Diagnosis of enterovirus and rhinovirus infections by RT-PCR and time-resolved fluorometry with lanthanide chelate labeled probes

Abstract

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.

Type 1 Diabetes Is Associated With Enterovirus Infection in Gut Mucosa

“…RNA was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed using two independent methods: a previously described method amplifying a sequence common to all known enterovirus serotypes (25) and a real-time RT-PCR using the same primers and probes. A sample was considered enterovirus positive if it gave a positive signal in at least one of these assays.…”

Enterovirus Infection and Progression From Islet Autoimmunity to Type 1 Diabetes

“…RNA was extracted from 140 μl serum and from 140 μl rectal swab solution according to the manufacturer's protocol (QIAamp viral RNA kit; Qiagen, Hilden, Germany). The presence of enterovirus RNA was detected with RT-PCR using primers specific for the 5′ noncoding region conserved among Picornaviridae and subsequent enterovirus-specific hybridization with lanthanide chelated probes, providing sensitive and specific detection of practically all known enterovirus serotypes (25). All samples with a RT-PCR signal five-fold or higher than a negative control were tested two more times, and a sample was interpreted as positive if at least two out of the three tests were five-fold or higher than the negative control.…”

Enterovirus RNA in Blood Is Linked to the Development of Type 1 Diabetes

“…Screening for enterovirus RNA was done by RT-PCR followed by hybridization of PCR amplicons using an enterovirus-specific probe. The detection limit for the method is <0.015 fg RNA, which is equivalent to fewer than four copies of enteroviral RNA genome (15). All RNA-positive samples were retested twice, and a result of at least two positive tests out of three tests was interpreted as a positive sample.…”