A LAMP–CRISPR/Cas12b rapid detection platform for canine parvovirus detection

IF 2.6 3区 化学 Q2 CHEMISTRY, ANALYTICAL
Yuting Chen, Xinyu Zhang, Gui Hu, Yueying Pan, Yuhong Guan, Jinquan Yang and Hui Chen
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Abstract

Canine parvovirus (CPV) is one of the main pathogens causing toxic diarrhea in Chinese dogs, is the cause of large-scale epidemic of dogs, and poses a great threat to the dog industry in China. Rapid, sensitive, and specific CPV testing facilitates the timely diagnosis and treatment of sick dogs. The aim of this study was to build a LAMP–CRISPR/Cas12b platform for CPV detection. The loop mediated isothermal amplification (LAMP) technique was combined with CRISPR-Cas12b analysis to establish a “two-step” and “one-tube” CRISPR/Cas12b rapid CPV method, respectively. The detection system was constructed with specific LAMP primers and single guide RNA (sgRNA) for the highly conserved short fragment of the CPV gene, which could be detected within 1 h without cross-reaction with the other viruses causing canine diarrhea. The detection limits of both “two-step” and “one-tube” CRISPR/Cas12b reactions were 10−1 copies per μL, which was 100 times more sensitive than qPCR and LAMP. In order to achieve point-of-care testing (POCT) of CPV, a one-tube LAMP–CRISPR/Cas12b nucleic acid extraction and detection platform based on magnetic nanoparticle enrichment technology was established to achieve “sample in-result out”. The results of this method for simulated samples were compared with those of quantitative real-time PCR; the results showed 100% consistency, and the time was shorter, which could be used to detect the diseased dogs earlier and provide a basis for clinical diagnosis. The LAMP–CRISPR/Cas12b method established in this study provides a sensitive and specific method for rapid detection of CPV, and provides technical support for rapid diagnosis of CPV.

Abstract Image

用于检测犬细小病毒的 LAMP-CRISPR/Cas12b 快速检测平台
犬细小病毒(CPV)是引起我国犬只中毒性腹泻的主要病原体之一,是造成犬只大规模流行的原因,对我国养犬业构成极大威胁。快速、灵敏、特异的 CPV 检测有助于及时诊断和治疗病犬。本研究旨在建立一个用于CPV检测的LAMP-CRISPR/Cas12b平台。将环介导等温扩增(LAMP)技术与CRISPR-Cas12b分析相结合,分别建立了 "两步法 "和 "一管法 "CRISPR/Cas12b CPV快速检测方法。该检测系统由特异性LAMP扩增引物和针对CPV基因高度保守短片段的单导RNA(sgRNA)构建而成,可在1小时内完成检测,且不会与引起犬腹泻的其他病毒产生交叉反应。为了实现CPV的床旁检测(POCT),建立了基于磁性纳米粒子富集技术的一管式LAMP-CRISPR/Cas12b核酸提取和检测平台,实现了 "样品进,结果出"。该方法对模拟样本的检测结果与实时定量 PCR 检测结果进行了比较,结果一致性达到 100%,且检测时间更短,可更早地发现病犬,为临床诊断提供依据。本研究建立的LAMP-CRISPR/Cas12b方法为CPV的快速检测提供了灵敏、特异的方法,为CPV的快速诊断提供了技术支持。
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
Analytical Methods
Analytical Methods CHEMISTRY, ANALYTICAL-FOOD SCIENCE & TECHNOLOGY
CiteScore
5.10
自引率
3.20%
发文量
569
审稿时长
1.8 months
期刊介绍: Early applied demonstrations of new analytical methods with clear societal impact
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