Contained Research Facility

This manual outlines a molecular method of identifying tomato leafminer without the need for specialized equipment. The method is based on the CRISPR-Cas (Clustered Regularly-Interspaced Short Palindromic Repeats – CRISPR associated proteins) system. In this system, the Cas12a enzyme has indiscriminate single-stranded DNA nuclease activity when it binds to target DNA. If a single-stranded oligonucleotide probe that has a fluorophore and quencher is present when the Cas12a enzyme binds to the target DNA, the single-stranded oligonucleotide probe will also be cleaved, releasing the fluorophore and quencher.

The fluorescent signal which is proportional to the number of target DNA molecules, can be measured. The Cas12a enzyme reaction is isothermal and occurs between 15°C and 50°C. Coupling this reaction with Recombinase Polymerase Amplification (RPA), an isothermal DNA amplification method, the target DNA from the tomato leafminer can be detected and visualized using a fluorimeter or an UV illuminator and cellphone camera. This method reduces the need for specialized equipment for identifying any life stage of the tomato leafminer.