Ultrasensitive and Visual Detection of Pseudorabies Virus based on CRISPR-Cas12b system.

IF 3.3 3区医学 Q3 IMMUNOLOGY

Microbial pathogenesis Pub Date : 2025-03-01 DOI:10.1016/j.micpath.2025.107447

Haoran Kang, Xintan Yang, Ruijiao Jiang, Peng Gao, Yongning Zhang, Lei Zhou, Xinna Ge, Jun Han, Xin Guo, Hanchun Yang

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引用次数: 0

Abstract

Aujeszky's disease (AD) is an acute infectious disease that infects pigs and other animals, resulting in significant economic losses and posing a threat to human health. Reliable and rapid detection methods are essential for the prevention of AD. In this study, a RAA-Cas12b assay based on recombinase-aided amplification (RAA) and CRISPR-Cas12b system was established, optimized and evaluated for the rapid detection of wild-type Pseudorabies Virus (PRV). The results can not only be detected by real-time fluorescence readout, but also can be visualized by a portable blue light instrument. There was no cross-reaction with PRV Bartha-K61 strain or other swine infectious viruses. The analytical sensitivities of the real-time PRV RAA-Cas12b assay and visual PRV RAA-Cas12b assay were determined to be 15 copies/μL with 95% confidence interval and 140 copies/μL with 95% confidence interval, respectively. A total of 31 clinical samples were detected and compared with PRV qPCR assay to evaluate the diagnostic performance of the PRV RAA-Cas12b assay. The diagnostic coincidence rate of the two assays was 100%. In summary, this convenient and reliable assay has great potential for rapid detection of wild type PRV in point-of-care testing (POCT).

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奥杰斯基病（AD）是一种感染猪和其他动物的急性传染病，会造成重大经济损失，并对人类健康构成威胁。可靠、快速的检测方法对于预防奥杰斯基病至关重要。本研究建立、优化并评估了一种基于重组酶辅助扩增（RAA）和 CRISPR-Cas12b 系统的 RAA-Cas12b 检测方法，用于快速检测野生型伪狂犬病病毒（PRV）。检测结果不仅可以通过实时荧光读数进行检测，还可以通过便携式蓝光仪进行观察。与 PRV Bartha-K61 株或其他猪传染性病毒没有交叉反应。实时 PRV RAA-Cas12b 检测法和可视 PRV RAA-Cas12b 检测法的分析灵敏度分别为 15 个拷贝/μL（置信区间为 95%）和 140 个拷贝/μL（置信区间为 95%）。共检测了 31 份临床样本，并与 PRV qPCR 检测法进行了比较，以评估 PRV RAA-Cas12b 检测法的诊断性能。两种检测方法的诊断吻合率均为 100%。总之，这种方便可靠的检测方法在床旁检测（POCT）中快速检测野生型 PRV 方面具有很大的潜力。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Microbial pathogenesis 医学-免疫学

CiteScore

7.40

自引率

2.60%

发文量

472

审稿时长

56 days

期刊介绍： Microbial Pathogenesis publishes original contributions and reviews about the molecular and cellular mechanisms of infectious diseases. It covers microbiology, host-pathogen interaction and immunology related to infectious agents, including bacteria, fungi, viruses and protozoa. It also accepts papers in the field of clinical microbiology, with the exception of case reports. Research Areas Include: -Pathogenesis -Virulence factors -Host susceptibility or resistance -Immune mechanisms -Identification, cloning and sequencing of relevant genes -Genetic studies -Viruses, prokaryotic organisms and protozoa -Microbiota -Systems biology related to infectious diseases -Targets for vaccine design (pre-clinical studies)