Luo Ran, Cheng Zhimeng, Wang Haoyu, Yang Qiyue, Zeng Yongping, Yang Yijun, Chen Yuankun, Li Wenting, Liu Xiao
College of Veterinary Medicine, Southwest University, Chongqing, China.
Division of Biliary Tract Surgery, Department of General Surgery, West China Hospital, Sichuan University, Chengdu, Sichuan, China.
Front Microbiol. 2024 Jul 25;15:1429486. doi: 10.3389/fmicb.2024.1429486. eCollection 2024.
Porcine deltacoronavirus (PDCoV) is a newly discovered porcine intestinal pathogenic coronavirus with a single-stranded positive-sense RNA genome and an envelope. PDCoV infects pigs of different ages and causes acute diarrhea and vomiting in newborn piglets. In severe cases, infection leads to dehydration, exhaustion, and death in sick piglets, entailing great economic losses on pig farms. The clinical symptoms of PDCoV infection are very similar to those of other porcine enteroviruses. Although it is difficult to distinguish these viral infections without testing, monitoring PDCoV is very important because it can spread in populations. The most commonly used methods for the detection of PDCoV is qPCR, which is time-consuming and require skilled personnel and equipment. Many farms cannot meet the conditions required for detection. Therefore, it is necessary to establish a faster and more convenient method for detecting PDCoV.
To establish a rapid and convenient detection method for PDCoV by combining RPA (Recombinase Polymerase Isothermal Amplification) with CRISPR/Cas13a.
Specific RPA primers and crRNA for PDCoV were designed, and the nucleic acids in the samples were amplified with RPA. Fluorescent CRISPR/Cas13a detection was performed. We evaluated the sensitivity and specificity of the RPA-CRISPR/Cas13a assay using qPCR as the control method.
CRISPR/Cas13a-assisted detection was completed within 90 min. The minimum detection limit of PDCoV was 5.7 × 10 copies/μL. A specificity analysis showed that the assay did not cross-react with three other porcine enteroviruses.
The RPA-CRISPR/Cas13a method has the advantages of high sensitivity, strong specificity, fast response, and readily accessible results, and can be used for the detection of PDCoV.
猪德尔塔冠状病毒（PDCoV）是一种新发现的猪肠道致病性冠状病毒，具有单链正链RNA基因组和包膜。PDCoV感染不同年龄的猪，可导致新生仔猪急性腹泻和呕吐。严重时，感染会导致患病仔猪脱水、衰竭和死亡，给猪场带来巨大经济损失。PDCoV感染的临床症状与其他猪肠道病毒非常相似。虽然未经检测很难区分这些病毒感染，但监测PDCoV非常重要，因为它可在猪群中传播。检测PDCoV最常用的方法是qPCR，该方法耗时且需要技术人员和设备。许多猪场无法满足检测所需条件。因此，有必要建立一种更快、更便捷的PDCoV检测方法。
通过将重组酶聚合酶等温扩增（RPA）与CRISPR/Cas13a相结合，建立一种快速便捷的PDCoV检测方法。
设计针对PDCoV的特异性RPA引物和crRNA，用RPA扩增样本中的核酸，进行荧光CRISPR/Cas13a检测。以qPCR作为对照方法，评估RPA-CRISPR/Cas13a检测方法的灵敏度和特异性。
CRISPR/Cas13a辅助检测在90分钟内完成。PDCoV的最低检测限为5.7×10拷贝/μL。特异性分析表明，该检测方法与其他三种猪肠道病毒无交叉反应。
RPA-CRISPR/Cas13a方法具有灵敏度高、特异性强、反应快速、结果易获取等优点，可用于PDCoV的检测。
