Establishment and validation of two duplex one-step real-time RT-PCR assays for diagnosis of foot-and-mouth disease - INRAE - Institut national de recherche pour l’agriculture, l’alimentation et l’environnement Access content directly
Journal Articles Journal of Virological Methods Year : 2016

Establishment and validation of two duplex one-step real-time RT-PCR assays for diagnosis of foot-and-mouth disease

K. Gorna
  • Function : Author
A. Relmy
  • Function : Author
A. Romey
S. Zientara
L. Bakkali-Kassimi

Abstract

Two duplex one-step TaqMan-based RT-PCR protocols for detection of foot-and-mouth disease virus (FMDV) were established and validated. Each RT-PCR test consists of a ready-to-use master mix for simultaneous detection of the well established 3D or IRES FMDV targets and incorporates the host (3 actin mRNA as an internal control target, in a single-tube assay. The two real-time RT-PCR 3D/beta-actin and IRES/beta-actin tests are highly sensitive and able to detect up to 7 TCID50/ml of FMDV and 10 copies/1 mu l of viral RNA. In field epithelium samples, the diagnostic sensitivity was 100% (95% CI; 91-100%) for the 3D/beta-actin test and 97% (95% CI; 87-100%) for the IRES/beta-actin test. The diagnostic specificity was 100% (95% CI; 95-100%) for both RT-PCRs. In addition, the two protocols proved to be robust, showing inter assay coefficients of variation ranging from 1.94% to 6.73% for the IRES target and from 2.33% to 5.42% for the 3D target for different RNA extractions and different RT-PCR conditions. The internally controlled one-step real-time RT-PCR protocols described in this study provide a rapid, effective and reliable method for the detection of FMDV and thus may improve the routine diagnosis for foot-and-mouth disease.
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Dates and versions

hal-03941538 , version 1 (23-06-2023)

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K. Gorna, A. Relmy, A. Romey, S. Zientara, S. Blaise-Boisseau, et al.. Establishment and validation of two duplex one-step real-time RT-PCR assays for diagnosis of foot-and-mouth disease. Journal of Virological Methods, 2016, 235, pp.168-175. ⟨10.1016/j.jviromet.2016.03.020⟩. ⟨hal-03941538⟩
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