1. Introduction
African swine fever (ASF) is a highly lethal contagious disease of swine caused by the African swine fever virus (ASFV). ASF affects both domestic and wild suids of all breeds and ages, with a high mortality rate of nearly 100% (Parker et al., 1969; Anderson et al., 1998). Normally, ASF presents with high fever, cyanosis of the skin and severe hemorrhages in the lymph nodes. ASFV is a large and complex double-stranded DNA arbovirus that is the only member of theAsfarviridae family, Asfivirus genus(Alonso et al., 2018).
At present, there is no treatment or effective vaccine commercially available (Penrith and Vosloo, 2009), ASFV usually causes acute infection and it causes death before the production of protective antibody. Therefore, the early detection of ASFV plays an important role in the prevention and control of the disease. Both conventional and quantitative PCR are recommended by the World Organization for Animal Health (OIE) as the gold standard for the detection of the ASFV genome (Aguero et al., 2003; King et al., 2003; Aguero et al., 2004; Zsak et al., 2005). However, these methods require an expensive instruments and skilled operators, which limits the application of these methods for on-site situations. Isothermal amplification techniques, such as recombinase polymerase amplification (RPA) (Wang et al., 2017; Miao et al., 2019; Fan et al., 2020; Zhai et al., 2020), loop-mediated isothermal amplification (LAMP) (James et al., 2010; Mee et al., 2020; Wang et al., 2020a) and cross-priming amplification (CPA) (Fraczyk et al., 2016), have been successfully used to detect ASFV. Moreover, those isothermal amplification assays in combination with immunochromatographic strips have also been developed for application in the field. The main drawback of these techniques is the lack of high specificity and sensitivity, which limits their application in the detection of ASFV.
Recently, nucleic acid detection techniques based on the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonucleases (CRISPR/Cas) systems have been developed (Chen et al., 2018; Gootenberg et al., 2018; Li et al., 2018). The detection relies on the cleavage preferences of Cas12 or Cas13 in a nonspecific way after binding to a specific target DNA or RNA via programmable guide RNAs. Combined with isothermal amplification RPA assay, the CRISPR system has been used for detecting ASFV(Bai et al., 2019; He et al., 2020; Li et al., 2020; Lu et al., 2020; Wang et al., 2020b; Wu et al., 2020; Ren et al., 2021). CRISPR/Cas-based diagnostic technology has been successfully applied to detect a variety of human viruses, such as Zika virus (ZIKV) (Gootenberg et al., 2018), Dengue virus (DENV) and human papillomavirus (HPV) (Tsou et al., 2019). However, the high cost of RPA assay limits its application in the field.
To improve the existing tools and to overcome the limitations for ASF diagnosis. Here, the low-cost LAMP amplification assay integrates with CRISPR Cas12a-based detection was developed. Compare with RPA-CRISPR based assays, LAMP-CRISPR uses less enzyme and less labor work, which is more efficient and time saving. This inexpensive, highly sensitive and specific, portable and visual method will be an alternative way for on-site ASFV detection, which might contribute to a timely monitoring and rapid strategy making for control of ASF.