Detection and rapid differentiation of human enteroviruses following genomic amplification

Abstract

By employing a nested PCR (n-PCR) with specific primers derived from the 5' nontranslated and consensus region of the human enterovirus genome, we detected enterovirus RNA from 32 different serotypes of prototypic strains. A specific 297-bp fragment was amplified by this method from all of these strains. Not only was the method highly sensitive, detecting enterovirus RNA extracted from 0.01 50% tissue culture infective dose/50 μl (which is more sensitive than our current routine method of enterovirus diagnosis, based on the virus isolation and secotypic neutralization), but it was also relatively rapid. By using this technology, we also detected enterovirus RNA in uncultured specimens (including throat swabs and stools) from patients with respiratory illness and acute flaccid paralysis syndrome. This method enabled us to rapidly and directly distinguish enterovirus-infected specimens from nonenterovirus specimens in laboratory diagnosis. Furthermore, restriction fragment length polymorphism was assessed as an alternative means of differentiating various serotypes of prototypical enteroviruses. Fourteen of 16 human enterovirus-infected specimens exhibited restriction patterns identical to those of the corresponding prototypes.