Diagnosis of enterovirus and rhinovirus infections by RT-PCR and time-resolved fluorometry with lanthanide chelate labeled probes.
Detection of enteroviruses and rhinoviruses has traditionally been based on laborious and time-consuming virus isolation. Recently, rapid and sensitive assays for detecting enterovirus and rhinovirus genomic sequences by reverse transcription-polymerase chain reaction (RT-PCR) have been introduced. An RT-PCR assay is described that amplifies both enteroviral and rhinoviral sequences, followed by liquid-phase hybridization carried out in a microtiter plate format. In the hybridization assay, amplicons are identified by enterovirus- or rhinovirus-specific probes carrying lanthanide chelate labels, which can be detected simultaneously by time-resolved fluorometry. The sensitivity and specificity of the RT-PCR-hybridization method were evaluated with a representative collection of enteroviruses and rhinoviruses and tested further its applicability to the clinical setting with cerebrospinal fluid samples and nasopharyngeal aspirates. The RT-PCR assay amplified all enteroviruses and rhinoviruses tested, and all but one amplicon gave a positive result in the subsequent hybridization assay. The RT-PCR-hybridization method was more sensitive than virus isolation for the detection of enteroviruses and rhinoviruses in the clinical samples. High sensitivity, rapidity, and easy performance make the assay suitable for the routine diagnosis of enterovirus and rhinovirus infections.
Lönnrot
M
M
Department of Virology, Medical School and University Hospital of Tampere University, Tampere, Finland. maria.lonnrot@uta.fi
Sjöroos
M
M
Salminen
K
K
Maaronen
M
M
Hyypiä
T
T
Hyöty
H
H
D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000821:virology; D002460:Cell Line; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000134:cerebrospinal fluid / Q000175:diagnosis* / Q000821:virology; D005470:Fluorometry / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007811:Lanthanum / Q000378:metabolism*; D010850:Picornaviridae Infections / Q000134:cerebrospinal fluid / Q000175:diagnosis* / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis; D012229:Rhinovirus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D012367:RNA, Viral; D007811:Lanthanum