PDF(Pubmed)
Abstract：
UNASSIGNED: 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.
UNASSIGNED: To establish a rapid and convenient detection method for PDCoV by combining RPA (Recombinase Polymerase Isothermal Amplification) with CRISPR/Cas13a.
UNASSIGNED: 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.
UNASSIGNED: CRISPR/Cas13a-assisted detection was completed within 90 min. The minimum detection limit of PDCoV was 5.7 × 101 copies/μL. A specificity analysis showed that the assay did not cross-react with three other porcine enteroviruses.
UNASSIGNED: 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.
UNASSIGNED: To establish a rapid and convenient detection method for PDCoV by combining RPA (Recombinase Polymerase Isothermal Amplification) with CRISPR/Cas13a.
UNASSIGNED: 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.
UNASSIGNED: CRISPR/Cas13a-assisted detection was completed within 90 min. The minimum detection limit of PDCoV was 5.7 × 101 copies/μL. A specificity analysis showed that the assay did not cross-react with three other porcine enteroviruses.
UNASSIGNED: 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.
摘要：
■猪deltacoronavirus（PDCoV）是一种新发现的猪肠道致病性冠状病毒，具有单链正义RNA基因组和包膜。PDCoV感染不同年龄的猪，并导致新生仔猪急性腹泻和呕吐。在严重的情况下,感染导致脱水,疲惫,和病态仔猪的死亡，给养猪场带来巨大的经济损失。PDCoV感染的临床症状与其他猪肠道病毒非常相似。尽管不进行测试很难区分这些病毒感染，监测PDCoV非常重要，因为它可以在人群中传播。最常用的检测PDCoV的方法是qPCR，这是耗时的，需要熟练的人员和设备。许多农场无法满足检测所需的条件。因此,有必要建立一种更快速、更方便的检测PDCoV的方法。
■通过将RPA（重组酶聚合酶等温扩增）与CRISPR/Cas13a相结合，建立一种快速便捷的PDCoV检测方法。
■设计了PDCoV的特异性RPA引物和crRNA，样品中的核酸用RPA扩增。进行荧光CRISPR/Cas13a检测。我们使用qPCR作为对照方法评估了RPA-CRISPR/Cas13a测定的灵敏度和特异性。
■CRISPR/Cas13a辅助检测在90分钟内完成。PDCoV的最低检测限为5.7×101拷贝/μL。特异性分析显示该测定不与其它三种猪肠道病毒发生交叉反应。
■RPA-CRISPR/Cas13a方法具有灵敏度高的优点，特异性强，快速反应,和容易获得的结果，并可用于PDCoV的检测。
■通过将RPA（重组酶聚合酶等温扩增）与CRISPR/Cas13a相结合，建立一种快速便捷的PDCoV检测方法。
■设计了PDCoV的特异性RPA引物和crRNA，样品中的核酸用RPA扩增。进行荧光CRISPR/Cas13a检测。我们使用qPCR作为对照方法评估了RPA-CRISPR/Cas13a测定的灵敏度和特异性。
■CRISPR/Cas13a辅助检测在90分钟内完成。PDCoV的最低检测限为5.7×101拷贝/μL。特异性分析显示该测定不与其它三种猪肠道病毒发生交叉反应。
■RPA-CRISPR/Cas13a方法具有灵敏度高的优点，特异性强，快速反应,和容易获得的结果，并可用于PDCoV的检测。
