Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcrip...

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Bibliographic Details
Published in:Journal of clinical microbiology 2007-07, Vol.46 (3)
Main Authors: Hindson, B J, Reid, S M, Baker, B R, Ebert, K, Ferris, N P, Bentley Tammero, L F, Lenhoff, R J, Naraghi-Arani, P, Vitalis, E A, Slezak, T R, Hullinger, P J, King, D P
Format: Article
Language:English
Subjects:
60 APPLIED LIFE SCIENCES
ACCURACY
AMPLIFICATION
ANIMALS
BASIC BIOLOGICAL SCIENCES
CATTLE
DETECTION
DIAGNOSIS
DIARRHEA
DISEASES
DNA
DOMESTIC ANIMALS
HYBRIDIZATION
MICROSPHERES
RNA
SCREENS
SENSITIVITY
SWINE
TRANSCRIPTION
VIRUSES
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Summary:A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.
ISSN:0095-1137
1098-660X