DOI: 10.1094/phyto-06-23-0195-r ISSN:

Sensitive and visual detection of brassica yellows virus using RT-LAMP-coupled CRISPR-Cas12 assay

Tengzhi Xu, Xiaolan Yang, Xia Feng, Hao Luo, Chun Luo, Meng-ao Jia, Lei Lei
  • Plant Science
  • Agronomy and Crop Science

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℃ in order to allow portable BrYV detection in a water bath. Additionally, 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 compared to the 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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