Table 1

PCR and RPA primer sequences"

Table 2

crRNA sequence"

Fig. 1

Construction of standard plasmids A. PCR amplification of the MSTN gene: Marker.1 000 bp DNA marker; 1. Ear sample from MSTN gene-edited homozygous pig; 2. Ear sample from MSTN wild-type pig; H2O. Negative control without PCR template; The size of the PCR product is approximately 370 bp. B. Sanger sequencing results of the GE standard plasmid. C. Sanger sequencing results of the WT standard plasmid"

Fig. 2

crRNA screening results A-C. The fluorescence curve, fluorescence intensity bar chart, and results of colloidal gold test strips for GEcrRNA are presented in sequence; D-F. The fluorescence curve, fluorescence intensity bar chart, and results of colloidal gold test strips for WTcrRNA are presented in sequence; NC. Negative control group without crRNA. ** indicates a highly significant difference compared with the NC group (P < 0.01); ns indicates no significant difference compared with the NC group (P>0.05), the same as below. GE represents the GE standard plasmid samples, WT represents the WT standard plasmid samples; T line represents the test line, C line represents the control line; A positive result is indicated by the appearance of color on the T line alone or on both the T and C lines; A negative result is indicated by the appearance of color on the C line alone, the same as below"

Fig. 3

Cas12a system specificity test and system optimization A, B. Fluorescence reporter system and colloidal gold test strip results for the specificity test of GEcrRNA2; C, D. Fluorescence reporter system and colloidal gold test strip results for the specificity test of WTcrRNA1; E. Fluorescence intensity of the CRISPR/Cas12a system at different molar ratios of Cas12a protein to GEcrRNA2; F. Fluorescence intensity of the CRISPR/Cas12a system at different concentrations of ssDNA probe; NC. Negative control without crRNA. GE+WT represents a 1∶1 mixture of GE standard plasmid and WT standard plasmid, the same as below. In panels E and F, different lowercase letters indicate a significant difference between groups (P < 0.05); The same lowercase letter indicates no significant difference between groups (P > 0.05), the same as below"

Fig. 4

Sensitivity test of the RPA-CRISPR/Cas12a system A, B. RPA amplification results of gradient-diluted GE and WT plasmid: H2O. Negative control without RPA template; C, D. Sensitivity test of the RPA-CRISPR/Cas12a fluorescence reporter system mediated by GEcrRNA2 and WTcrRNA1; E, F. Sensitivity test of the RPA-CRISPR/Cas12a colloidal gold test strip system mediated by GEcrRNA2 and WTcrRNA1; NC. Negative control without crRNA. Lane 1 to 11 represent 4.1×1010, 4.1×109, 4.1×108, 4.1×107, 4.1×106, 4.1×105, 4.1×104, 4.1×103, 4.1×102, 4.1×101, 4.1×100 copies·μL-1, respectively"

Fig. 5

Validation of the RPA-CRISPR/Cas12a system by using pig ear tissue samples A, B. Detection results of the RPA-CRISPR/Cas12a fluorescence reporter system mediated by GEcrRNA2 and WTcrRNA1; C, D. Detection results of the RPA-CRISPR/Cas12a colloidal gold test strip system mediated by GEcrRNA2 and WTcrRNA1; NC. Negative control without crRNA"

Fig. 6

Application of the RPA-CRISPR/Cas12a system A, B. Detection results of the RPA-CRISPR/Cas12a fluorescence reporter system mediated by GEcrRNA2 and WTcrRNA1; C, D. Detection results of the RPA-CRISPR/Cas12a colloidal gold test strip system mediated by GEcrRNA2 and WTcrRNA1; NC. Negative control without crRNA"

Table 3

Summary of genotype testing results for pig ear tissue samples"

Fig. 7

Sanger sequencing validation of the partial pig ear tissue samples"