PubMed · 4285024
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G H Schaap. 1965-10-02. [Enteroviruses].. https://pubmed.ncbi.nlm.nih.gov/4285024/
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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 microl (which is more sensitive than our current routine method of enterovirus diagnosis, based on the virus isolation and serotypic 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.
The AMPLICOR Enterovirus Test was evaluated with 103 cerebrospinal fluid (CSF) specimens. Twenty-seven CSF specimens were culture positive. With the AMPLICOR test, enterovirus RNA was detected in 34 specimens. Compared with culture, the AMPLICOR test gave a sensitivity of 96.3% and a specificity of 100%. The sensitivity of culture was 79.4% in comparison with the AMPLICOR test.
In this study we present a modified diagnostic routine PCR (polymerase chain reaction) technique for the detection of specific enteroviral nucleic acid sequences in human body fluids, the use of which would reduce the number of false-positive results, the costs and the concentration of reagents required in PCR. Cerebrospinal samples, pharyngeal swabs and faeces were tested. To this end, general primers, selected in the highly conserved 5' non-coding region were used, with a closed-tube RT semi-nested PCR protocol, and the PCR product was analysed using a hybridization in solid phase. A total of 32 patients with suspected enteroviral infection, 17 with suspected viral meningitis and 15 with a possible acute enteroviral infection different from meningitis were analysed, and 80% of the patients with possible acute enteroviral infection were PCR-positive. A broad range of enteroviruses can be detected and false-positive results can be reduced. The availability of results in less than 12 h and the lack of need for radiolabelled probes also increase the convenience of this protocol.