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Sensitive and Visual Detection of Brassica Yellows Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification-Coupled CRISPR-Cas12 Assay

文献类型： 外文期刊

作者： Xu, Tengzhi 1 ; Yang, Xiaolan 1 ; Feng, Xia 1 ; Luo, Hao 1 ; Luo, Chun 1 ; Jia, Meng-Ao 2 ; Lei, Lei 3 ;

作者机构： 1.Guizhou Univ, Inst Crop Protect, Coll Agr, Guiyang 550025, Peoples R China

2.Guizhou Acad Tobacco Sci, Guiyang 550001, Guizhou, Peoples R China

3.Guizhou Acad Agr Sci, Guizhou Rapeseed Inst, Guiyang 550008, Peoples R China

关键词： brassica yellows virus; colorimetric and fluorimetric; one-pot detection; on-site detection; RT-LAMP-CRISPR/Cas12a

期刊名称：PHYTOPATHOLOGY （ 影响因子：3.2； 五年影响因子：3.9 ）

ISSN： 0031-949X

年卷期： 2024 年 114 卷 2 期

页码：

收录情况： SCI

摘要： Brassica yellows virus (BrYV) is an economically important virus on cruciferous species. In this study, a one-pot reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was developed for the detection of BrYV. The limit of detection of this method reached 32.8 copies of the BrYV ORF5, which is 100-fold more sensitive than the RT-LAMP method. Moreover, there was no cross-reactivity with other rapeseed-infecting RNA viruses or poleroviruses. We dried the CRISPR/Cas12a reagent in a trehalose and pullulan mixture to retain its efficacy at the RT-LAMP temperature of 63 degrees C in order to allow portable BrYV detection in a water bath. The entire process can be performed in about 1 h, and a positive result can be rapidly and conveniently detected using a handheld UV lamp. In the field, the RT-LAMP-CRISPR/Cas12a assay was accurate and had higher sensitivity than RT-LAMP and reverse transcription-polymerase chain reaction assays. The novel RT-LAMP-CRISPR/Cas12a assay allows convenient, portable, rapid, low-cost, highly sensitive, and specific detection of BrYV and has great potential for on-site monitoring of BrYV.

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