Team:Shanghai HS United/Model

Model

1.Get a target sequence

Our aim is to develop a rapid detection method based on the DNA sequence of ASFv. In China, Biosafety level 3 and level 4 laboratories are engaged in the isolation and identification of African swine fever virus, live virus culture, animal inoculation (infection) test and other experimental activities. However, the testing activities can be at the biosafety level 2 or above of laboratory biosafety protection. So, we plan to synthesis a DNA fragment to test our new method in biosafety level 2 lab.
So, we search the papers to get a fragment which fits our experiement. For Real-time detection method, primer sets and probes used in these molecular techniques are usually designed within the VP72 coding region, a well-characterized and highly conserved region of the ASFV genome
(1). Also, C-terminal end of the VP72 coding gene was used to study the evolution relationship among the strains
(2). Then, we plan to use N-terminal end of the VP72 coding gene sequence (also named b464 fragment) as a detection target sequence. Before using, we use software Snapgene to align some typical stains, including GA/2007 (Genbank: FR682468.1), PoL/2017 (Genbank: MG939588.1) and ASFV-SY2018 (Genbank: MH766894.1) which is first idenitified in China Shenyang.

2.crRNA design

For Cas12a detection method, crRNA is a RNA to guide Cas12a recognize the target sequence. We design five crRNA, and then, to see the RNA folding form, we use the online software (http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form). The inappropriate structure of crRNA may effect the activity.

3.LAMP primer design

Next, we use LAMP primer designing software PrimerExplorer http://primerexplorer.jp/e/index.html to assist the LAMP primer design.
LAMP-primer-4

LAMP-primer-5

Reference
(1)Beltran-Alcrudo D , Arias M , Gallardo C , et al. African swine fever: detection and diagnosis – A manual for veterinarians[M]. 2017.
(2)Gallardo C, Fernández-Pinero J, Pelayo V, et al. Genetic variation among African swine fever genotype II viruses, eastern and central Europe[J]. Emerging infectious diseases, 2014, 20(9): 1544.