Rapid detection of insect pest with CRISPR-Cas13a-Based Lateral Flow Strip: Using Locusta migratoria manilensis

The Clustered Regularly Interspaced Short Palindromic Repeats-associated (CRISPR-Cas) system has been used as a powerful diagnostic tool because of its trans-cleavage ability to accurately recognize and cleave specific nucleic acid targets. However, its application in insect pest detection has rarely been reported. The aim of the current study was to develop a new molecular identification method for insect based on CRISPR-Cas13a system. In this study, we have developed a highly specific and sensitive detection system that combines polymerase chain reaction, CRISPR-Cas13a system and lateral flow dipstick (PCR-Cas13a-LFD) to rapidly detect the presence of Locusta migratoria manilensis (Orthoptera: Acrididae) DNA and discriminate it from other species, by using material from different stages of development. A pair of specific primers and crRNA were designed based on the Gene DEAD-box DDX10 mRNA sequence. For visual readout, a FAM-RNA-biotin reporter was used for detection. The CRISPR-Cas13a-based detection method utilizes the visual identification of L. migratoria manilensis at different developmental stages or with the use of incomplete samples. The test results could be distinguished within 2 h. The sensitivity of the PCR-Cas13a-LFD detection system reached 0.1 ng/mu L, a 10-fold increase compared with that of the PCR with electrophoresis. The LFD coupled with CRISPR-Cas13a (Cas13a-LFD) is promising for the portable, onsite and fast identification of insect pests. This study provides a reference for the development of a new method for insect identification.