2.6 LAMP-CRISPR fluorescence assay
LAMP-CRISPR fluorescence assays include LAMP amplification of the dsDNA template and an optimized Cas12a cleavage assay. The LAMP reaction was added to the bottom of the PCR tube and Cas12a reaction was placed into the cap of the tube (Figure 1). Reactions were firstly incubated at optimized LAMP reaction temperature for 30 min. Then, the PCR tube was centrifuged thoroughly to mix the LAMP reaction and Cas12a reaction. The mixed reaction was performed and the fluorescent intensity was measured using ABI. The fluorescence can be observed with a transilluminator.
The p3xFLAG-CMV-7.1-p72 dsDNA was serially diluted from 7×106 to 7×100 copies/μl. The sensitivity of the LAMP-CRISPR was analyzed with those of different dilutions of p72 dsDNA template. The fluorescent intensity was measured by ABI. To further determine the specificity of the developed LAMP-CRISPR assay, six porcine viruses including Classical Swine Fever Virus (CSFV), Foot-and-Mouth Disease Virus (FMDV), Senecavirus A (SVA), Porcine Circovirus 2 (PCV2), Porcine Epidemic Diarrhea Virus (PEDV) and Porcine Reproductive and Respiratory Syndrome (PRRSV) were tested.