Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (5): 1073-1080.doi: 10.3864/j.issn.0578-1752.2021.05.018

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles    

Development of a TaqMan Real-Time PCR Targeting the MGF360-13L Gene of African Swine Fever Virus

Tao WANG(),Yu HAN,Li PAN,Bing WANG,MaoWen SUN,Yi WANG,YuZi LUO,HuaJi QIU(),Yuan SUN()   

  1. Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences/Key Laboratory of Veterinary Biotechnology, Harbin 150069
  • Received:2019-12-31 Accepted:2021-01-11 Online:2021-03-01 Published:2021-03-09
  • Contact: HuaJi QIU,Yuan SUN E-mail:wangtaocaas@163.com;qiuhuaji@caas.cn;sunyuan@caas.cn

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Abstract:

【Objective】The objective of this study is to develop a TaqMan real-time PCR for detection of African swine fever virus (ASFV) MGF360-13L gene, providing technical support for diagnosis, virus purification, differential diagnosis of MGF360-13L deletion ASFV strains, and gene function research of ASFV.【Method】In this study, the TaqMan real-time PCR primers and probes were designed based on the ASFV MGF360-13L (GenBank: MK333180.1), and the TaqMan real-time quantitative PCR assay of ASFV was established. A pair of specific primers, 13L-F/13L-R was designed to construct ASFV standard plasmid. The standard curve was generated for quantitative analysis using the ten-fold dilution of the ASFV standard plasmid, and the sensitivity and repeatability of the system were also evaluated. The ten-fold dilution of the ASFV standard was also detected by PCR to determine the sensitivity of the method. Thirty clinical samples collected from an outbreak ASF pig farm in Heilongjiang province were simultaneously tested by this TaqMan real-time PCR, and another one previously established in our laboratory to compare the coincidence rate of these two detection methods. Differential diagnosis of parent ASFV and MGF gene deletion strain after infection with PAM cell【Result】A specific band of about 800 bp was amplified using primer 13L-F/13L-R, and no band was found in the negative control, and the standard plasmid was successfully constructed. The standard curve had a good linear relationship between real-time PCR cycle threshold (Ct) and template copies. The linear regression equation was: y = -3.295 lg(x) + 45.995 with correlation coefficient of 0.997, and the minimum detection limit was 15.6 copies/μL of ASFV nucleic acid. There was no cross-reaction with classical swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine transmissible gastroenteritis virus, porcine circovirus type 1, and porcine circovirus type 2. In the clinical sample test, the results of the two methods in McNemar test, P = 0.5>0.05, indicating that there was no statistical difference between the two methods and in Kappa test, Kappa = 0.867>0.75, P<0.001, suggesting it was a good agreement and could effectively identify parent ASFV or MGF gene deletion ASFV infection. 【Conclusion】The TaqMan real-time PCR for detection of ASFV MGF360-13L established in this study had highly specific, sensitive, reproducible, and high coincidence rate. It not only enriched the detection method of ASFV, but also provided technical support for researching the gene function of MGF360-13L, identification of the MGF360-13L gene-deleted ASFV, and differential diagnosis of related gene deletion vaccine strains.

Key words: African swine fever virus, MGF360-13L, TaqMan real-time PCR, detection

Fig. 1

Amplification of target gene M：DL2000 DNA Marker;1：MGF360-13L Gene;2：Negative control"

Fig. 2

Standard curve for TaqMan Real-time PCR of ASFV MGF360-13L"

Fig. 3

TaqMan Real-time PCR specific detection of ASFV MGF360-13L"

Table 1

Intra-assay and inter-assay reproducibility of TaqMan Real-time PCR using a MGF360-13L standard"

标准品（拷贝）
Standard plasmid template (copies/μL)		 组内重复性 Intra-assay 组间重复性 Inter-assay
平均值Mean ± SD 变异系数 CV(%) 平均值 Mean ± SD 变异系数CV(%)
1.56 × 109 16.33 ± 0.09 0.54 16.23 ± 0.26 1.63
1.56 × 108 19.96 ± 0.09 0.28 20.0 ± 0.12 0.6
1.56 × 107 22.89 ± 0.08 0.35 22.56 ± 0.23 1.0
1.56 × 106 26.13 ± 0.08 0.85 25.76 ± 0.5 1.93

Fig. 4

Results of MGF360-13L detected by PCR M: 2000 bp 分子量标准M：DL2000 DNA Marker;1-10：质粒标准品1.56×1010-1.56×101拷贝/μL 1.56×1010-1.56×101 copies/μL standard plasmid;11：阴性对照 Negative control;12：阳性对照 Positive control"

Fig. 5

Amplification curve of PAM cells infected with ASFV or ASFV-ΔMGF"

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