基于 RT-RPA 和 CRISPR/Cas12a-LFS 的诺罗病毒 GII.2 基因型核酸检测方法的建立

Polish journal of microbiology Pub Date : 2024-06-20 eCollection Date: 2024-06-01 DOI:10.33073/pjm-2024-023
Ting Wang, Hao Zeng, Jie Kang, Lanlan Lei, Jing Liu, Yuhong Zheng, Weidong Qian, Cheng Fan
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引用次数: 0

摘要

为了建立诺如病毒GII.2基因型的快速检测方法,本研究采用了反转录重组聚合酶扩增(RT-RPA)结合CRISPR/Cas12a和侧流条带(RT-RPA-Cas12a-LFS)的方法。通过比较诺如病毒GII.2基因型的基因组,确定了高度保守的序列,从而设计出针对诺如病毒GII.2保守区的特异性RT-RPA引物和crRNA。随后,对 RT-RPA 的反应参数进行了优化,并使用琼脂凝胶电泳和 LFS 进行了评估。结果表明,诺如病毒GII.2的保守序列在37℃、25分钟的RT-RPA反应中被成功扩增。此外,以扩增产物为模板,在 37°C 下 10 分钟内通过 LFS 实现了 CRISPR/Cas12a 介导的裂解检测。包括等温扩增反应时间在内,总时间为 35 分钟。已建立的 RT-RPA-Cas12a-LFS 方法能特异性检测诺如病毒 GII.2,对其他病毒基因组的检测结果为阴性,检测限为 10 拷贝/μl。通过分析 60 份食品污染样本,进一步比较了 RT-RPA-Cas12a-LFS 方法与 qRT-PCR 方法。阳性符合率为 100%,阴性符合率为 95.45%,总体符合率达到 98.33%。这种诺如病毒 GII.2 基因型的检测方法具有成本低、灵敏度高、特异性强、操作简便等特点,为现场检测诺如病毒 GII.2 基因型提供了一种可行的技术方案。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Establishment of a Nucleic Acid Detection Method for Norovirus GII.2 Genotype Based on RT-RPA and CRISPR/Cas12a-LFS.

To establish a rapid detection method for norovirus GII.2 genotype, this study employed reverse transcription recombinase polymerase amplification (RT-RPA) combined with CRISPR/Cas12a and lateral flow strip (RT-RPA-Cas12a-LFS). Here, the genome of norovirus GII.2 genotype was compared to identify highly conserved sequences, facilitating the design of RT-RPA primers and crRNA specific to the conserved regions of norovirus GII.2. Subsequently, the reaction parameters of RT-RPA were optimized and evaluated using agar-gel electrophoresis and LFS. The results indicate that the conserved sequences of norovirus GII.2 were successfully amplified through RT-RPA at 37°C for 25 minutes. Additionally, CRISPR/Cas12a-mediated cleavage detection was achieved through LFS at 37°C within 10 minutes using the amplification products as templates. Including the isothermal amplification reaction time, the total time is 35 minutes. The established RT-RPA-Cas12a-LFS method demonstrated specific detection of norovirus GII.2, yielding negative results for other viral genomes, and exhibited an excellent detection limit of 10 copies/μl. The RT-RPA-Cas12a-LFS method was further compared with qRT-PCR by analyzing 60 food-contaminated samples. The positive conformity rate was 100%, the negative conformity rate was 95.45%, and the overall conformity rate reached 98.33%. This detection method for norovirus GII.2 genotype is cost-effective, highly sensitive, specific, and easy to operate, offering a promising technical solution for field-based detection of the norovirus GII.2 genotype.

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