Identification of enteroviruses in clinical specimens by competitive PCR followed by genetic typing using sequence analysis

Abstract

A sensitive method based on competitive PCR was developed to detect and quantitate enteroviral RNA in clinical specimens, with special emphasis on controlling contamination and the presence of potential inhibitory factors in the specimens. Oligonucleotide primers from the conserved parts of the 5' untranslated and VP2 capsid protein-coding regions were selected to differentiate between enteroviruses and rhinoviruses on the basis of the length of the cDNA amplicons. RNA transcribed from a truncated cDNA copy of the echovirus 11 genome was used as an internal control for the reverse transcription reaction and PCR. This allowed simple differentiation of the control and viral PCR products from each other by agarose gel electrophoresis and nonradioactive quantitation of the viral RNA in the clinical specimens. By direct sequencing of the PCR products and subsequent computer analysis of the data, potential laboratory contaminations could be monitored and enteroviruses from clinical samples could be grouped into four distinct clusters, thus enabling genetic typing of the viruses. The described method can be applied to the diagnosis and epidemiology of enteroviral infections.