Establishment and validation of two duplex one-step real-time RT-PCR assays for diagnosis of foot-and-mouth disease.
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 β-actin mRNA as an internal control target, in a single-tube assay. The two real-time RT-PCR 3D/β-actin and IRES/β-actin tests are highly sensitive and able to detect up to 7TCID50/ml of FMDV and 10 copies/1μl of viral RNA. In field epithelium samples, the diagnostic sensitivity was 100% (95% CI; 91-100%) for the 3D/β-actin test and 97% (95% CI; 87-100%) for the IRES/β-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.
Université Paris EST, ANSES, Laboratoire de Santé Animale, UMR Virologie 1161 (ANSES INRA ENVA), Laboratoire National et OIE de référence pour la fièvre aphteuse, 14 rue Pierre et Marie Curie, 94706 Maisons-Alfort, France.
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Gorna
Université Paris EST, ANSES, Laboratoire de Santé Animale, UMR Virologie 1161 (ANSES INRA ENVA), Laboratoire National et OIE de référence pour la fièvre aphteuse, 14 rue Pierre et Marie Curie, 94706 Maisons-Alfort, France.
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Relmy
Université Paris EST, ANSES, Laboratoire de Santé Animale, UMR Virologie 1161 (ANSES INRA ENVA), Laboratoire National et OIE de référence pour la fièvre aphteuse, 14 rue Pierre et Marie Curie, 94706 Maisons-Alfort, France.
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Romey
Université Paris EST, ANSES, Laboratoire de Santé Animale, UMR Virologie 1161 (ANSES INRA ENVA), Laboratoire National et OIE de référence pour la fièvre aphteuse, 14 rue Pierre et Marie Curie, 94706 Maisons-Alfort, France.
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Zientara
Université Paris EST, ANSES, Laboratoire de Santé Animale, UMR Virologie 1161 (ANSES INRA ENVA), Laboratoire National et OIE de référence pour la fièvre aphteuse, 14 rue Pierre et Marie Curie, 94706 Maisons-Alfort, France.
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Blaise-Boisseau
Université Paris EST, ANSES, Laboratoire de Santé Animale, UMR Virologie 1161 (ANSES INRA ENVA), Laboratoire National et OIE de référence pour la fièvre aphteuse, 14 rue Pierre et Marie Curie, 94706 Maisons-Alfort, France. Electronic address: Labib.bakkali-kassimi@anses.fr.
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Bakkali-Kassimi
D000199:Actins / Q000235:genetics; D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000821:virology; D005536:Foot-and-Mouth Disease / Q000175:diagnosis* / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000302:isolation & purification*; D015511:Goat Diseases / Q000175:diagnosis / Q000821:virology; D006041:Goats; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis / Q000821:virology; D014059:Tongue / Q000166:cytology / Q000821:virology
D000199:Actins; D012367:RNA, Viral
Diagnosis; Duplex assay; Foot-and-mouth disease virus; One-step real-time RT-PCR; Validation