Porcine epidemic diarrhea virus: Viral RNA detection and quantification using a validated one-step real time RT-PCR

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) 10⁸ 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.

Keywords: NF U47-600; Porcine epidemic diarrhea virus; RT-qPCR; Validation.