Chinese Bulletin of Botany ›› 2022, Vol. 57 ›› Issue (3): 308-319.DOI: 10.11983/CBB21225

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

Establishment and Application of RPA-CRISPR/Cas12a Detection System for Potato Virus Y

Yulong He1, Jiage Wang1, Shanshan Zhao1, Jin Gao1,3, Yingying Chang1, Xiting Zhao1,2, Bihua Nie4, Qingxiang Yang1,3, Jiangli Zhang1,2,*(), Mingjun Li1,2,*()   

  1. 1College of Life Sciences, Henan Normal University, Xinxiang 453007, China
    2Engineering Technology Research Center of Nursing and Utilization of Genuine Chinese Crude Drugs of Henan Province/Engineering Laboratory of Green Medicinal Material Biotechnology of Henan Province, Xinxiang 453007, China
    3Henan International Joint Laboratory of Agricultural Microbial Ecology and Technology, Xinxiang 453007, China
    4Key Laboratory of Potato Biology and Biotechnology (HZAU), Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China
  • Received:2021-12-23 Accepted:2022-03-18 Online:2022-05-01 Published:2022-05-18
  • Contact: Jiangli Zhang,Mingjun Li


Plant virus disease is an important factor restricting the safe production of crops. Virus detection can identify viruses and determine the types of viruses, which is the key to disease monitoring, early warning and prevention in crops production. In this study, a detection system based on Recombinase Polymerase Amplification-Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 12a (RPA-CRISPR/Cas12a) was established for potato virus Y (PVY). The results showed that: (1) Cas12a and other components in the CRISPR/Cas12a detection system were necessary for the detection; (2) The target location of crRNA had a significant effect on Cas12a nuclease activity, and the reaction efficiency was the highest when the target of crRNA contained part of PAM site sequence; (3) The minimum detection limit of RPA-CRISPR/Cas12a was 3×102 copies∙μL-1, which was higher than that of PCR and qPCR methods; (4) The combination of RPA-CRISPR/Cas12a system with crude extraction of nucleic acid and reverse transcription could detect PVY in a non-laboratory setting and the whole process took about 60 minutes. The RPA-CRISPR/Cas12a detection system of PVY established in this study provides an effective method for real-time and rapid visual detection of plant viruses under non-laboratory conditions.

Key words: CRISPR/Cas12a, potato virus Y, rapid detection, recombinase polymerase isothermal amplification