1B, 52 and 87 hybridized with the probe to give a real-time EV positive result.  PCR testing conducted on clinical specimens in 2007 resulted in ninety-one RVs and 57 EV PCR positive reactions.  11/11 amplicons from specimens that were positive in the real-time PCR (EVs) and 23/23 real-time PCR negative, gel electrophoresis positive (RVs) were identified by nucleotide sequence as EV and RV, respectively.  The assay repeatedly detected 0.1 TCID₅₀ of CBV-3 virus. 

Conclusion:  We report results of a PCR assay for Real-time detection of EV and gel detection of RV.  AS EVand RV are increasingly recognized as important viruses that can cause severe illness rapid sensitive detection that can differentiate between EV and RV is needed.