The way #CRISPRDx has flourished in the recent years is unbelievable with nearly 7-8 different technologies developed in the last 5 years. Among them the #DETECTRassay is one of the simplest to perform and implement and holds great promise!
Principle
LAMP is performed beforehand with the 3 pairs of primers, #Bstpolymerase and a buffer system to proceed to the DETECTR assay.The #LAMP PCR products can be specifically cleaved by #Cas12a protein, thereby activating its collateral cutting activity ultimately chopping fluorophore-labelled #ssDNA reporter molecules. These cut reporter molecules are lastly read out with the help of #LateralFlowDipsticks.
The CRISPR effector used here is the Lba #Cas12a from Lachnospiraceae bacterium ND2006. It is a site-specific DNA endonuclease which is used along with a single 41-44bp long guide RNA (#gRNA). #Cas12a recognizes a complementary DNA sequence of its gRNA and a 5’ TTTV protospacer adjacent motif (#PAM) on the DNA strand opposite the target sequence. #Cas12a cuts ~18 bases 3′ of the PAM and generates 5′ overhanging ends. On formation of the ternary complex between Cas12a, #gRNA and specific LAMP PCR products, the trans cleavage property of the Cas activates and it incessantly and non-specifically cleaves nearby environmental #ssDNA probes with #fluorescent labels. The reaction can be completed in 10 mins at 37C.
The cleaved/ intact #ssDNA reporters then climb through a fluorescence label-specific #LateralFlowDipstick to give unbiased and quick #chromatographic results.
Protocol
RT reaction and Isothermal amplification (62°C, 20 min): This step converts the sample #RNA into dsDNA PCR products specific to the gene of interest. Alternately DNA samples can also be used without the RT reaction step.
Cas12a detection (37°C, 10 min): In this step #LbaCas12a from Lachnospiraceae bacterium ND2006, specific guide RNAs (gRNAs) and substrate reporter molecules are mixed and incubated at 37°C for 10 min with the PCR products. On successful detection of specific sequences by Cas12a, the trans/ collateral cleavage of reporter DNA occurs.
Lateral flow strip result analysis (RT, 2-3mins): Lateral flow strip (compatible with fluorescent labels) is inserted directly into reaction, until test band(s) (control and/ or sample) appears on the strip.
Merits
Demerits
Lateral flow strips has to be of the highest quality as there is no intermediate troubleshooting point.
Applications
Molecular diagnostics of different viral and bacterial diseases.
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