Anses, Laboratory of Ploufragan-Plouzané-Niort, BP53, 22440, Ploufragan, France.
Anses, Laboratory of Ploufragan-Plouzané-Niort, BP53, 22440, Ploufragan, France.
J Virol Methods. 2020 Sep;283:113906. doi: 10.1016/j.jviromet.2020.113906. Epub 2020 May 31.
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 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.
自 2014 年以来，猪流行性腹泻病毒（PEDV）在欧洲再次出现。已经描述了用于检测 PEDV 的 RT-PCR 方法，但没有一种方法是根据规范进行验证的。在这项研究中，我们根据法国标准 NF U47-600 描述了一种 SYBR™ Green 一步法 RT-qPCR 的开发和验证，用于检测和定量 PEDV 病毒 RNA。该方法从样品制备（粪便或空肠）到核酸提取和 RT-qPCR 检测进行了验证。使用转录 RNA 和用病毒污染的粪便和空肠基质评估了特异性和灵敏度、检测限 (LoD)、定量限 (LQ)、线性、内和间分析变异性。在本研究中，该 RT-qPCR 的分析和诊断特异性和灵敏度均为 100%。从粪便基质中用病毒或 RNA 转录物污染的 50 个基因组拷贝/5 μl 提取物以及从空肠基质中用病毒污染的 100 个基因组拷贝/5 μl 提取物中获得 LoD。下限定量 (LLQ) 为 100 个基因组拷贝/5 μl，上限定量 (ULQ) 为 10 个拷贝/5 μl。该方法是第一个根据规范验证的 PEDV 方法，可作为在现场和实验环境中检测和定量 PEDV 病毒 RNA 的全球参考方法。
