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A novel multiplex real-time RT-PCR assay with FRET hybridization probes for the detection and quantitation of 13 respiratory viruses.

ABSTRACT: Quantitative multiplex real-time RT-PCR assays utilizing fluorescence resonance energy transfer (FRET) hybridization probes were developed for the detection of 13 respiratory viruses, including well recognized viral causes (respiratory syncytial virus, influenza viruses A and B, parainfluenza viruses types 1, 2, and 3, adenovirus) as well as viruses described recently as causes of acute respiratory tract infections (human coronaviruses NL63, HKU1, 229E, and OC43, human bocavirus, and human metapneumovirus). FRET probes have an improved toleration for single base mismatches than other probe chemistries, reducing the chances of missing highly variable RNA viruses. The assay could detect 2.5-25 DNA/RNA copies/microl (2.5 x 10(3)-2.5 x 10(4) copies/ml). Validation on 91 known positive respiratory specimens indicated similar specificity as commercial direct immunofluorescence assays (IFA) or single-round PCRs used in initial identification. Screening of 270 IFA negative respiratory specimens identified new viruses in 40/270 (14.8%) cases and additional 79/270 (29.3%) well recognized viruses missed by routine diagnostic assays including 6.7% co-infections. All viruses could be detected in the clinical screening panel. The assays demonstrates an improved sensitivity and scope of detecting respiratory viruses relative to routine antigen detection assays while the quantitative utility may facilitate investigation of the role of co-infections and viral load in respiratory virus pathogenesis.

SUBMITTER: Lassauniere R

PROVIDER: S-EPMC7112774 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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A novel multiplex real-time RT-PCR assay with FRET hybridization probes for the detection and quantitation of 13 respiratory viruses.

Lassaunière R R Kresfelder T T Venter M M

Journal of virological methods 20100211 2

Quantitative multiplex real-time RT-PCR assays utilizing fluorescence resonance energy transfer (FRET) hybridization probes were developed for the detection of 13 respiratory viruses, including well recognized viral causes (respiratory syncytial virus, influenza viruses A and B, parainfluenza viruses types 1, 2, and 3, adenovirus) as well as viruses described recently as causes of acute respiratory tract infections (human coronaviruses NL63, HKU1, 229E, and OC43, human bocavirus, and human metap ...[more]

PMID: 20153377

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Project description:Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction.An rRT-PCR assay targeting the 5' untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log?? cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1-4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested.This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this assay in dengue-endemic areas has the potential to improve both dengue diagnosis and epidemiologic surveillance.

Dataset Information

A novel multiplex real-time RT-PCR assay with FRET hybridization probes for the detection and quantitation of 13 respiratory viruses.

Publications

A novel multiplex real-time RT-PCR assay with FRET hybridization probes for the detection and quantitation of 13 respiratory viruses.

Similar Datasets

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