Rapid detection of human rhinoviruses in nasopharyngeal aspirates by a microwell reverse transcription-PCR-hybridization assay

Abstract

A rapid and sensitive microwell reverse transcription (RT)-PCR-hybridization assay was developed to detect human rhinoviruses in clinical specimens and cell culture suspensions. Two hundred three nasopharyngeal aspirates collected from children with symptoms of respiratory disease were analyzed by a classical rolling-tube cell culture method, microwell culture of HeLa Ohio cell monolayers, and RT-PCR with detection of the amplicons in a microwell hybridization assay. The RT-PCR was also done with harvests of the microwell cultures. RNA was extracted with a commercial kit, and the RT-PCR procedure was carried out with microtiter-format equipment. A confirmatory test that exploited a blocking oligonucleotide at the hybridization step was developed to reliably identify marginally positive specimens. Of the 203 nasopharyngeal aspirate specimens, rhinovirus or rhinoviral RNA was detected in 111 specimens (55%). Ninety-eight specimens (48%) were found to be positive by RT-PCR of the original nasopharyngeal aspirates, while the conventional rolling-tube cell culture method yielded 52 (26%) positive specimens. This RT-PCR method with solid-phase hybridization is easy to perform, sensitive, and specific and will be especially useful for analysis of large numbers of clinical specimens.

FIG. 1

Detection of different prototype rhinoviruses and enteroviruses in a 2% agarose gel after RT-PCR. Lane M, DNA molecular size marker (Life Technologies); rhinovirus lanes 1 to 12, rhinovirus serotypes 1B, 2, 9, 11, 12, 13A, 13B, 14, 29, 38, 39, and 48, respectively; lane N, negative control; enterovirus lanes 1 to 17, polioviruses types 1, 2, and 3, enterovirus type 70, coxsackievirus types A9, A16, A21, B1, B3, B4, and B5, and echovirus types 1, 6, 7, 11, 22, and 30, respectively.