Back to Search
Start Over
Porcine epidemic diarrhea virus: Viral RNA detection and quantification using a validated one-step real time RT-PCR.
- Source :
-
Journal of virological methods [J Virol Methods] 2020 Sep; Vol. 283, pp. 113906. Date of Electronic Publication: 2020 May 31. - Publication Year :
- 2020
-
Abstract
- Since 2014, porcine epidemic diarrhea virus (PEDV) has reemerged in Europe. RT-PCR methods have been described for the detection of PEDV, but none have been validated according to a norm. In this study we described the development and validation of a SYBR™ Green one-step RT-qPCR according to the French norm NF U47-600, for the detection and quantification of PEDV viral RNA. The method was validated from sample preparation (feces or jejunum) through to nucleic acid extraction and RT-qPCR detection. Specificity and sensitivity, limit of detection (LoD), limit of quantification (LQ), linearity, intra and inter assay variability were evaluated using transcribed RNA and fecal and jejunum matrices spiked with virus. The analytical and diagnostic specificities and sensitivities of this RT-qPCR were 100% in this study. A LoD of 50 genome copies/5 μl of extract from fecal matrices spiked with virus or RNA transcript and 100 genome copies/5 μl of extract from jejunum matrices spiked with virus were obtained. The Lower LQ (LLQ) was 100 genome copies/5 μl and the Upper LQ (ULQ) 10 <superscript>8</superscript> copies/5 μl. This method is the first, validated according a norm for PEDV and may serve as a global reference method to harmonize detection and quantification of PEDV viral RNA in both field and experimental settings.<br />Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.<br /> (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Subjects :
- Animals
Coronavirus Infections diagnosis
Coronavirus Infections virology
Diarrhea virology
Europe
Feces virology
Limit of Detection
Porcine epidemic diarrhea virus genetics
RNA, Viral genetics
Sensitivity and Specificity
Sequence Alignment
Swine
Swine Diseases diagnosis
Swine Diseases virology
Microbiological Techniques methods
Porcine epidemic diarrhea virus isolation & purification
RNA, Viral isolation & purification
Real-Time Polymerase Chain Reaction methods
Subjects
Details
- Language :
- English
- ISSN :
- 1879-0984
- Volume :
- 283
- Database :
- MEDLINE
- Journal :
- Journal of virological methods
- Publication Type :
- Academic Journal
- Accession number :
- 32485176
- Full Text :
- https://doi.org/10.1016/j.jviromet.2020.113906