LAMP assay coupled with CRISPR/Cas12a system for portable detection of African swine fever virus
Bo Yang, Zhengwang Shi, Yuan Ma, Lijuan Wang, Liyan Cao, Juncong Luo, Ying Wan, Rui Song, Yiyong Yan, kehu yuan, Hong Tian, Haixue Zheng
Abstract
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/μl 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.