A novel system with robust compatibility and stability for detecting Sugarcane yellow leaf virus based on CRISPR-Cas12a.

IF 3.7 2区 生物学 Q2 MICROBIOLOGY Microbiology spectrum Pub Date : 2024-09-03 Epub Date: 2024-08-09 DOI:10.1128/spectrum.01149-24
Ting Wang, Anzhen Li, Hong Zhao, Qibin Wu, Jinlong Guo, Helei Tian, Jingwen Wang, Youxiong Que, Liping Xu
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Abstract

Sugarcane yellow leaf virus (SCYLV) can reduce sugarcane productivity. A novel detection system based on reverse transcription-multienzyme isothermal rapid amplification (RT-MIRA) combined with CRISPR-Cas12a, named RT-MIRA-CRISPR-Cas12a, was developed. This innovative approach employs crude leaf extract directly as the reaction template, streamlining the extraction process for simplicity and speed. Combining RT-MIRA and CRISPR-Cas12a in one reaction tube increases the ease of operation while reducing the risk of aerosol contamination. In addition, it exhibits sensitivity equivalent to qPCR, boasting a lower detection limit of 25 copies. Remarkably, the entire process, from sample extraction to reaction completion, requires only 52-57 minutes, just a thermostat water bath. The result can be observed and judged by the naked eye.IMPORTANCESugarcane yellow leaf disease (SCYLD) is an important viral disease that affects sugarcane yield. There is an urgent need for rapid, sensitive, and stable detection methods. The reverse transcription-multienzyme isothermal rapid amplification combined with CRISPR-Cas12a (RT-MIRA-CRISPR-Cas12a) method established in this study has good specificity and high sensitivity. In addition, the system showed good compatibility and stability with the crude leaf extract, as shown by the fact that the crude extract of the positive sample could still be stably detected after 1 week when placed at 4°C. RT-MIRA-CRISPR-Cas12a, reverse transcription polymerase chain reaction (RT-PCR), and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect SCYLV on 33 sugarcane leaf samples collected from the field, and it was found that the three methods reached consistent conclusions. This Cas12a-based detection method proves highly suitable for the rapid on-site detection of the SCYLV.

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基于 CRISPR-Cas12a 的新型甘蔗黄化曲叶病毒检测系统,兼容性强,稳定性高。
甘蔗黄花叶病毒(SCYLV)会降低甘蔗的产量。研究人员开发了一种基于反转录-多酶等温快速扩增(RT-MIRA)与CRISPR-Cas12a相结合的新型检测系统,命名为RT-MIRA-CRISPR-Cas12a。这种创新方法直接采用粗叶片提取物作为反应模板,简化了提取过程,简单快捷。将 RT-MIRA 和 CRISPR-Cas12a 结合在一个反应管中,既增加了操作的简便性,又降低了气溶胶污染的风险。此外,它的灵敏度与 qPCR 相当,检测限低至 25 个拷贝。值得注意的是,从样品提取到反应完成,整个过程仅需 52-57 分钟,只需一个恒温水浴。重要意义甘蔗黄叶病(SCYLD)是影响甘蔗产量的重要病毒病。目前迫切需要快速、灵敏、稳定的检测方法。本研究建立的反转录-多酶等温快速扩增结合CRISPR-Cas12a(RT-MIRA-CRISPR-Cas12a)方法具有良好的特异性和较高的灵敏度。此外,该系统与叶片粗提取物具有良好的兼容性和稳定性,阳性样品的粗提取物在 4°C 下放置 1 周后仍能稳定检测。利用 RT-MIRA-CRISPR-Cas12a、反转录聚合酶链式反应(RT-PCR)和反转录定量聚合酶链式反应(RT-qPCR)检测了从田间采集的 33 份甘蔗叶片样本中的 SCYLV,发现三种方法得出的结论一致。这种基于 Cas12a 的检测方法证明非常适合现场快速检测 SCYLV。
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来源期刊
Microbiology spectrum
Microbiology spectrum Biochemistry, Genetics and Molecular Biology-Genetics
CiteScore
3.20
自引率
5.40%
发文量
1800
期刊介绍: Microbiology Spectrum publishes commissioned review articles on topics in microbiology representing ten content areas: Archaea; Food Microbiology; Bacterial Genetics, Cell Biology, and Physiology; Clinical Microbiology; Environmental Microbiology and Ecology; Eukaryotic Microbes; Genomics, Computational, and Synthetic Microbiology; Immunology; Pathogenesis; and Virology. Reviews are interrelated, with each review linking to other related content. A large board of Microbiology Spectrum editors aids in the development of topics for potential reviews and in the identification of an editor, or editors, who shepherd each collection.
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