7种猪场常见高致死病原GeXP检测方法的建立
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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10~3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。
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 10~3 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.
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 10~3 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.
引文
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[6] 于吉锋,王文贵,魏勇,等.副猪嗜血杆菌四川株生物学特性及药物敏感性研究[J].中国畜牧兽医,2015,42(6):1566-1570.YU J F,WANG W G,WEI Y,et al.Study on biological characteristics and drug sensitivity of Haemophilus parasuis isolated in Sichuan province[J].China Animal Husbandry & Veterinary Medicine,2015,42(6):1566-1570.(in Chinese)
[7] SHIBATA I,YAZAWA S,ONO M,et al.Experimental dual infection of specific pathogen free pigs with porcine reproductive and respiratory syndrome virus and pseudorabies virus[J].Journal of Veterinary Medicine B Infectious Diseases & Veterinary Public Health,2003,50(1):14-19.
[8] CHEN N,CAO Z,YU X,et al.Emergence of novel European genotype porcine reproductive and respiratory syndrome virus in mainland China[J].Journal of General Virology,2011,92(8):880-892.
[9] 刘晓丹,孟祥莉,徐凝,等.多重PCR方法快速检测3种消化道病原菌[J].中国预防兽医学报,2013,35(4):308-311.LIU X D,MENG X L,XU N,et al.Development of a multiplex PCR for simultaneous detection of three conditioned pathogen bacteria of alimentary tract diseases[J].Chinese Journal of Preventive Veterinary Medicine,2013,35(4):308-311.(in Chinese)
[10] 范晴,谢芝勋,谢志勤,等.同时监测8种牛常见病原体的GeXP高通量快速检测技术[J].畜牧兽医学报,2017,48(10):1920-1931.FAN Q,XIE Z X,XIE Z Q,et al.Development of a GeXP assay for simultaneous differentiation of 8 pathogens of bovine infectious diseases[J].Acta Veterinaria et Zootechnica Sinica,2017,48(10):1920-1931.(in Chinese)
[11] RAI A J,KAMATH R M,GERALD W,et al.Analytical validation of the GeXP analyzer and design of a workflow for cancer-biomarker discovery using multiplexed gene-expression profiling[J].Analytical and Bioanalytical Chemistry,2009,393(5):1505-1511.
[12] HU X,ZHANG Y,ZHOU X,et al.Simultaneously typing nine serotypes of enteroviruses associated with hand food,and mouth disease by a GeXP analyzer based multiplex reverse transcription-PCR assy[J].Journal of Clinical Microbiology,2012,50(2):288-293.
[13] DREW J E,MAYER C D,FARQUHARSON A J,et al.Custom design of a GeXP multiplexed assay used to assess expression profiles of inflammatory gene targets in normal colon,polyp and tumor tissue[J].Journal of Molecular Diagnostics,2011,13(2):233-242.
[14] 张民秀,谢芝勋,邓显文,等.8种猪呼吸道和繁殖障碍病病原体GeXP检测方法的建立[J].中国农业科学,2015,48(24):4996-5006.ZHANG M X,XIE Z X,DENG X W,et al.Establishment of a GeXP analyser-based multiplex PCR assay for detection of eight reproductive and respiratory swine pathogens[J].Scientia Agricultura Sinica,2015,48(24):4996-5006.(in Chinese)
[15] 仲继武.猪常见疾病混合感染的防治策略探讨[J].中国畜牧兽医文摘,2017,33(4):123.ZHONG J W.Discussion on the prevention and treatment strategy of mixed infection of common swine diseases[J].Chinese Abstracts of Animal Husbandry and Veterinary Medicine,2017,33(4):123.(in Chinese)
[16] 周珍辉,向双云,杨久仙,等.规模猪场猪瘟、猪蓝耳病、猪圆环病毒病的抗体检测和防治[J].黑龙江畜牧兽医,2013,10:92-93.ZHOU Z H,XIANG S Y,YANG J X,et al.Antibody detection and prevention of swine fever,porcine reproductive and respiratory syndrome and porcine circovirus disease in large-scale hoggery[J].Heilongjiang Animal Science and Veterinary Medicine,2013,10:92-93.(in Chinese)
[17] 夏叶,郭佳宏,廖学,等.猪圆环病毒2d亚型病毒样颗粒的制备[J].上海农业学报,2017,33(6):43-48.XIA Y,GUO J H,LIAO X,et al.Preparation of porcine circovirus subtype 2d virus-like particles[J].Acta Agriculturae Shanghai,2017,33(6):43-48.(in Chinese)
[18] 夏道伦.猪常见混合感染疾病的判断要点及防治方案[J].广东饲料,2011,20(5):47-48.XIA D L.The prevention and control of common mixed infection disease in swine[J].Guangdong Feed,2011,20(5):47-48.(in Chinese)
[19] 张建楼,徐瑞涛,霍珊珊,等.猪细小病毒NS1基因的克隆、表达及密码子优化提高表达水平[J].中国兽医学报,2018,38(10):1840-1845.ZHANG J L,XU R T,HUO S S,et al.Cloning and expression of porcine parvivirus NS1 gene and its codon optimization for enhancing the expression[J].Chinese Journal of Veterinary Science,2018,38(10):1840-1845.(in Chinese)
[20] 罗思思,谢芝勋,谢志勤,等.H6N1亚型禽流感病毒GeXP检测方法的建立[J].畜牧与兽医,2018,50(11):101-105.LUO S S,XIE Z X,XIE Z Q,et al.Establishment of a GeXP assay for detection of the H6N1 subtype avian influenza virus[J] Animal Husbandry and Veterinary Medicine,2018,50(11):101-105.(in Chinese)
[21] MENG Q,WANG D Y,HUANG F,et al.Detection of pandemic influenza A H1N1 virus by multiplex reverse transcription-PCR with a GeXP analyzer[J].Journal of Virological Methods,2010,168(1/2):255-258.
[22] 张艳芳,谢芝勋,谢丽基,等.9种鸭病毒病GeXP多重PCR检测方法的建立及其应用[J].畜牧兽医学报,2016,47(12):2457-2468.ZHANG Y F,XIE Z X,XIE L J,et al.Development and application of a GeXP-multiplex PCR assay for detection of nine duck viruses[J].Acta Veterinaria et Zootechnica Sinica,2016,47(12):2457-2468.(in Chinese)
[2] 姜永厚,徐辉,商晗武.多重PCR同时检测猪圆环病毒2型、猪细小病毒、猪繁殖与呼吸综合征病毒和猪瘟病毒[J].中国兽医学报,2009,29(10):1237-1241.JIANG Y H,XU H,SHANG H W.A multiplex PCR for simultaneous detection of porcine circovirus type 2,porcine parvovirus,porcine reproductive and respiratory syndrome virus,and classical swine fever virus in pig[J].Chinese Journal of Veterinary Science,2009,29(10):1237-1241.(in Chinese)
[3] 李娇,祖立闯,王金良,等.我国4省猪瘟、猪蓝耳病、猪伪狂犬病、猪圆环病毒病的血清流行病学调查[J].养猪,2014,4:123-124.LI J,ZU L C,WANG J L,et al.Sero epidemiological investigation of classical swine fever,swine blue ear disease,pseudorabies and porcine circovirus disease in 4 provinces of China[J].Swine Production,2014,4:123-124.(in Chinese)
[4] 梁晟,郑敏,郑列丰,等.猪群常见疫病混合感染情况调查[J].中国畜牧兽医,2013,40(6):221-223.LIANG S,ZHENG M,ZHENG L F,et al.Investigation of the mixture infection of common swine diseases[J].China Animal Husbandry & Veterinary Medicine,2013,40(6):221-223.(in Chinese)
[5] 韦显凯,蒋晓霞,苏姣秀,等.2014年广西部分地区常见猪群疫病感染情况监测分析[J].中国畜牧兽医,2016,43(4):1079-1085.WEI X K,JIANG X X,SU J X,et al.Monitoring analysis of common swine disease in partial areas of Guangxi in 2014[J].China Animal Husbandry & Veterinary Medicine,2016,43(4):1079-1085.(in Chinese)
[6] 于吉锋,王文贵,魏勇,等.副猪嗜血杆菌四川株生物学特性及药物敏感性研究[J].中国畜牧兽医,2015,42(6):1566-1570.YU J F,WANG W G,WEI Y,et al.Study on biological characteristics and drug sensitivity of Haemophilus parasuis isolated in Sichuan province[J].China Animal Husbandry & Veterinary Medicine,2015,42(6):1566-1570.(in Chinese)
[7] SHIBATA I,YAZAWA S,ONO M,et al.Experimental dual infection of specific pathogen free pigs with porcine reproductive and respiratory syndrome virus and pseudorabies virus[J].Journal of Veterinary Medicine B Infectious Diseases & Veterinary Public Health,2003,50(1):14-19.
[8] CHEN N,CAO Z,YU X,et al.Emergence of novel European genotype porcine reproductive and respiratory syndrome virus in mainland China[J].Journal of General Virology,2011,92(8):880-892.
[9] 刘晓丹,孟祥莉,徐凝,等.多重PCR方法快速检测3种消化道病原菌[J].中国预防兽医学报,2013,35(4):308-311.LIU X D,MENG X L,XU N,et al.Development of a multiplex PCR for simultaneous detection of three conditioned pathogen bacteria of alimentary tract diseases[J].Chinese Journal of Preventive Veterinary Medicine,2013,35(4):308-311.(in Chinese)
[10] 范晴,谢芝勋,谢志勤,等.同时监测8种牛常见病原体的GeXP高通量快速检测技术[J].畜牧兽医学报,2017,48(10):1920-1931.FAN Q,XIE Z X,XIE Z Q,et al.Development of a GeXP assay for simultaneous differentiation of 8 pathogens of bovine infectious diseases[J].Acta Veterinaria et Zootechnica Sinica,2017,48(10):1920-1931.(in Chinese)
[11] RAI A J,KAMATH R M,GERALD W,et al.Analytical validation of the GeXP analyzer and design of a workflow for cancer-biomarker discovery using multiplexed gene-expression profiling[J].Analytical and Bioanalytical Chemistry,2009,393(5):1505-1511.
[12] HU X,ZHANG Y,ZHOU X,et al.Simultaneously typing nine serotypes of enteroviruses associated with hand food,and mouth disease by a GeXP analyzer based multiplex reverse transcription-PCR assy[J].Journal of Clinical Microbiology,2012,50(2):288-293.
[13] DREW J E,MAYER C D,FARQUHARSON A J,et al.Custom design of a GeXP multiplexed assay used to assess expression profiles of inflammatory gene targets in normal colon,polyp and tumor tissue[J].Journal of Molecular Diagnostics,2011,13(2):233-242.
[14] 张民秀,谢芝勋,邓显文,等.8种猪呼吸道和繁殖障碍病病原体GeXP检测方法的建立[J].中国农业科学,2015,48(24):4996-5006.ZHANG M X,XIE Z X,DENG X W,et al.Establishment of a GeXP analyser-based multiplex PCR assay for detection of eight reproductive and respiratory swine pathogens[J].Scientia Agricultura Sinica,2015,48(24):4996-5006.(in Chinese)
[15] 仲继武.猪常见疾病混合感染的防治策略探讨[J].中国畜牧兽医文摘,2017,33(4):123.ZHONG J W.Discussion on the prevention and treatment strategy of mixed infection of common swine diseases[J].Chinese Abstracts of Animal Husbandry and Veterinary Medicine,2017,33(4):123.(in Chinese)
[16] 周珍辉,向双云,杨久仙,等.规模猪场猪瘟、猪蓝耳病、猪圆环病毒病的抗体检测和防治[J].黑龙江畜牧兽医,2013,10:92-93.ZHOU Z H,XIANG S Y,YANG J X,et al.Antibody detection and prevention of swine fever,porcine reproductive and respiratory syndrome and porcine circovirus disease in large-scale hoggery[J].Heilongjiang Animal Science and Veterinary Medicine,2013,10:92-93.(in Chinese)
[17] 夏叶,郭佳宏,廖学,等.猪圆环病毒2d亚型病毒样颗粒的制备[J].上海农业学报,2017,33(6):43-48.XIA Y,GUO J H,LIAO X,et al.Preparation of porcine circovirus subtype 2d virus-like particles[J].Acta Agriculturae Shanghai,2017,33(6):43-48.(in Chinese)
[18] 夏道伦.猪常见混合感染疾病的判断要点及防治方案[J].广东饲料,2011,20(5):47-48.XIA D L.The prevention and control of common mixed infection disease in swine[J].Guangdong Feed,2011,20(5):47-48.(in Chinese)
[19] 张建楼,徐瑞涛,霍珊珊,等.猪细小病毒NS1基因的克隆、表达及密码子优化提高表达水平[J].中国兽医学报,2018,38(10):1840-1845.ZHANG J L,XU R T,HUO S S,et al.Cloning and expression of porcine parvivirus NS1 gene and its codon optimization for enhancing the expression[J].Chinese Journal of Veterinary Science,2018,38(10):1840-1845.(in Chinese)
[20] 罗思思,谢芝勋,谢志勤,等.H6N1亚型禽流感病毒GeXP检测方法的建立[J].畜牧与兽医,2018,50(11):101-105.LUO S S,XIE Z X,XIE Z Q,et al.Establishment of a GeXP assay for detection of the H6N1 subtype avian influenza virus[J] Animal Husbandry and Veterinary Medicine,2018,50(11):101-105.(in Chinese)
[21] MENG Q,WANG D Y,HUANG F,et al.Detection of pandemic influenza A H1N1 virus by multiplex reverse transcription-PCR with a GeXP analyzer[J].Journal of Virological Methods,2010,168(1/2):255-258.
[22] 张艳芳,谢芝勋,谢丽基,等.9种鸭病毒病GeXP多重PCR检测方法的建立及其应用[J].畜牧兽医学报,2016,47(12):2457-2468.ZHANG Y F,XIE Z X,XIE L J,et al.Development and application of a GeXP-multiplex PCR assay for detection of nine duck viruses[J].Acta Veterinaria et Zootechnica Sinica,2016,47(12):2457-2468.(in Chinese)