4. Discussion
The prevalence of ASF in a variety of countries causes a serious social and economic impact, which is limiting the trade of swine products and affecting food security. At present, molecular diagnostic techniques of ASF are mainly relying on two OIE-recommended conventional and real-time qPCR technique methods. Although these techniques have been widely validated and are useful tools for detecting of the disease, there is still lack of convenience because of the expensive instrument and professional operation system. In this study, a convenient, highly sensitive LAMP coupled with CRISPR-Cas12a assay was established for the rapid detection of ASFV.
LAMP assay has been used to detect ASFV with high sensitivity and efficiency (James et al., 2010; Wang et al., 2020a). The design of appropriate primers is one of the most important factors in optimizing the LAMP reaction. Four primers that recognize six distinct regions on the target are required (Notomi et al., 2000). In our study, five sets of primers were designed based on the conserved regions of the p72 gene, which is the most essential structural component of the virion, accounting for 31%-33% of the total mass of the virion (Carrascosa et al., 1984; Garcia-Escudero et al., 1998). According to the amplification plot of those primers, finally, one set of primers was selected to establish the LAMP assay. Moreover, analytical sensitivity indicated that the LAMP can detect 7 copies/μl of DNA template at 63oC for 30 min. Although LAMP assay has high analytical sensitivity, cross-contamination LAMP product into pre-reaction mixtures can readily cause false-positive results. Thus, combining with the CRISPR system is effective to avoid the false-positive cases.
The fluorescence reporter is quite important for visualizing nucleic acids in the CRISPR Cas12a reaction. In this study, two FAM modified ssDNA-FQ reporters were tested. The results revealed that different ssDNA-FQ has a prodigious difference for visualization. Therefore, the most effective ssDNA-FQ reporter was selected in our study. More new types will be used to compare the effectiveness in the near future. Five crRNAs were designed to target ASFV p72, and one of which exhibited the highest activity based on the fluorescent signal. Parinaz et al have demonstrated that combinations of crRNA can increase the sensitivity of Cas13a detection by activating more Cas13a per target RNA (Fozouni et al., 2020). Furthermore, the use of multiple crRNAs that target different parts of the gene also safeguards against a potential loss of detection because of naturally occurring viral mutations. Future work will entail a combination of crRNAs to improve the sensitivity of nucleic acid detection through enhanced crRNA activity.
Previously, RPA coupled with CRISPR system has been established for on-site viral detection owing to the similar optimal reaction temperature between two steps. However, the cost of the RPA reaction is too high to be applied in the field. Therefore, in our study, LAMP combined with CRISPR Cas12a was developed at a single test tube. The LAMP reaction reagents can be first put in the bottom of the tube, the CRISPR reaction reagents remain stable within the cap of the tube. When the LAMP reaction is completed, the CRISPR reagents can be spun down into the tube for detection of ASFV. At this moment, it is important to notice that the temperature control of the test tube is essential, because the Cas12a can be inactivated under high temperature. In the near future, the annular tube will be used to contain LAMP and CRISPR reactions with different optimal temperatures.
The sensitivity of the LAMP-CRISPR method has demonstrated its capability of detecting ASFV. Cas12a-based nucleic acid fluorescence reporting system reached a sensitivity level of 7× 108copies/μl without amplification of DNA targets. In combination with LAMP amplification, the LAMP-CRISPR assay detected the DNA target at a sensitivity level of 7 copies/μl. Moreover, when diluted the DNA target as 1 copies/μl, 6 out of 10 can be detected, which demonstrated its high sensitivity. In addition, the crude RNA extraction methods (e.g. by using high temperature or lysis buffer to release nucleic acid) that could be performed in the field has been tested. Indeed, the sensitivity of this method can be slightly decreased. The impact is not that significant, but a more appropriate and optimized extraction methods is necessary to developed for the on-site sample detection in the future. Furthermore, the results of the clinical samples test illustrated that the LAMP-CRISPR assay not only requires less time than real-time qPCR but also simplifies the detection process.
In conclusion, LAMP-CRISPR was established and used for the rapid, low-cost, sensitive, specific and portable detection of ASFV. LAMP-CRISPR has great potential for on-site ASFV detection, which could be an effective way for timely monitoring of ASFV to prevent the occurrence and spread of ASFV at an early stage.