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. 2021 Nov 29:8:753967.
doi: 10.3389/fvets.2021.753967. eCollection 2021.

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

Dan Yin  1   2   3 , Renhao Geng  1   2   3 , Hui Lv  1   2   3 , Chunhui Bao  1   2   3 , Hongxia Shao  1   2   3 , Jianqiang Ye  1   2   3 , Kun Qian  1   2   3 , Aijian Qin  1   2   3
Affiliations

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

Dan Yin et al. Front Vet Sci. .

Abstract

African swine fever virus (ASFV) can infect domestic pigs and wild boars and causes huge economic losses in global swine industry. Therefore, early diagnosis of ASFV is important for the control and eradication of African swine fever (ASF). In this study, a SYBR Green-based real-time polymerase chain reaction (PCR) assay targeting the viral encoded A137R gene was established for the detection of ASFV infection. For the evaluation of the established real-time PCR, 34 clinical samples were assessed by both the A137R gene-based real-time PCR and OIE-recommended TaqMan PCR. The results showed that 85.29% (29/34) were detected by A137R gene-based real-time PCR, but only 79.41% (27/34) positive using OIE-recommended TaqMan PCR. Moreover, no cross-reaction with other common swine pathogens was found in the A137R gene-based real-time PCR. These results demonstrated that the established real-time PCR assay in this study showed better performance than the OIE-recommended method in detecting ASFV from clinical samples, which could be applied for control and eradication programs of ASF.

Keywords: A137R gene; African swine fever virus; SYBR Green; detection; real-time PCR.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
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.
Figure 2
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.
Figure 3
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.
Figure 4
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.
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