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Journal of Integrative Agriculture  2023, Vol. 22 Issue (5): 1603-1607    DOI: 10.1016/j.jia.2023.02.035
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Developing a duplex ARMS-qPCR method to differentiate genotype I and II African swine fever viruses based on their B646L genes

DING Lei-lei1*, REN Tao1, 2*, HUANG Lian-yu1, Weldu TESFAGABER1, ZHU Yuan-mao1, LI Fang1, SUN En-cheng1#, BU Zhi-gao1#, ZHAO Dong-ming1, 2#

1 State Key Laboratory for Animal Disease Control and Prevention, National High Containment Facilities for Animal Diseases Control and Prevention, National African Swine Fever Para-reference Laboratory, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, P.R.China

2 College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, P.R.China

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非洲猪瘟(African swine feverASF)是由非洲猪瘟病毒(African swine fever virus, ASFV)引起的家猪和野猪的一种急性、烈性、高度接触性传染病,发病率和致死率可高达100%,是全球危害猪群最严重的传染病之一,严重威胁


20188中国首次爆发ASF疫情2021年,高毒力基因II型、低毒力基因II型和基因I型三种不同类型的ASFV陆续被报道。这使得ASFV感染的鉴别诊断更具挑战性。截止目前,仍无有效的预防疫苗和特效的治疗药物,因此,能够明确区分基因I型和IIASFV的鉴别诊断方法对于ASF有效预防和控制至关重要。在此,我们基于基因I型和IIASFV编码P72蛋白B646L基因C两个单核苷酸多态性(SNP)位点,利用突变阻滞扩增系统(ARMS)和定量实时PCRqPCR)技术开发了一种双重ARMS-qPCR方法。进一步研究显示,该方法标准曲线R2在0.992 - 0.999,线性关系良好;与PRRSV、CSFV、PRV、PCV2、PEDV、TGEV 和 PoRV 七种病原无交叉反应,具有较高的特异性;能够稳定的检测含有10个拷贝数的基因I型和IIASFV B646L基因的标准品质粒,敏感性较好;组间和组内变异系数均小于2.2%,重复性良好。此外,我们用18个基因I型和18个基因IIASFV感染样本,包括血液、口腔和直肠拭子、组织和细胞培养物对其进一步的验证并与动物世界卫生组织(WOAH)推荐的qPCR方法进行比对。结果显示,ARMS-qPCR方法成功鉴别诊断出36个基因I型和IIASFV感染样本且与WOAH推荐的qPCR方法检测的结果一致。因此,我们建立的ARMS-qPCR方法可用于检测和区分基因I型和基因II


African swine fever (ASF), caused by the African swine fever virus (ASFV), is an acute, hemorrhagic, and contagious disease of domestic pigs and wild boars.  The disease is notifiable and listed by the World Organization for Animal Health (WOAH) (Wang N et al. 2019).  The outcomes of ASF infection can be peracute, acute, subacute, and chronic, depending on the virulence of ASFVs.  According to the report of WOAH (, from January 2020 to December 2022, ASF led to more than  2 million pig losses.  Currently, ASFV persists continuously in more than 23 countries and poses a serious threat to the global swine industry.  ASF invaded China on 3 August, 2018, caused by genotype II virulent Georgia-07-like ASFVs (Wen et al. 2019; Zhao et al. 2019; Wang et al. 2020; Wang L et al. 2022).  An experimental study showed that Georgia-07-like ASFV HLJ/18 isolated in China is highly lethal and efficiently transmissible in domestic pigs (Zhao et al. 2019; Jiang et al. 2021).  During the past four years, genotype II Georgia-07-like ASFVs dominantly spread in China.  However, the low virulent genotype II and I ASFVs have been successively reported in China in 2020 and 2021, respectively (Sun et al. 2021a, b; Shi et al. 2022).  Compared with the high virulent genotype II HLJ/18 strain, the low virulent genotype I and II ASFVs had lower virulence and high transmissibility in pigs and induced persistent and chronic infection showing irregular virus shedding at low levels (Sun et al. 2021a, b; Tsegay et al. 2022; Wang P et al. 2022).  Notably, when different genotype I and genotype II viruses infect the same pig in the field, a novel virus may be generated through viral genome recombination, which brings new problems and challenges for the prevention and control of ASF in China.  Thus, a diagnostic method that differentiates genotype I and II ASFVs with high sensitivity and stability is urgently needed and will be helpful for the prevention and control of ASF in China.  

ASFVs have been divided into at least 24 genotypes based on the C-terminus of the B646L gene with 478 nt (Bastos et al. 2003).  B646L gene is one of the most used target genes for ASF diagnosis, which is also the target gene for the WOAH recommended PCR and fluorescent quantitative PCR assays (Agüero et al. 2003; King et al. 2003).  Sanger sequencing of targeted amplification of the B646L genes is the main genotyping approach for ASFVs.  Recently, Li et al. (2022) developed the duplex real-time PCR assay based on the ASFV E296R gene, and Cao et al. (2022) established the TaqMAN-MGB probe assay based on the N-terminal sequences of the B646L gene (Cao et al. 2022; Li et al. 2022), which could distinguish genotype I and II ASFVs with detection limits of 10 copies.  However, the target genes or regions in their methods were out of ASFV genotyping regions.  

Single nucleotide polymorphism (SNP) is a single base change at a specific position in the genome of different individuals and can be used as a genotyping marker for the detection of different individual genotypes (Gut 2001).  The amplification refractory mutation system (ARMS), also named Allele-specific PCR (AS-PCR), relies on the extension of primer only when its 3´ end has a perfect complement to the template (Wang M et al. 2019).  ARMS-qPCR technology has been developed and widely used in SNP detection and genotyping (Ochsenreither et al. 2010; Shi et al. 2013; Wang M et al. 2019).  Compared with other assays for SNP detection and genotyping, ARMS-qPCR has the advantage of low-cost, simple operation, high sensitivity, and rapid and real-time detection.

Here, 126 complete or partial B646L genes of ASFVs, including 78 genotype I and 48 genotype II viruses, were obtained from the GenBank database, and their information is shown in Appendix A.  After analyzing these genes by the MegAlign Software (DNAStar), there were 4 SNPs in the C-terminus of the B646L gene, differentiating genotype I viruses from genotype II viruses (Fig. 1-A).  Two SNPs at sites 1 656 and 1 710 were used to design primers and probes for differential detection of genotype I and II ASFVs (Fig. 1-A).  As previously described (Huang et al. 1992; Liu et al. 2012), primers (I F, II F and R) and probes (probe 1 and probe 2) were designed with the targeted gene sequences using Primer 5 Software (Fig. 1-B; Appendix B).  The duplex ARMS-qPCR reaction system volume was 25 μL: 12.5 μL of 2× HyperProbe Mixture (GENFINE), 0.5 μL of I F, II F and R primers (10 μmol L–1), 0.5 μL of probe 1 and probe 2 (10 μmol L–1), 5 μL of template DNA, and 5 μL of ddH2O.  The duplex ARMS-qPCR was performed by using the Bio-Rad CFX96 Touch Real-Time PCR Detection System with the following reaction conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s.  Fluorescence signal was detected at the end of each cycle of extension step.  For the positive sample of genotype I ASFV, FAM and Cy5 fluorophores could be detected; however, for the positive sample of genotype II ASFV, only FAM fluorophore could be detected (Fig. 1-B).  

The standard curve test revealed that for the standard plasmids of genotype I ASFV, the slopes were –3.3825 for Cy5 and –3.1906 for FAM; the correlation coefficient R2 was 0.999 for Cy5 and 0.998 for FAM; the amplification efficiency was 97.53% for Cy5 and 100.06% for FAM, respectively (Fig. 1-C); for the standard plasmids of genotype II ASFV, the slope was –3.2983 for FAM, the correlation coefficient R2 was 0.992 for FAM, the amplification efficiency was 100.01% for FAM, whereas Cy5 fluorophore could not be detected (Fig. 1-C).  In addition, the sensitivity of the duplex ARMS-qPCR was 10 copies per reaction for both genotype I and II ASFVs (Fig. 1-D).  Thus, these results indicated that the duplex ARMS-qPCR assay has high efficiency and sensitivity.  

We then evaluated the specificity of the duplex ARMS-qPCR.  The nucleic acids of 7 other swine viruses, including PRRSV, CSFV, PRV, PCV2, PEDV, TGEV, and PoRV, were used as templates.  There were 3 amplification curves obtained for genotype I ASFV (FAM and Cy5 signals) and II ASFV (FAM signal), whereas no amplification curve was recorded for the nucleic acids of PRRSV, CSFV, PRV, PCV2, PEDV, TGEV, and PoRV, as well as genotype II ASFV (Cy5 signal) and ddH2O (Fig. 1-E).  The results demonstrated that the duplex ARMS-qPCR has a good specificity without cross-reactivity with other swine viruses.

The results of the stable detection limit test showed that for the standard plasmids of genotype I ASFV, all 12 replicates were tested positive at the dilution of 10 copies, while 7/12 replicates were tested positive at the dilution of 5 copies (Fig. 1-F); for the standard plasmids of genotype II ASFV, all 12 replicates were tested positive at the dilution of 10 copies, while 6/12 replicates were tested positive at the dilution of 1 copy (Fig. 1-F).  Thus, the stable detection limit of the duplex ARMS-qPCR was 10 copies per reaction for both genotype I and II ASFVs (Fig. 1-F).

We further assessed the repeatability and reproducibility of the duplex ARMS-qPCR.  The assay tested the standard plasmids of 3 concentrations (106, 104, and 102 copies).  For the standard plasmids of genotype I ASFV, the intra- and inter-assay variation of Ct value for the duplex ARMS-qPCR ranged from 0.07 to 0.93% and 1.2 to 2.17% in FAM fluorescence channel and from 0.38 to 1.02% and 0.85 to 1.27% in Cy5 fluorescence channel, respectively (Table 1).  For the standard plasmids of genotype II ASFV, the intra- and inter-assay variation of Ct value for the duplex ARMS-qPCR ranged from 0.27 to 0.61% and 0.77 to 1.07% in FAM fluorescence channel (Table 1).  These findings suggested that the duplex ARMS-qPCR assay has satisfactory repeatability and reproducibility.

Finally, we evaluated the duplex ARMS-qPCR compared with WOAH-qPCR.  A total of 40 samples were detected using both assays, including blood, oral and rectal swabs, tissues, and cell cultures from pigs or PAMs infected by genotype I and II ASFVs.  Animal studies have evaluated the virulence and transmissibility of genotype I ASFV SD/DY-I/21 and genotype II virus HLJ/18 (Zhao et al. 2019; Sun et al. 2021a), respectively.  The results showed that 36 samples, including 18 of genotype I ASFV and 18 of genotype II ASFV were detected to be positive and differentiated by the duplex ARMS-qPCR, which were consistent with the results of the WOAH-qPCR (Appendix C).  

In summary, we developed a duplex ARMS-qPCR assay based on ASFV genotyping region of B646L gene, which can effectively differentiate genotype I and II ASFVs.  The assay had high sensitivity and specificity and exhibited good results in detecting samples, including blood, oral and rectal swabs, tissues, and cell culture.  Whether our method could be used for differentiating other genotypes of ASFVs is needed for further evalution.  However, just genotype I and II ASFVs are spreading outside Africa.  Thus, our method will provide an additional epidemiological investigation tool to implement effective ASFV control and prevention.

Received: 06 January 2023   Accepted: 10 February 2023
Fund: This work was supported by the National Key R&D Program of China (2021YFD1800101 and 2019YFE0107300), the Applied Technology Research and Development Project of Heilongjiang Province, China (GA19B301), and the Central Public-Interest Scientific Institution Basal Research Fund, China (1610302022003).
About author:  DING Lei-lei, E-mail:; REN Tao, E-mail:; #Correspondence ZHAO Dong-ming, E-mail:; BU Zhi-gao, E-mail:; SUN En-cheng, E-mail: *These authors equally contributed to this study.

Cite this article: 

DING Lei-lei, REN Tao, HUANG Lian-yu, Weldu TESFAGABER, ZHU Yuan-mao, LI Fang, SUN En-cheng, BU Zhi-gao, ZHAO Dong-ming. 2023. Developing a duplex ARMS-qPCR method to differentiate genotype I and II African swine fever viruses based on their B646L genes. Journal of Integrative Agriculture, 22(5): 1603-1607.

Agüero M, Fernández J, Romero L, Sánchez Mascaraque C, Arias M, Sánchez-Vizcaíno J M. 2003. Highly sensitive PCR assay for routine diagnosis of African swine fever virus in clinical samples. Journal of Clinical Microbiology41, 4431–4434.

Bastos A D, Penrith M L, Cruciere C, Edrich J L, Hutchings G, Roger F, Couacy-Hymann E, Thomson G R. 2003. Genotyping field strains of African swine fever virus by partial p72 gene characterisation. Archives of Virology48, 693–706.

Cao S, Lu H, Wu Z, Zhu S. 2022. A duplex fluorescent quantitative PCR assay to distinguish the genotype I and II strains of African swine fever virus in Chinese epidemic strains. Frontiers in Veterinary Science9, 998874.

Gut I G. 2001. Automation in genotyping of single nucleotide polymorphisms. Human Mutation17, 475–492.

Huang M M, Arnheim N, Goodman M F. 1992. Extension of base mispairs by Taq DNA polymerase: Implications for single nucleotide discrimination in PCR. Nucleic Acids Research20, 4567–4573.

Jiang C, Sun Y, Zhang F, Ai X, Feng X, Hu W, Zhang X, Zhao D, Bu Z, He X. 2021. Viricidal activity of several disinfectants against African swine fever virus. Journal of Integrative Agriculture20, 3084–3088.

King D P, Reid S M, Hutchings G H, Grierson S S, Wilkinson P J, Dixon L K, Bastos A D, Drew T W. 2003. Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus. Journal of Virological Methods107, 53–61.

Li X, Hu Y, Liu P, Zhu Z, Liu P, Chen C, Wu X. 2022. Development and application of a duplex real-time PCR assay for differentiation of genotypes I and II African swine fever viruses. Transboundary and Emerging Diseases69, 2971–2979.

Liu J, Huang S, Sun M, Liu S, Liu Y, Wang W, Zhang X, Wang H, Hua W. 2012. An improved allele-specific PCR primer design method for SNP marker analysis and its application. Plant Methods8, 34.

Ochsenreither S, Reinwald M, Thiel E, Burmeister T. 2010. Melting point assay for the JAK2 V617F mutation, comparison with amplification refractory mutation system (ARMS) in diagnostic samples, and implications for daily routine. Molecular Diagnosis & Therapy14, 185–190.

Shi K, Liu H, Yin Y, Si H, Long F, Feng S. 2022. Molecular characterization of African swine fever virus from 2019–2020 outbreaks in Guangxi Province, Southern China. Frontiers in Veterinary Science9, 912224.

Shi X, Zhang C, Shi M, Yang M, Zhang Y, Wang J, Shen H, Zhao G, Ma X. 2013. Development of a single multiplex amplification refractory mutation system PCR for the detection of rifampin-resistant Mycobacterium tuberculosisGene530, 95–99.

Sun E, Huang L, Zhang X, Zhang J, Shen D, Zhang Z, Wang Z, Huo H, Wang W, Huangfu H, Wang W, Li F, Liu R, Sun J, Tian Z, Xia W, Guan Y, He X, Zhu Y, Zhao D, et al. 2021a. Genotype I African swine fever viruses emerged in domestic pigs in China and caused chronic infection. Emerging Microbes & Infections10, 2183–2193.

Sun E, Zhang Z, Wang Z, He X, Zhang X, Wang L, Wang W, Huang L, Xi F, Huangfu H, Tsegay G, Huo H, Sun J, Tian Z, Xia W, Yu X, Li F, Liu R, Guan Y, Zhao D, et al. 2021b. Emergence and prevalence of naturally occurring lower virulent African swine fever viruses in domestic pigs in China in 2020. Science China (Life Sciences), 64, 752–765.

Tsegay G, Tesfagaber W, Zhu Y, He X, Wang W, Zhang Z, Sun E, Zhang J, Guan Y, Li F, Liu R, Bu Z, Zhao D. 2022. Novel P22-monoclonal antibody based blocking ELISA for the detection of African swine fever virus antibodies in serum. Biosafety and Health4, 234–243.

Wang J, Shi X, Sun H, Chen H. 2020. Insights into African swine fever virus immunoevasion strategies. Journal of Integrative Agriculture19, 11–22.

Wang L, Fu D, Tesfagaber W, Li F, Chen W, Zhu Y, Sun E, Wang W, He X, Guo Y, Bu Z, Zhao D. 2022. Development of an ELISA method to differentiate animals infected with wild-type African swine fever viruses and attenuated HLJ/18-7GD vaccine candidate. Viruses14, 1731.

Wang M, Hang J, Abuzeid A M I, Huang Y, Fu Y, Yan X, Zhang P, Huo C, Liu Y, Ran R, Sun Y, Li G. 2019. Development of multi-ARMS-qPCR method for detection of hookworms from cats and dogs. Parasitology International73, 101974.

Wang N, Zhao D, Wang J, Zhang Y, Wang M, Gao Y, Li F, Wang J, Bu Z, Rao Z, Wang X. 2019. Architecture of African swine fever virus and implications for viral assembly. Science366, 640–644.

Wang P, Wang M, Shi Z, Sun Z, Wei L, Liu Z, Wang S, He X, Wang J. 2022. Development of a recombinant pB602L-based indirect ELISA assay for detecting antibodies against African swine fever virus in pigs. Journal of Integrative Agriculture21, 819–825.

Wen X, He X, Zhang X, Zhang X, Liu L, Guan Y, Zhang Y, Bu Z. 2019. Genome sequences derived from pig and dried blood pig feed samples provide important insights into the transmission of African swine fever virus in China in 2018. Emerg Microbes Infect8, 303–306.

Zhao D, Liu R, Zhang X, Li F, Wang J, Zhang J, Liu X, Wang L, Zhang J, Wu X, Guan Y, Chen W, Wang X, He X, Bu Z. 2019. Replication and virulence in pigs of the first African swine fever virus isolated in China. Emerging Microbes & Infections8, 438–447.

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