Development of a PCR-RFLP assay for differentiation between African swine fever virus genotypes Ⅰ and Ⅱ

doi:10.3969/j.issn.1004-1524.20250064

Acta Agriculturae Zhejiangensis ›› 2025, Vol. 37 ›› Issue (5): 1017-1028.DOI: 10.3969/j.issn.1004-1524.20250064

• Animal Science • Previous Articles Next Articles

Development of a PCR-RFLP assay for differentiation between African swine fever virus genotypes Ⅰ and Ⅱ

SUN Renjie¹(), XU Huiling², SUN Siqi¹, CHAI Juan¹, YU Yicong¹, XIE Ronghui¹, LI Xiaoliang²^,³, ZHAO Lingyan¹, ZHANG Chuanliang¹^,^*()

1. Zhejiang Provincial Center for Animal Disease Prevention & Control, Hangzhou 311199, China
2. ZJU-Xinchang Joint Innovation Centre (Tianmu Laboratory), Xinchang 312500, Zhejiang, China
3. College of Animal Sciences, Zhejiang University, Hangzhou 310058, China

Received:2025-01-22 Online:2025-05-25 Published:2025-06-11

Abstract

Abstract:

The aim of this study was to establish a PCR-RFLP assay for differentiating genotype Ⅰ and Ⅱ African swine fever virus(ASFV). By comparing the genomic sequences of ASFV genotype Ⅰ and Ⅱ, a pair of specific PCR-RFLP primers was designed targeting the conserved regions of the B385R and B125R genes, which showed no cross-reactivity with other nine important porcine viruses. The detection target was the specific amplification product covering the full length of the B646L gene. Combined with BmgBⅠ restriction enzyme digestion and subsequent RFLP analysis, this method effectively differentiated ASFV genotype Ⅰ and Ⅱ with a detection limit of 5 pg·mL^-1 of DNA. The results of detecting artificially spiked samples demonstrated that the developed method can effectively address co-infections of genotype Ⅰ and Ⅱ or contamination of their nucleic acids in field. In conclusion, the method described here is characterized by its simplicity, high accuracy, strong specificity, high sensitivity, and low cost. These attributes render it highly suitable for practical application. It provides a valuable tool for frontline veterinary technicians to conduct surveillance and epidemiological studies of African swine fever (ASF), thereby contributing to the effective monitoring and prevention of ASF outbreaks.

Key words: African swine fever virus, PCR-RFLP, genotype Ⅰ, genotype Ⅱ

CLC Number:

S855.3

SUN Renjie, XU Huiling, SUN Siqi, CHAI Juan, YU Yicong, XIE Ronghui, LI Xiaoliang, ZHAO Lingyan, ZHANG Chuanliang. Development of a PCR-RFLP assay for differentiation between African swine fever virus genotypes Ⅰ and Ⅱ[J]. Acta Agriculturae Zhejiangensis, 2025, 37(5): 1017-1028.

Figures/Tables 7

Fig.1 PCR-RFLP results of ASFV gene type Ⅰ and Ⅱ plasmid standards A, Results of ASFV gene type Ⅰ plasmid standard p-Ⅰ 2227 and gene type Ⅱ plasmid standard p-Ⅱ 2225; M, 250 bp DNA Ladder; 1-3, PCR product of p-Ⅰ 2227, p-Ⅱ 2225, and negative control digested with BmgBⅠ, respectively; 4-6, PCR product of p-Ⅰ 2227, p-Ⅱ 2225, and negative control, respectively. B, Results of gene type Ⅰ plasmid standard p-Ⅰ 2226 and gene type Ⅱ plasmid standard p-Ⅱ 2225; M, 250 bp DNA ladder; 1-3, PCR product of p-Ⅰ 2226, p-Ⅱ 2225, and negative control digested with BmgBⅠ, respectively; 4-6: PCR product of p-Ⅰ 2226, p-Ⅱ 2225, and negative control, respectively.

Fig.2 Results of specificity test for the developed PCR-RFLP assay A, Results of specificity test for PCR amplification; B, RFLP pattern of BmgBⅠ digestion. M, 250 bp DNA ladder; 1, p-Ⅰ 2227; 2, p-Ⅱ 2225; 3-11, 9 other common swine viruses; 12, Negative control.

Fig.3 Results of sensitivity test for the developed PCR-RFLP assay A, Results of sensitivity test for PCR amplification; 1-8, Different dilutions of p-Ⅰ 2227; 10-17, Different dilutions of p-Ⅱ 2225; 9, Negative control. B and C, RFLP patterns of PCR products of different dilutions of p-Ⅰ 2227 and p-Ⅱ 2225 after BmgBⅠ digestion; 1-8, Different dilutions of p-Ⅰ 2227 or p-Ⅱ 2225; 9, Negative control. M, 250 bp DNA ladder.

Fig.4 Results of repeatability test for the developed PCR-RFLP assay A, PCR amplification results; 1-4, p-Ⅰ 2227; 6-9, p-Ⅱ 2225; 11-14, Negative control. B, RFLP pattern after BmgBⅠ digestion; 1, 4, 7 and 10, PCR products of p-Ⅰ 2227 digested with BmgBⅠ; 2, 5, 8 and 11, PCR products of p-Ⅱ 2225 digested with BmgBⅠ; 3, 6, 9 and 12, Negative control. M, 250 bp DNA ladder.

Table 1 Cycle threshold of artificially spiked samples by qPCR assay

样本编号 The number of samples	样本类型 Sample type	循环阈值 Cycle threshold	结果判定 Result determination
1	肌肉组织样本Muscle tissue sample	31.72	+
2	淋巴结组织样本Lymph node tissue sample	30.85	+
3	环境拭子样本Environmental swab sample	33.50	+
4	环境拭子样本Environmental swab sample	31.65	+
5	血液样本Blood sample	26.41	+
6	肌肉组织样本Muscle tissue sample	—	-
7	淋巴结组织样本Lymph node tissue sample	—	-
8	环境拭子样本Environmental swab sample	—	-
9	环境拭子样本Environmental swab sample	—	-
10	血液样本Blood sample	—	-

Fig.5 Detection of artificially spiked samples 1, 3, 5, 7, 9, Blood samples, tissue samples, and environmental swab samples from pigs negative for ASFV nucleic acid; 2, 4, 6, 8 and 10, artificially spiked samples; 11, Negative control; M, DL5000 DNA marker.

Fig.6 The illustration of developed PCR-RFLP assay

References 35

[1]	RUEDAS-TORRES I, THI TO NGA B, SALGUERO F J. Pathogenicity and virulence of African swine fever virus[J]. Virulence, 2024, 15(1): 2375550.
[2]	WILLIAMS D T, METTENLEITER T C, BLOME S. African swine fever: advances and challenges[J]. Revue Scientifique et Technique(International Office of Epizootics), 2024, Special Edition: 58-69.
[3]	张振江, 孙恩成, 朱远茂, 等. 中国非洲猪瘟研究进展[J]. 中国科学: 生命科学, 2023, 53(12): 1767-1779.
	ZHANG Z J, SUN E C, ZHU Y M, et al. Research progress on African swine fever in China[J]. Scientia Sinica(Vitae), 2023, 53(12): 1767-1779. (in Chinese with English abstract)
[4]	WANG N, ZHAO D M, WANG J L, et al. Architecture of African swine fever virus and implications for viral assembly[J]. Science, 2019, 366(6465): 640-644.
[5]	LIU S, LUO Y Z, WANG Y J, et al. Cryo-EM structure of the African swine fever virus[J]. Cell Host & Microbe, 2019, 26(6): 836-843.
[6]	ZHU Y S, ZHANG M, JIE Z J, et al. Strategic nucleic acid detection approaches for diagnosing African swine fever (ASF): navigating disease dynamics[J]. Veterinary Research, 2024, 55(1): 131.
[7]	QU H L, GE S Q, ZHANG Y Q, et al. A systematic review of genotypes and serogroups of African swine fever virus[J]. Virus Genes, 2022, 58(2): 77-87.
[8]	URBANO A C, FERREIRA F. African swine fever control and prevention: an update on vaccine development[J]. Emerging Microbes & Infections, 2022, 11(1): 2021-2033.
[9]	VU H L X, MCVEY D S. Recent progress on gene-deleted live-attenuated African swine fever virus vaccines[J]. NPJ Vaccines, 2024, 9: 60.
[10]	HU Z Q, TIAN X G, LAI R R, et al. Current detection methods of African swine fever virus[J]. Frontiers in Veterinary Science, 2023, 10: 1289676.
[11]	LABADIE-BRACHO M Y, ADHIN M R. Advocating for PCR-RFLP as molecular tool within malaria programs in low endemic areas and low resource settings[J]. PLoS Neglected Tropical Diseases, 2023, 17(11): e0011747.
[12]	MU X R, GUO J C, WANG H C, et al. Establishment and preliminary application of PCR-RFLP genotyping method for Giardia duodenalis in goats[J]. BMC Veterinary Research, 2024, 20(1): 527.
[13]	AL DAHOUK S, TOMASO H, PRENGER-BERNINGHOFF E, et al. Identification of Brucella species and biotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)[J]. Critical Reviews in Microbiology, 2005, 31(4): 191-196.
[14]	赵灵燕, 张璐, 安慧婷, 等. 猪圆环病毒3型和猪伪狂犬病病毒双重荧光定量PCR方法的建立和应用[J]. 中国兽医科学, 2023, 53(1): 16-22.
	ZHAO L Y, ZHANG L, AN H T, et al. Development and application of a duplex real-time PCR for detection of porcine circovirus type 3 and pseudorabies virus[J]. Chinese Veterinary Science, 2023, 53(1): 16-22. (in Chinese with English abstract)
[15]	GALLARDO C, FERNÁNDEZ-PINERO J, ARIAS M. African swine fever (ASF) diagnosis, an essential tool in the epidemiological investigation[J]. Virus Research, 2019, 271: 197676.
[16]	WEN X X, HE X J, ZHANG X, et al. 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[J]. Emerging Microbes & Infections, 2019, 8(1): 303-306.
[17]	SUN E C, ZHANG Z J, WANG Z L, et al. Emergence and prevalence of naturally occurring lower virulent African swine fever viruses in domestic pigs in China in 2020[J]. Science China Life Sciences, 2021, 64(5): 752-765.
[18]	SUN E C, HUANG L Y, ZHANG X F, et al. Genotype I African swine fever viruses emerged in domestic pigs in China and caused chronic infection[J]. Emerging Microbes & Infections, 2021, 10(1): 2183-2193.
[19]	ZHAO D M, SUN E C, HUANG L Y, et al. Highly lethal genotype I and Ⅱ recombinant African swine fever viruses detected in pigs[J]. Nature Communications, 2023, 14: 3096.
[20]	JAING C, ROWLAND R R R, ALLEN J E, et al. Gene expression analysis of whole blood RNA from pigs infected with low and high pathogenic African swine fever viruses[J]. Scientific Reports, 2017, 7: 10115.
[21]	O'DONNELL V K, GRAU F R, MAYR G A, et al. Rapid sequence-based characterization of African swine fever virus by use of the Oxford nanopore MinION sequence sensing device and a companion analysis software tool[J]. Journal of Clinical Microbiology, 2019, 58(1): e01104-19.
[22]	TORMA G, TOMBÁCZ D, CSABAI Z, et al. Combined short and long-read sequencing reveals a complex transcriptomic architecture of African swine fever virus[J]. Viruses, 2021, 13(4): 579.
[23]	樊晓旭, 吴晓东, 赵洋, 等. 一种对非洲猪瘟病毒基因Ⅰ型和Ⅱ型的快速鉴别诊断方法:CN110438265B[P]. 2022.12.16.
[24]	沈海燕, 张春红, 刘志成, 等. 一种快速区分非洲猪瘟病毒基因Ⅱ型与其它基因型的引物、探针及其检测方法:CN111676316B[P]. 2021-08-31.
[25]	LI X D, HU Y X, LIU P G, et al. Development and application of a duplex real-time PCR assay for differentiation of genotypes I and Ⅱ African swine fever viruses[J]. Transboundary and Emerging Diseases, 2022, 69(5): 2971-2979.
[26]	陈雪蓉, 徐林, 王远微, 等. 恒温隔绝式荧光PCR检测非洲猪瘟病毒核酸方法的建立[J]. 中国动物检疫, 2022, 39(3): 80-84.
	CHEN X R, XU L, WANG Y W, et al. Development of an insulated isothermal PCR assay for detecting African swine fever virus[J]. China Animal Health Inspection, 2022, 39(3): 80-84. (in Chinese with English abstract)
[27]	DING L L, REN T, HUANG L Y, et al. Developing a duplex ARMS-qPCR method to differentiate genotype I and Ⅱ African swine fever viruses based on their B646L genes[J]. Journal of Integrative Agriculture, 2023, 22(5): 1603-1607.
[28]	LUGO-TRAMPE Á, DEL C TRUJILLO-MURILLO K, RODRIGUEZ-SANCHEZ I P, et al. A PCR-RFLP method for typing human papillomavirus type 16 variants[J]. Journal of Virological Methods, 2013, 187(2): 338-344.
[29]	ZHANG C M, YU Y L, YANG H Y, et al. Development of a PCR-RFLP assay for the detection and differentiation of canine parvovirus and mink enteritis virus[J]. Journal of Virological Methods, 2014, 210: 1-6.
[30]	KAWAUCHI K, TAKAHASHI C, ISHIHARA R, et al. Development of a novel PCR-RFLP assay for improved detection and typing of bovine papillomaviruses[J]. Journal of Virological Methods, 2015, 218: 23-26.
[31]	VERNA F, GIORDA F, MICELI I, et al. Detection of morbillivirus infection by RT-PCR RFLP analysis in cetaceans and carnivores[J]. Journal of Virological Methods, 2017, 247: 22-27.
[32]	OCHIAI C, KATAGIRI Y, KOBAYASHI S, et al. Development of a microchip electrophoresis-based, high-throughput PCR-RFLP method to type Tax 233 variants of bovine leukemia virus in Japan[J]. Archives of Virology, 2020, 165(12): 2961-2966.
[33]	CONESA A, DIESER S, BARBERIS C, et al. Differentiation of non-aureus staphylococci species isolated from bovine mastitis by PCR-RFLP of groEL and gap genes in comparison to MALDI-TOF mass spectrometry[J]. Microbial Pathogenesis, 2020, 149: 104489.
[34]	ROSA N M, PENATI M, FUSAR-POLI S, et al. Species identification by MALDI-TOF MS and gap PCR-RFLP of non-aureus Staphylococcus, Mammaliicoccus, and Streptococcus spp. associated with sheep and goat mastitis[J]. Veterinary Research, 2022, 53(1): 84.
[35]	LEITE I G C, BENARD G, CAVALCANTI S C, et al. Comparison between PCR-RFLP and sequencing techniques in the analysis of Paracoccidioides spp. biodiversity: limitations and insights into species and variant differentiation[J]. Mycopathologia, 2024, 189(6): 97.

Development of a PCR-RFLP assay for differentiation between African swine fever virus genotypes Ⅰ and Ⅱ

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