Polymerase Chain Reaction for Human Picornaviruses

Abstract

SUMMARYWe have used enzymic amplification of specific nucleic acid sequences followed by hybridization, for the rapid detection and typing of human picornaviruses after cell culture isolation. The test is based on the synthesis of cDNA, the polymerase chain reaction and the use of oligonucleotide probes. The primers were selected from the 5' non-coding region of the genome representing highly conserved regions. Sequences specific to enteroviruses and rhinoviruses were used as probes. The assay was able to identify all the picornavirus reference strains analysed and it was also possible to discriminate between enteroviruses and rhinoviruses by the hybridization procedure. When 29 picornavirus clinical isolates were analysed, all except one were detected by gel electrophoresis and a specific hybridization signal was obtained with all except three strains using the oligonucleotide probes. INTRODUCTIONHuman picornaviruses consist of approximately 200 serologically distinct members which are important pathogens. Human rhinoviruses (HRVs) are the most frequent cause of the common cold whereas members of enterovirus subgroups (coxsackie-, echo-and polioviruses) are responsible for e.g. meningitis, paralysis, myositis and myocarditis. Because of the large number of serotypes, specific virological diagnosis of these infections is problematic. Enteroviruses are currently detected using isolation in cell culture followed by neutralization typing with antiserum pools. Rhinoviruses are identified after isolation on the basis of their characteristic acid lability. These methods are laborious and time-consuming because at least two cycles in cell culture are needed for specific recognition of the virus. Since these viruses are common causes of human disease there is an obvious need for new rapid and reliable diagnostic procedures.The nucleotide sequences of approximately a dozen human picornaviruses are already known and these data can be used for the selection of nucleic acid probes for diagnostic purposes. The 5'-terminal non-coding part and regions of the ssRNA genome encoding non-structural proteins are highly conserved among human picornaviruses. Both cloned cDNA (Hyypi~t et al., 1984;Rotbart et al., 1984; A1-Nakib et al., 1986) and synthetic oligonucleotides (Rotbart et al., 1988) have been used successfully as broadly reacting probes in hybridization tests. However, these assays require a passage of the virus in cell culture for amplification of the target. Because it would be desirable to have an assay that could directly demonstrate the presence of virus in clinical samples, we have studied the possibility of using a combination of the polymerase chain reaction (PCR) (Saiki et al., 1988) and oligonucleotide hybridization, for human picornavirus detection. With this aim, we have first considered an assay system and reagents which can be used for identification of picornaviruses after a passage in cell culture. Our results indicate that the system based on these methods can be used for broad detection and typing of ...

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