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

•Establishment of Internally controlled Pan-FMD one-step duplex real-time RT-PCR.•Validation according to OIE and ISO/IEC 17025 standards.•Rapid, effective and reliable method for the detection of FMDV. Two duplex one-step TaqMan-based RT-PCR protocols for detection of foot-and-mouth disease virus (...

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Bibliographic Details
Published in:Journal of virological methods 2016-09, Vol.235, p.168-175
Main Authors: Gorna, K., Relmy, A., Romey, A., Zientara, S., Blaise-Boisseau, S., Bakkali-Kassimi, L.
Format: Article
Language:English
Subjects:
Actins - genetics
Animals
Cattle
Cattle Diseases - diagnosis
Cattle Diseases - virology
Diagnosis
Duplex assay
Foot-and-Mouth Disease - diagnosis
Foot-and-Mouth Disease - virology
Foot-and-mouth disease virus
Foot-and-Mouth Disease Virus - genetics
Foot-and-Mouth Disease Virus - isolation & purification
Goat Diseases - diagnosis
Goat Diseases - virology
Goats
Life Sciences
One-step real-time RT-PCR
Real-Time Polymerase Chain Reaction - methods
Reverse Transcriptase Polymerase Chain Reaction - methods
RNA, Viral - genetics
Sensitivity and Specificity
Sheep
Sheep Diseases - diagnosis
Sheep Diseases - virology
Tongue - cytology
Tongue - virology
Validation
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Summary:•Establishment of Internally controlled Pan-FMD one-step duplex real-time RT-PCR.•Validation according to OIE and ISO/IEC 17025 standards.•Rapid, effective and reliable method for the detection of FMDV. 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.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2016.03.020