Team:Shanghai HS United/Design
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.
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.
Different from the typical and current detection method by using the PCR, LAMP (Loop-mediated isothermal amplification) requires only one temperature throughout the entire process. Without constantly altering between 3 different temperatures, as in the PCR, LAMP amplifies the targeted DNA fragment under one temperature, therefore it satisfies our goal to keep the detection simple and quick. Moreover, the PCR happens in one professional machine that needs to be operated by well-trained staffs, but our detection method requires a simple JULABO, specially designed test strips, and reagents prepared in advance. Additionally, our method can be operated by regular personnels such as the pig owners who are desperate to know whether their pig populations are affected by the ASFv or not. Since our method can be operated easily, it also reduces the time consumed during the samples’ transportation process from farms to the one and only authorized laboratory within China.
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
- (1)Test best candidate site
We plan to test five different candidate sites on ASFv DNA (b464 gene fragment) and choose the best one. - (2)Test the best primers
LAMP amplification needs six primers in one reaction, and different primer sets have various amplification efficiency. - (3)Test the best reaction temperature for LAMP amplification
Though the recommend temperature for LAMP amplification is 65 ℃ (NEB), the fittest temperature is around 65 ℃ according to the primers and templates. - (4)Determine the reaction time (both LAMP amplification and Cas12a detection)
We plan to set a shortest reaction time under the condition of ensuring the sensitivity.
- (1)Sensitivity
We plan to gradient dilute the known concentration of the template DNA (synthetic DNA fragement of ASFv) to test the sensitivity. - (2)Specificity
We will use other bateria genomes (Escherichia coli and Staphylococcus aureus) to test the specificity.
- (1)We will determine the activity of Cas12a protein by testing the fluorescence detection.
- (2)Milenia Genline HybriDetect 1 will use as lateral flow test papers
Our instructor told us, LAMP reaction products produce aerosols and influence the next reaction, leading to false positives. Adding uracil-DNA Glycosylase and dUTP to the reaction system may reduce the risk of false positives.
Uracil-DNA Glycosylase (UDG) catalyses the release of free uracil from uracil-containing DNA. The principle of the UDG in LAMP reaction shows as following figure.
We plan to add synthetic DNA fragments which mimic ASFv DNA into pig blood or pig flodder. And then, using these samples to test our method.
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.