Ultra-rapid and sensitive detection of African swine fever virus using multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor

Figures

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  Figure 1. The mechanism of the ASFV-MCDA-LFB assay. The entire ASFV-MCDA-LFB assay procedure includes MCDA amplification (A) and SA-GNPs-LFB biosensor detection (B). The DNA templates were prepared from simulated blood samples (A, step 1), and the target regions on the template were amplified exponentially with primers driven by the Bst DNA polymerase (A, steps 2 and 3). 0.6–1.4 μL of FAM- and biotin-labeled MCDA amplicons are added to the designated sample well of the SA-GNPs-LFB biosensor, followed by the addition of 100–150 μL of running buffer to the sample pad (B, step 1). Subsequently, the biotinylated amplicons bind to the streptavidin-conjugated gold nanoparticles (SA-GNPs) on the conjugate pad, forming FAM/target/biotin/SA-GNPs complexes. These complexes were detected by anti-FITC and produced red TL lines on the NC membrane (B, steps 2 and 3).

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  Figure 2. ASFV-specific gene MCDA-LFB primer sequence and position. Sequence and location of B646L gene used to design multiple cross displacement amplification primers. The nucleotide sequences of the sense strand of B646L are listed. Right arrows and left arrows indicate forward and reverse complementary sequences that are used. ASFV, African swine fever virus; MCDA, multiple cross displacement amplification; LFB, nanoparticle-based lateral flow biosensor.

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  Figure 3. Confirmation experiments for ASFV-MCDA-LFB. Detection sensitivity of the ASFV-MCDA assay using a DNA template (recombinant standard plasmids). Two detection methods, including LFB biosensors (A) and MG chromogenic reagents (B), were used to analyze the ASFV-MCDA products. DW was used as a blank control. MCDA, multiple cross displacement amplification; LFB, nanoparticle-based lateral flow biosensor; ASFV, African swine fever virus; TL, test line; CL, control line; DW, distilled water.

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  Figure 4. The volume dependency of the LFB assay. The LFB 1–5 represents that the sample loading amount of the amplified products were 0.6 μL, 0.8 μL, 1.0 μL, 1.2 μL, 1.4 μL in turn. LFB, nanoparticle-based lateral flow biosensor; TL, test line; CL, control line; distilled water (DW).

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  Figure 5. ASFV-MCDA amplification results validation assay. Monitoring the MCDA reaction was carried out using four methods: LFB biosensors (A), MG chromogenic reagent (B), agarose gel electrophoresis (C), and real-time turbidimetry (D). The standard plasmid DNA of ASFV was diluted 10-fold and added to the MCDA reaction test. Four different reaction methods were tested and compared at 63°C; LFB biosensors 1 to 8 represented DNA levels of 2.0 × 106 copies/μL, 2.0 × 105 copies/μL, 2.0 × 104 copies/μL, 2.0 × 103 copies/μL, 2.0 × 102 copies/μL, 2.0 × 101 copies/μL, 2.0 copies/μL and distilled water. ASFV, African swine fever virus; TL, test line; CL, control line; LFB, nanoparticle-based lateral flow biosensor; DW, distilled water.

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  Figure 6. Optimal temperature for ASFV-MCDA assay. The optimal temperature of the ASFV-MCDA assay was determined by a real-time turbidity (LA-500). The results of isothermal amplification ranging from 60°C to 67°C and the corresponding amplification results from (A) to (H) are presented. The threshold value of real-time turbidity is 0.1. If the value is greater than 0.1, it indicates a positive result. Otherwise, it indicates a negative result. These curves showed that the ASFV-MCDA assay has better amplification efficiency at temperatures ranging from 62°C to 65°C. ASFV, African swine fever virus.

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  Figure 7. Optimal time for ASFV-MCDA-LFB assay. The standard plasmid DNA of ASFV was diluted 10-fold and added to the MCDA reaction test. Four reaction times (A) 15 min; (B) 25 min; (C) 35 min; and (D) 45 min, were tested and compared at optimal temperature (63°C); LFB 1 to 8 represented DNA levels of 2.0 × 106 copies/μL, 2.0 × 105 copies/μL, 2.0 × 104 copies/μL, 2.0 × 103 copies/μL, 2.0 × 102 copies/μL, 2.0 × 101 copies/μL, 2.0 copies/μL, and distilled water. The best sensitivity was obtained when the amplification lasted for 25 min (B). TL, test line; CL, control line; ASFV, African swine fever virus; LFB, nanoparticle-based lateral flow biosensor.

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  Figure 8. The specificity assay of ASFV-MCDA-LFB. The ASFV-MCDA-LFB was tested using different genomic DNAs. The LFB 1–32 represented African swine fever virus (ASFV); foot-and-mouth disease virus type A (FMDV-A); foot-and-mouth disease virus type O (FMDV-O); foot-and-mouth disease virus type C (FMDV-C); porcine reproductive and respiratory syndrome virus (PRRSV); classical swine fever virus (CSFV); porcine pseudorabies virus (PRV); distilled water (DW); respiratory syncytial virus (RSV); herpes simplex virus (HSV); influenza A virus (IAV); rotavirus (RV); sendai virus (SV); Neisseria gonorrhoeae type 3 (N. gonorrhoeae); Bacillus anthracis (B. anthracis); Shigella dysenteriae (S. dysenteriae); Vibrio cholerae (V. cholerae); Haemophilus parainfluenzae (H. parainfluenzae); Streptococcus pneumoniae (S. pneumoniae); Klebsiella pneumoniae (K. pneumoniae); Streptococcus suis (S. suis); Mycobacterium tuberculosis 37Rv (M. tuberculosis H37Rv); Staphylococcus aureus (Staph. aureus); dengue virus (DENV); Measles virus (MV); Epstein–Barr virus (EB); Pseudomonas aeruginosa (P. aeruginosa); Brucella melitensis strain M5 (B. melitensis M5); Brucella abortus strain A19 (B. abortus A19); Brucella suis S2…

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  Figure 9. Verification of the feasibility of ASFV-MCDA-LFB assay. The field applicability of ASFV-MCDA-LFB validated by simulated specimens. LFB biosensors 1–12 tested a negative specimen, and the result remained negative. LFB biosensors 13–60 tested a positive specimen, and the result remained positive. TL, test line; CL, control line.