Rapid routine detection of enterovirus RNA in cerebrospinal fluid by a one-step real-time RT-PCR assay

Abstract

Background and objectives This study provides a one-step transcription/real-time (TaqMan probe) PCR assay (TM-PCR) with new consensus primer and probe sequences for generic detection of human pathogenic enteroviruses including difficult to detect ones like for instance Echovirus 30. The amplicon included parts of domain IV and V of the highly conserved internal ribosomal entry site. Generic detection was confirmed by testing a panel of 41 prototypes representing all five human enterovirus/poliovirus species. Study design and results The 95% detection limit was found to be 100 copies per run using in vitro transcribed coxsackievirus B3 RNA. TM-PCR was compared to an in house nested-PCR assay implemented in detecting enterovirus RNA from CSF samples of patients suffering from meningitis and encephalitis. Concordant results were obtained in all samples (11 positive, 101 negative). Specificity was confirmed with laboratory strains of other neurotropic viruses, and by testing 76 CSF samples of patients with encephalomyelitis disseminata, which all gave negative results. Conclusions The new TM-PCR is a convincing alternative to conventional PCR protocols for the diagnosis of enterovirus meningitis. The one-step strategy limits hands on time and cross contamination risk combined with accelerated assay procedure of only 100 min.