Detection of enteroviruses and rhinoviruses in clinical specimens by PCR and liquid-phase hybridization

Abstract

A sensitive method based on PCR followed by liquid-phase hybridization for detection of enterovirus and rhinovirus RNAs in clinical specimens and cell culture supernatants is described. RNA was extracted from stool samples, throat swabs, nasopharyngeal aspirates, cerebrospinal fluid, urine, and plasma with a commercial phenol-guanidinium-chloroform reagent and purified on a polysulfone membrane, on which the reverse transcriptase reaction was also done. Two sets of oligonucleotide primers from the 5 noncoding region of picornaviruses were selected for DNA amplification of 153-bp (enterovirus) and 120-bp (rhinovirus) regions. Double-stranded amplicons were digested into single strands with T7 gene 6 exonuclease and quantitated by an assay using a europium-labeled probe, streptavidin-and biotinylated probe-coated microtitration wells, and time-resolved fluorometry. The sensitivity of the assay was about one template molecule when purified coxsackievirus A9 RNA was used. All enterovirus prototype strains, except echoviruses 22 and 23, and clinical isolates grown in cell culture or suckling mice were strongly positive by the enterovirus PCR-hybridization, as were selected prototype strains and untyped isolates of rhinoviruses by the rhinovirus PCR-hybridization. In a series of 100 clinical specimens tested, the results for 92 agreed with virus culture results. The detection method described will be useful in etiopathogenic studies on enteroviruses and rhinoviruses.Conventional laboratory diagnosis of enterovirus infections includes isolation of the virus in cell culture, less frequently in suckling mice, followed by neutralization typing. The diagnosis can be supported by serology, mainly on the basis of a titer increase between acute-and convalescent-phase serum specimens or detection of immunoglobulin M antibody. Rhinovirus diagnosis is done almost exclusively by culture of the virus followed by acid lability testing. Serology is complicated by more than 100 serotypes. Diagnosis can be delayed because many enteroviruses and rhinoviruses cause cytopathogenic changes in cell cultures only during prolonged incubation, particularly in the first passage. Increased information on enterovirus and rhinovirus sequences (26, 35) has made possible a new and more rapid approach to this diagnostic problem by detection of the viral genome directly in clinical specimens by the PCR. The first application of this technology was for detection of rhinovirus in nasal washes (9, 10). Later, methods were developed to amplify highly conserved sequences present in both viruses followed by a hybridization assay that differentiated between the groups, first with cell culture isolates (15) and then with clinical specimens (25). A number of groups have now reported the use of PCR for direct detection of enteroviruses (1,14,18,24,27,31,33,38,41,43) and rhinoviruses (3,17,18,39) in clinical specimens.In the present report, we describe the development of a highly sensitive nonnested PCR assay followed by liquid-phase hybridization for detection ...

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