非洲猪瘟病毒基因Ⅰ型和Ⅱ型PCR-RFLP鉴别方法的建立

doi:10.3969/j.issn.1004-1524.20250064

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (5): 1017-1028.DOI: 10.3969/j.issn.1004-1524.20250064

非洲猪瘟病毒基因Ⅰ型和Ⅱ型PCR-RFLP鉴别方法的建立

孙仁杰¹(), 徐慧玲², 孙思琪¹, 柴娟¹, 虞一聪¹, 谢荣辉¹, 李肖梁²^,³, 赵灵燕¹, 张传亮¹^,^*()

1.浙江省动物疫病预防控制中心,浙江杭州 311199
2.浙江大学-新昌联合创新中心(天姥实验室),浙江新昌 312500
3.浙江大学动物科学学院,浙江杭州 310058

收稿日期:2025-01-22 出版日期:2025-05-25 发布日期:2025-06-11
作者简介:孙仁杰(1993—),男,浙江杭州人,博士,兽医师,主要从事病毒感染生物学研究与动物疫病监测工作。E-mail: rjsun@zju.edu.cn
通讯作者: *张传亮,E-mail: 417270733@qq.com
基金资助:
浙江省自然科学基金(LQ23C180001);浙江省万人计划(2021R52041);浙江省“三农九方”科技协作项目(2023SNJF059);浙江省农业重大技术协同推广计划项目(2023ZDXT14-5)

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

摘要/Abstract

摘要：

本研究旨在建立一种非洲猪瘟病毒(African swine fever virus, ASFV)基因Ⅰ型和Ⅱ型的PCR-RFLP鉴别方法。通过对ASFV基因Ⅰ型与基因Ⅱ型基因组序列的比对分析,针对B385R基因和B125R基因保守区设计1对特异性PCR-RFLP引物,与其他临床上常见的9种猪病病毒均无交叉反应。通过该引物扩增获得覆盖B646L基因全长的DNA片段作为检测靶标,结合BmgBⅠ酶切进行RFLP图谱分析,即可实现对B646L基因全序列的快速检测,有效区分ASFV基因Ⅰ型和Ⅱ型,最低检测DNA质量浓度可达到5 pg·mL^-1。模拟临床样本的研究结果表明,本方法还能够有效应对实际检测中基因Ⅰ型和Ⅱ型的混合感染。综上,本方法操作简便、准确性高、特异性强、灵敏度高、成本较低,具有良好的应用前景,为基层兽医技术人员开展非洲猪瘟疫情的监测和流行病学研究提供了一种新方法。

关键词: 非洲猪瘟病毒, 聚合酶链式反应-限制性片段长度多态性, 基因Ⅰ型, 基因Ⅱ型

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

中图分类号:

S855.3

孙仁杰, 徐慧玲, 孙思琪, 柴娟, 虞一聪, 谢荣辉, 李肖梁, 赵灵燕, 张传亮. 非洲猪瘟病毒基因Ⅰ型和Ⅱ型PCR-RFLP鉴别方法的建立[J]. 浙江农业学报, 2025, 37(5): 1017-1028.

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.

图/表 7

图1 ASFV基因Ⅰ型和Ⅱ型质粒标准品的PCR-RFLP结果 A,ASFV基因Ⅰ型质粒标准品p-Ⅰ 2227和基因Ⅱ型质粒标准品p-Ⅱ 2225的结果;M为250 bp DNA Ladder;1~3依次为p-Ⅰ 2227、p-Ⅱ 2225和阴性对照的PCR产物经BmgBⅠ酶切;4~6依次为p-Ⅰ 2227、p-Ⅱ 2225和阴性对照的PCR产物。B,基因Ⅰ型质粒标准品p-Ⅰ 2226和基因Ⅱ型质粒标准品p-Ⅱ 2225的结果;M,250 bp DNA ladder;1~3依次为p-Ⅰ 2226、p-Ⅱ 2225和阴性对照PCR产物经BmgBⅠ酶切;4~6依次为p-Ⅰ 2226、p-Ⅱ 2225和阴性对照的PCR产物。

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.

图2 PCR-RFLP方法的特异性试验结果 A,PCR扩增的特异性试验结果;B,BmgBⅠ酶切的RFLP图谱。M,250 bp DNA ladder;1,p-Ⅰ 2227;2,p-Ⅱ 2225;3~11,9种其他常见猪病毒病病原;12,阴性对照。

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.

图3 PCR-RFLP方法的敏感性试验结果 A,PCR扩增的敏感性试验结果;1~8,不同稀释梯度的p-Ⅰ 2227;10~17,不同稀释梯度的p-Ⅱ 2225;9,阴性对照。B和C,不同稀释梯度p-Ⅰ 2227和p-Ⅱ 2225的PCR产物经BmgBⅠ酶切的RFLP图谱;1~8,不同稀释梯度的p-Ⅰ 2227或p-Ⅱ 2225;9,阴性对照。M,250 bp DNA ladder。

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.

图4 PCR-RFLP方法的重复性试验结果 A,PCR扩增结果;1~4,p-Ⅰ 2227;6~9,p-Ⅱ 2225;11~14,阴性对照。B,BmgBⅠ酶切的RFLP图谱;1、4、7、10,p-Ⅰ 2227的PCR产物经BmgBⅠ酶切;2、5、8、11,p-Ⅱ 2225的PCR产物经BmgBⅠ酶切;3、6、9、12,阴性对照。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.

表1 qPCR方法检测模拟临床样本的循环阈值

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	—	-

图5 模拟临床样本检测结果 1、3、5、7、9,非洲猪瘟病毒核酸阴性猪的血液、组织和环境拭子样本;2、4、6、8、10,模拟临床样本;11,阴性对照;M,DL5000 DNA marker。

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.

图6 建立的PCR-RFLP鉴别方法的图示

Fig.6 The illustration of developed PCR-RFLP assay

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非洲猪瘟病毒基因Ⅰ型和Ⅱ型PCR-RFLP鉴别方法的建立

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

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