Development of Real-Time PCR Based on A137R Gene for the Detection of African Swine Fever Virus

Figures

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  Figure 1. Alignment of sequences of the ASFV A137R gene and B646L gene in GenBank. (A) Locations of the target sequences of the primers A137R-F/A137R-R designed in the present study. (B) Locations of the target sites of the primers OIE-recommended TaqMan PCR method (19). Dots (.) indicate identical bases. The primer-binding sequences are boxed.

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  Figure 2. Standard curve and sensitivity tests for A137R gene-based real-time PCR assay of ASFV. (A) Establishment of standard curve for A137R gene-based real-time PCR. The 10-fold serial dilutions ranging from 1.0 × 108 to 1.0 × 101 copies/μL of DNA plasmid were tested in the real-time PCR. Each point corresponds to the mean value of three replicates. The optimal standard formula is y = −3.252x + 36.26, and the correlation coefficient is 0.9992. (B) Sensitivity tests for A137R gene-based real-time PCR assay of ASFV. Ten-fold serial dilutions of the DNA plasmid were used to perform the real-time PCR to obtain the expanded curve of the assay.

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  Figure 3. Sensitivity of different real-time PCR assays for detection of ASFV DNA. Ten-fold serial dilutions of the ASFV DNA were used to perform the real-time PCR to obtain the expanded curve of the assays. (A) Detection limit of the A137R gene-based real-time PCR assay. (B) Detection limit of the OIE-recommended TaqMan PCR assay.

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  Figure 4. Specificity analysis of the A137R gene-based real-time PCR. Only ASFV showed a positive fluorescence signal, and no positive signal was observed with other swine viruses.