非洲猪瘟病毒LNA引物PCR检测方法的建立及优化

doi:10.11924/j.issn.1000-6850.casb2020-0643

中国农学通报 ›› 2021, Vol. 37 ›› Issue (29): 107-113.doi: 10.11924/j.issn.1000-6850.casb2020-0643

所属专题：畜牧兽医

• 畜牧·动物医学·蚕·蜂 • 上一篇下一篇

非洲猪瘟病毒LNA引物PCR检测方法的建立及优化

李艳(), 张煜轩, 楚品品, 蒋智勇, 席振军, 蔡汝健, 勾红潮, 张昆丽, 宋帅, 卞志标, 李春玲()

广东省农业科学院动物卫生研究所,广东省畜禽疫病防治研究重点实验室,农业农村部兽用药物与诊断技术广东科学观测实验站,广州 510640

收稿日期:2020-11-10 修回日期:2021-02-24 出版日期:2021-10-15 发布日期:2021-10-29
通讯作者: 李春玲
作者简介:李艳,女,1980年出生,山东招远人,高级兽医师,博士,研究方向：动物传染病病原分子生物学。通信地址：510640 广州市天河区五山白石岗广东省农业科学院动物卫生研究所,Tel：020-85291976,E-mail： 371273724@qq.com。
基金资助:
广东省重点领域研发计划项目“非洲猪瘟精准检测技术研究及应用”(2019B020211005);国家十三五重点研发项目“猪重要疫病远程诊断技术研究”(2016YFD0500709);广东省自然科学基金面上项目“副猪嗜血杆菌OMVs对小鼠单核巨噬细胞RAW264.7的致炎机制研究”(2020A1515010475)

Establishment of PCR with LNA Modified Primers for Detection of African Swine Fever Virus

Li Yan(), Zhang Yuxuan, Chu Pinpin, Jiang Zhiyong, Xi Zhenjun, Cai Rujian, Gou Hongchao, Zhang Kunli, Song Shuai, Bian Zhibiao, Li Chunling()

Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture, Guangzhou 510640

Received:2020-11-10 Revised:2021-02-24 Online:2021-10-15 Published:2021-10-29
Contact: Li Chunling

摘要/Abstract

摘要：

为了建立一种便捷、快速且精准的非洲猪瘟病毒的检测方法,本研究根据GenBank中公布的ASFV p72基因序列,设计了一对特异性引物,并对引物中的适当碱基进行锁核酸修饰,通过优化退火温度、引物浓度,建立了非洲猪瘟病毒的锁核酸修饰引物PCR检测方法。结果表明,该方法具有良好的敏感性和特异性,检测灵敏度可以达到3×10¹ copies/uL,比常规引物PCR方法的灵敏度提高了100倍,比real-time PCR方法的灵敏度提高了10倍,对猪瘟病毒、猪圆环病毒2型、猪伪狂犬病毒等病原基因组均无扩增,特异性良好。72份临床样品的检测结果与OIE推荐的qPCR方法检测结果一致,符合率为100%。本研究成功建立了非洲猪瘟病毒的LNA引物PCR检测方法,方法的特异性强、敏感性高,操作简单,为非洲猪瘟病毒的检测提供了一种新的、更加敏感的检测技术。

关键词: 非洲猪瘟病毒, p72基因, LNA引物, PCR检测方法

Abstract:

In order to establish a simple, rapid and accurate method for detecting African swine fever virus, in this study a pair of specific primers were designed according to the p72 gene sequence of ASFV published in GenBank, and the appropriate bases in the primers were modified with locked nucleic acid. By optimizing the annealing temperature and the primers concentration, a PCR method with LNA modified primers for detecting ASFV was established. The results show that this method is highly sensitive and specific, and the sensitivity can reach 3×10¹ copies/uL, which is 100 times higher than that of PCR with conventional primers and 10 times higher than that of real-time PCR. The method has no amplification of the genomes of classical swine fever virus, porcine circovirus type 2 and pseudorabies virus, with good specificity. The results of 72 clinical samples are consistent with the results of qPCR recommended by OIE, and the coincidence rate is 100%. In this study, a LNA primer PCR method for detecting ASFV is established, with high specificity, sensitivity and simple operation, providing a novel, sensitive and specific detection technique for the detection of ASFV.

Key words: ASFV, p72 gene, LNA primer, PCR method

中图分类号:

S855.3

李艳, 张煜轩, 楚品品, 蒋智勇, 席振军, 蔡汝健, 勾红潮, 张昆丽, 宋帅, 卞志标, 李春玲. 非洲猪瘟病毒LNA引物PCR检测方法的建立及优化[J]. 中国农学通报, 2021, 37(29): 107-113.

Li Yan, Zhang Yuxuan, Chu Pinpin, Jiang Zhiyong, Xi Zhenjun, Cai Rujian, Gou Hongchao, Zhang Kunli, Song Shuai, Bian Zhibiao, Li Chunling. Establishment of PCR with LNA Modified Primers for Detection of African Swine Fever Virus[J]. Chinese Agricultural Science Bulletin, 2021, 37(29): 107-113.

图/表 8

引物名称	序列(5＇-3＇)	产物大小
P1	GGTAATGTGATCGGATACGTAACG	147 bp
P2	TTGGCACAAGTTCGGACATGTTGT
P3	GGTAATGTGAtCGGaTACGtAACG
P4	TTGGCACAaGTtCGGaCATGTTGT

表1. 引物信息

图1. 不同退火温度的扩增结果 A：LNA引物不同退火温度扩增结果;B：常规引物不同退火温度扩增结果;M：DL500;1-12：60.0℃,60.3℃,61.2℃,62.6℃,64.6℃,66.5℃,68.4℃,70.3℃,72.2℃,73.7℃,74.6℃,75.0℃;13：阴性对照

图2. 引物浓度的优化 M：DL500;1：0.2 uL;2：0.4 uL;3：0.6 uL;4：0.8 uL;5：1.0 uL;6：1.2 uL;7：1.4 uL;8：阴性对照

图3. LNA引物PCR敏感性 M：DL500;1-6：不同稀释度重组质粒(3×105~3×100 copies/uL);7：阴性对照

图4. 常规引物PCR敏感性 M：DL500;1-6：不同稀释度重组质粒(3×105~3×100 copies/uL);7：阴性对照

图5. Real-time PCR敏感性实验结果 A：扩增曲线;B：溶解曲线;1-6：不同稀释度重组质粒(3×106~3×101copies/uL);7：阴性对照

图6. 特异性试验结果 M：DL500;1-2：重组质粒pUC57-ASFV-p72;3：PRV;4：PRRSV;5：CSFV;6：阴性对照

检测结果	LNA引物 PCR	OIE推荐real-time PCR
阳性数	0	0
阴性数	72	72
总计	72	72

表2. LNA引物PCR和OIE推荐real-time PCR方法检测结果比对

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非洲猪瘟病毒LNA引物PCR检测方法的建立及优化

Establishment of PCR with LNA Modified Primers for Detection of African Swine Fever Virus

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