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.