基于CRISPR-Cas12a的禽流感病毒的快速检测
Xu Zhou1, Siwen Wang1, Yue Ma1
1State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Virology journal
|November 14, 2023
概括
一种新的反转录重组酶聚合酶放大 (RT-RPA) /CRISPR方法提供了快速,敏感的禽流感病毒 (AIV) 检测. 这种用户友好的平台非常适合于临床检测,克服了传统实验室诊断的局限性.
科学领域:
- 分子生物学分子生物学
- 病毒学 病毒学
- 生物技术是生物技术.
背景情况:
- 禽流感病毒 (AIV) 对家禽构成重大威胁,并可能感染其他物种.
- 目前的诊断方法,如实时逆转录聚合酶连锁反应 (rRT-PCR),需要实验室设置,限制了快速医疗点检测 (POCT).
- 对于AIV来说,非常需要敏感,特定和可在现场部署的诊断工具.
研究的目的:
- 开发一种改进的诊断方法来检测禽流感病毒 (AIV).
- 创建一个快速,灵敏和特定的测试,不依赖实验室设备.
- 为AIV诊断建立一个临床检测 (POCT) 解决方案.
主要方法:
- 开发了一种新的反转录重组酶聚合酶放大 (RT-RPA) 与CRISPR-Cas12a跨裂系统相结合.
- 该系统使用特定的原始体和CRISPRRNA (crRNA) 准了AIV的M和NP基因.
- 检测是通过光读数或侧流带来实现的,通过Cas12a蛋白质净化和基因查进行.
主要成果:
- RT-RPA/CRISPR方法显示出高灵敏度,M基因的检测极限为6.7副本/μL,NP基因的检测极限为12副本/μL.
- 该试验表现出极好的特异性,没有与其他禽类病毒如NDV,IBDV和IBV交叉反应.
- 开发的方法与现场样本的确定的rRT-PCR和病毒隔离技术相一致97.5%.
结论:
- 成功开发了一种快速,灵敏和特定的RT-RPA/CRISPR方法来检测禽流感病毒 (AIV).
- 这一创新的系统提供了一个用户友好的,准确的,成本效益高的点诊所测试 (POCT) 平台.
- 该方法具有在AIV监测和诊断中的实地应用的巨大潜力.
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