传染性胃肠炎病毒(TGEV)是一种引起腹泻的高度传染性的胃肠道病毒,呕吐,厌食症,脱水,和仔猪的体重减轻。在临床实践中,它通常发生在与其他病原体的混合感染中,因此难以诊断和预防。主要危害2周龄左右的仔猪,给农场造成巨大损失。TGEV的临床确认通常需要实验室诊断,但传统的PCR和免疫荧光检测方法有一定的局限性。此外,中国大多数农场都没有能力准确诊断这种疾病。因此,需要一种新的检测方法,该方法具有高灵敏度和特异性,并且对仪器的依赖性较小。
■我们使用重组酶聚合酶扩增(RPA),结合活化Cas13a蛋白的核酸酶特征,通过向系统中添加具有荧光和猝灭部分的报告RNA,建立TGEV的视觉CRISPR-Cas13a辅助检测方法。
■我们选择了最佳的RPA引物和最佳的CRISPRRNA(crRNA)。优化了反应体系,特异性,和灵敏度验证。TGEV检测系统未与其他常见腹泻病毒发生交叉反应,它的检测限是101份,这与qPCR的灵敏度相似。我们成功建立了RPA-CRISPR-Cas13a辅助检测方法,并使用该检测系统分析了123份猪血样本。qPCR用作金标准方法。敏感性,特异性,正符合率,新方法的负符合率分别为100、98.93、96.66和100%,分别。
UNASSIGNED: Transmissible gastroenteritis virus (TGEV) is a highly contagious gastrointestinal virus that causes diarrhea, vomiting, anorexia, dehydration, and weight loss in piglets. In clinical practice, it often occurs in mixed infections with other pathogens, and is therefore difficult to diagnose and prevent. It mainly harms piglets of about 2 weeks old, causing huge losses on farms. The clinical confirmation of TGEV usually requires a laboratory diagnosis, but traditional PCR and immunofluorescence assays have some limitations. Moreover, most farms in China are ill-equipped to accurately diagnose the disease. Therefore, a new detection method with high sensitivity and specificity and less dependence on instrumentation is required.
UNASSIGNED: We used recombinase polymerase amplification (RPA), combined with the nuclease characteristics of the activated Cas13a protein to establish a visual CRISPR-Cas13a-assisted detection method for TGEV by adding a reporter RNA with fluorescent and quenching moieties to the system.
UNASSIGNED: We selected the optimal RPA primer and best CRISPR RNA (crRNA). The reaction system was optimized and its repeatability, specificity, and sensitivity verified. The TGEV detection system did not cross-react with other common diarrhea viruses, and its detection limit was 101 copies, which is similar with the sensitivity of qPCR. We successfully established an RPA-CRISPR-Cas13a-assisted detection method, and used this detection system to analyze 123 pig blood samples. qPCR was used as the gold standard method. The sensitivity, specificity, positive coincidence rate, and negative coincidence rate of the new method were 100, 98.93, 96.66, and 100%, respectively.