《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (5): 1283-1289.doi: 10.16431/j.cnki.1671-7236.2019.05.004

• 生物技术 • 上一篇    下一篇

7种猪场常见高致死病原GeXP检测方法的建立

王秋实, 李江凌, 赵素君, 刘锐   

  1. 四川省畜牧科学研究院, 动物遗传育种四川省重点实验室, 成都 610066
  • 收稿日期:2018-12-10 出版日期:2019-05-20 发布日期:2019-05-20
  • 作者简介:王秋实(1985-),男,四川成都人,学士,研究方向:动物疫病,E-mail:podleader@163.com
  • 基金资助:

    四川省畜牧科学研究院基本科研业务费专项资金(SASA2017A05)

Establishment of GeXP Detection Method for 7 Common Hyper Lethal Pathogens in Pig Farms

WANG Qiushi, LI Jiangling, ZHAO Sujun, LIU Rui   

  1. 1. Key Laboratory of Animal Genetic and Breeding of Sichuan Province, Sichuan Animal Science Academy, Chengdu 610066, China
  • Received:2018-12-10 Online:2019-05-20 Published:2019-05-20

摘要:

本研究旨在建立能同时检测猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、伪狂犬病病毒(PRV)、猪细小病毒(PPV)、猪圆环病毒2型(PCV2)、坏死梭杆菌(Fn)和副猪嗜血杆菌(Hps)7种猪场常见高致死性流行病原的多重PCR检测方法。利用7种病原体的保守序列设计7对特异性引物,同时合成了Cy-5标记的通用引物。将通用引物分别连接到特异性引物的5'端形成7对特异性嵌合引物。优化反应条件,分别使用7组嵌合引物和通用引物混合,扩增7种病原的混合cDNA/DNA,验证其单重PCR的特异性。利用GeXP多重基因表达遗传分析系统,混合7种嵌合引物和通用引物,扩增单一病原的cDNA/DNA,验证其多重PCR特异性;将其他常见猪病病原的基因组作为干扰的阳性标本,利用7对混合嵌合引物和通用引物进行多重PCR分析,扩增加入了阳性标本的混合模板,验证其多重PCR的抗干扰能力。利用重组质粒和体外转录的RNA进行梯度稀释,确定GeXP多重检测体系的灵敏度。结果表明,7种不同引物分别进行GeXP单重及多重检测,均能检测出特异性目的片段的信号,无明显的干扰片段信号出现;GeXP多重检测抗干扰试验结果显示,在混入3种干扰病原模板后,依然可同时特异性检测出7种病原;GeXP多重检测灵敏度分析显示,在103拷贝/μL浓度条件下能检测到7种不同基因的特异性结果。本研究建立的同时检测7种猪场常见高致死性流行病原的GeXP检测方法具有高通量、高特异性和高灵敏度的特点,为快速诊断猪流行性疾病的交叉感染和混合感染提供了新型的检测方法。

关键词: 高致死疾病; 流行病原; GeXP; 特异性嵌合引物; 多重PCR

Abstract:

A new multiplex PCR method was established to simultaneously detecting classical swine fever virus (CSFV),porcine reproductive and respiratory syndrome virus (PRRSV),pseudorabies virus (PRV),porcine parvovirus (PPV),porcine circovirus type 2 (PCV2), Fusobacterium necrophorum (Fn) and Haemophilus parasuis (Hps) seven kinds of common highly lethal epidemic pathogen in hoggery.Seven specific primers refer to each pathogen were designed,and one pair Cy-5 conjugated universal primers was designed.In this study,the universal primers were respectively fused in the 5'end of each specific primer to form seven pairs of specific chimeric primers.In optimized reaction condition,each chimeric primer was mixed with universal primer respectively and amplified cDNA/DNA of seven pathogens to verify the specificity of single PCR.This study further relied on GenomeLab Genetic Analysis System (GeXP),we mixed all the seven specific chimeric primers and universal primers,amplified cDNA/DNA of a single pathogen to verify multiplex PCR specificity.The genomes of other common pig disease pathogenes were taken as positive internal.Seven pairs of specific chimeric primers and universal primers were used to amplify cDNA/DNA of seven pathogens,then we mixed templates with positive samples to determine their anti-interference.The sensitivity of GeXP multiplex PCR assay was determined by gradient recombinant plasmid dilution.The results showed that GeXP multiplex and single detection both could detect the signal of specific target fragments,no other obvious interference signals were detected.GeXP multiplex anti-interference showed that mixed 3 pathogen genomes of other common pig diseases,the assay could also detect the seven pathogens simultaneously.GeXP multiplex sensitivity analysis showed that specific results of seven genes were detected,and the detection limit was 103 copies/μL.The GeXP multiplex assay was established in this study for simultaneous detection of 7 kinds of common and highly lethal epidemic pathogens in pig farms,and it had the characteristics of high throughput,specificity and sensitivity,providing a new detection method for rapid diagnosis of cross-infection and mixed infection of swine epidemic diseases.

Key words: highly lethal disease, epidemic pathogen, GeXP, specific chimeric primers, multiplex PCR

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