Reverse transcription-recombinase-aided amplification and CRISPR/Cas12a-based visual detection of maize chlorotic mottle virus

Xueyan Duan; Wendi Ma; Zhiyuan Jiao; Yiying Tian; Ragab Gomaa Ismail; Tao Zhou; Zaifeng Fan

doi:10.1186/s42483-022-00128-y

Reverse transcription-recombinase-aided amplification and CRISPR/Cas12a-based visual detection of maize chlorotic mottle virus

Phytopathol Res. 2022;4(1):23. doi: 10.1186/s42483-022-00128-y. Epub 2022 Jun 20.

Authors

Xueyan Duan^{1

2}, Wendi Ma¹, Zhiyuan Jiao^{1

2}, Yiying Tian^{1

2}, Ragab Gomaa Ismail^{1

3}, Tao Zhou^{1

2}, Zaifeng Fan^{1

2}

Affiliations

¹ State Key Laboratory of Agrobiotechnology, MARA Key Laboratory of Surveillance and Management for Plant Quarantine Pests, College of Plant Protection, China Agricultural University, Beijing, 100193 China.
² Sanya Institute of China Agricultural University, Building 8, Yongyou Industrial Park, Yazhou Bay Science and Technology City, Yazhou District, Sanya, 572025 Hainan China.
³ Department of Plant Pathology, College of Agriculture, Alexandria University, El-Shatby, Alexandria, 21545 Egypt.

Abstract

Maize chlorotic mottle virus (MCMV) is one of the important quarantine pathogens in China. It often co-infects with one or two viruses in the family Potyviridae and causes maize lethal necrosis disease. Therefore, an accurate and sensitive method for the detection of MCMV is urgently needed. Combined with reverse transcription and recombinase-aided amplification, we developed a CRISPR/Cas12a-based visual nucleic acid detection system targeting the MCMV coat protein gene. The whole process can be completed within 45 min with high sensitivity. This system could detect cDNAs diluted up to 10^-5 when 2000 ng of total RNA was used for reverse transcription. The Cas12a/crRNA complex designed for MCMV detection could recognize and cleave the targeted double-stranded DNA, and ultimately cleave the single-stranded DNA probes and produce fluorescent signals. The green fluorescence produced under blue light (440-460 nm) in this procedure could be observed by the naked eye. Since this novel method is specific, rapid, sensitive and does not require special instruments and technical expertise, it should be suitable for on-site visual detection of MCMV in seeds, plants of maize and potentially in its insect vectors.

Keywords: CRISPR/Cas12a; MCMV; On-site detection; RT-RAA.