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.