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使用RAA-CRISPR/Cas12b单检测试验 (Rcod) 快速和灵敏地检测核酸

Kangfeng Lin¹, Kaihu Yao², Xiao Li¹

¹State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network and Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.

|January 26, 2024

概括

一种新的单测定方法结合了复合酶辅助放大 (RAA) 和CRISPR/Cas12b,用于快速检测波德氏菌百日咳. 这种方法在不用提取DNA的情况下在30分钟内提供了灵敏,具体的结果,改善了咳诊断.

科学领域:

分子生物学分子生物学
生物技术是生物技术.
传染病诊断传染病诊断传染病诊断

背景情况:

核酸检测方法对于诊断诸如百日咳等传染病至关重要.
目前的CRISPR/Cas12b测定通常需要单独的放大和检测步骤,增加复杂性和污染风险.
一些Cas12b蛋白的热性质需要温度变化,阻碍了单步测试.

研究的目的:

开发一种快速,灵敏和特定的单核酸检测测试剂,用于 Bordetella pertussis.
将复合酶辅助放大 (RAA) 与CRISPR/Cas12b检测集成到一个单一的反应中.
为了利用热友Cas12b变体来实现稳定,同热检测系统.

主要方法:

一种称为RAA-CRISPR/Cas12b单检测 (Rcod) 的单测定被设计用于 Bordetella pertussis.
该试验使用了Alicyclobacillus acidoterrestris Cas12b蛋白 (AacCas12b) 以其在37°C的特定跨裂变活性.
在临床样本上使用无核酸提取方法评估Rcod系统,并与实时PCR进行比较.

主要成果:

在没有核酸提取的情况下,Rcod试验从样本到结果的检测时间不到30分钟.
达到0.2副本/μL的高灵敏度,证明了该试验能够检测低病原体负载.
在临床样本中,Rcod系统与实时PCR相比,具有97.96%的灵敏度和99.19%的特异性.

结论:

开发的RAA-CRISPR/Cas12b单检测 (Rcod) 试验提供了一种快速,灵敏和特定的方法来检测Bordetella pertussis.
在恒温下使用AacCas12b简化了测试,并减少了潜在的干扰.
这种无核酸提取的一步式方法为医疗诊断提供了一个有前途的平台,并简化了病原体检测过程.

关键词:

基于CRISPR/Cas12b的检测方式一步检测检测的一步检测.RAA RAA RAA RAA RAA RAA RAA RAA RAA RAA RAA RAA RAA RAA RAA RAA 视觉检测视觉检测是一种视觉检测.

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