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基于CRISPR/Cas12a的一管RT-RAA测试用于PoRV基因型鉴定

Mingfang Bi^1,2, Zunbao Wang¹, Kaijie Li¹

¹College of Veterinary Medicine, State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, and Institute of Zoonosis, Jilin University, Changchun 130062, China.

International journal of molecular sciences

|July 29, 2025

概括

一个新的诊断工具可以快速识别猪轮状病毒基因型G4,G5和G9. 这一进步有助于控制小猪的病毒性腹,并评估疫苗的有效性.

科学领域:

兽医病毒学兽医病毒学
分子诊断学分子诊断学
公共卫生微生物学

背景情况:

猪轮状病毒 (PoRV) 由于病毒性腹,在养猪场造成重大经济损失.
由于PoRV的遗传多样性和重组潜力,通过跨物种传播,对公众健康构成风险.
由于PoRV基因型之间的交叉保护性较差,现有疫苗的疗效受到限制,这凸显了持续监测的必要性.

研究的目的:

开发一个快速有效的诊断平台,同时对常见的PoRV菌株进行基因定型.
在速度和技术要求方面克服传统分子分析的局限性.
支持针对猪轮状病毒的及时疾病控制和疫苗开发战略.

主要方法:

开发一种新的诊断平台,将逆转录重组酶辅助放大 (RT-RAA) 与CRISPR-Cas12a技术相结合.
用于G4,G5和G9 PoRV基因型的同时放大.
采用特定序列的CRISPR-Cas12a识别,用于准确和快速的基因造型.

主要成果:

开发的RT-RAA-CRISPR-Cas12a系统在37°C时50分钟内实现了PoRV G4/G5/G9的同时基因定型.
证明了高灵敏度,检测极限为10^0副本/μL.
临床验证显示与已建立的逆转录定量聚合酶链反应 (RT-qPCR) 方法具有很高的一致性.

结论:

新型RT-RAA-CRISPR-Cas12a平台为快速的PoRV基因型定型提供了一个有效的解决方案.
这项技术可以在评估疫苗兼容性和优化流行病控制策略方面发挥重要作用.
该平台为实时监测流通的猪轮状病毒菌株提供了有价值的工具.

关键词:

这就是CRISPR/Cas12a.在 PoRVV 里面.土耳其人土耳其人RT-RAAA 基因定型是指基因的定型.

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CRISPR and crRNAs

CRISPR and crRNAs

Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...