LAMP assay coupled with CRISPR/Cas12a system for portable detection of African swine fever virus

African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a loop-mediated isothermal amplification (LAMP) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was established in one tube for the detection of the African swine fever virus (ASFV) p72 gene. The single-stranded DNA-fluorophore quencher reporter and CRISPR-derived RNA were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/mu l of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples; a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.

Automated summary

A method for the convenient, portable, low cost, highly sensitive and specific detection of African swine fever virus (ASFV) was established using a combined LAMP-CRISPR assay. The detection limit reached 7 copies/microliter of p72 gene per reaction with no cross-reactivity with other porcine viruses. The performance of this method showed good consistency with real-time qPCR tests, demonstrating a great potential for on-site monitoring of ASFV in the field.