基于实时荧光定量PCR技术的猪伪狂犬病活疫苗含毒量评价
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摘要
为定量分析贵州省临床用猪伪狂犬病(pseudorabies,PR)活疫苗中病毒含量,根据GenBank中公布的伪狂犬病病毒(pseudorabies virus,PRV)gB基因序列(登录号:M17321.1)设计1对特异性引物,建立实时荧光定量PCR方法,并对来自6个不同厂家的猪伪狂犬病活疫苗进行含毒量分析。结果显示,试验成功建立了可用于PRV检测的实时荧光定量PCR方法,标准曲线中标准品各稀释度质粒拷贝数与Ct值呈良好的线性关系,标准曲线方程为:Y=-0.97X+33.66(R~2=0.999),该方法最低检测病毒含量为3.19×10~1拷贝/μL,敏感性高且具有良好的重复性和特异性。应用所建立的实时荧光定量PCR方法对贵州省临床用6个厂家生产的猪伪狂犬病活疫苗的病毒含量进行检测,结果显示,6种市售猪伪狂犬病活疫苗(A～F)中病毒含量分别为1.67×10~7、4.83×10~5、2.64×10~6、4.27×10~7、3.39×10~6和3.68×10~5拷贝/μL,来自不同厂家的疫苗病毒含毒量存在明显差异,最大差异可达116倍,其中来自A、D厂家的两种猪伪狂犬病活疫苗具有较高的含毒量。本试验所建立的实时荧光定量PCR方法可用于猪伪狂犬病活疫苗病毒含量的快速检测和评价,对猪伪狂犬病活疫苗的质控和临床用疫苗选择具有一定的指导意义。
In order to quantitatively analyze the virus content of clinical live pseudorabies(PR) vaccine in Guizhou province,a pair of specific primers was designed according to the sequence of pseudorabies virus(PRV) gB gene in GenBank(accession No.:M17321.1),a Real-time quantitative PCR method was established,and the virus content of live PR vaccine from 6 different manufacturers was analyzed.The results showed that a Real-time quantitative PCR method was successfully established for detection of PRV.There was a good linear relationship between the number of plasmid copies diluted in the standard curve and Ct value,the standard curve equation was Y=-0.97 X+33.66(R~2=0.999).The method had a minimum detection virus content of 3.19×10~1 copies/μL,high sensitivity,good repeatability and specificity.The Real-time quantitative PCR method was applied to detect the viral content of live PR vaccine produced by 6 manufacturers in Guizhou province.The results showed that the virus content of 6 live PR vaccine(A-F) sold in 6 markets were 1.67×10~7,4.83×10~5,2.64×10~6,4.27×10~7,3.39×10~6 and 3.68×10~5 copies/μL,the viral content of the vaccine from different manufacturers was significantly different,the biggest difference could be 116 times.Two kinds of live PR vaccine from A and D manufacturers had higher viral content.The Real-time quantitative PCR method established in this experiment could be used for rapid detection and evaluation of the viral content of live vaccine against porcine PR,it had a certain guiding significance for the quality control of the live vaccine of PR and the selection of clinical vaccine.
In order to quantitatively analyze the virus content of clinical live pseudorabies(PR) vaccine in Guizhou province,a pair of specific primers was designed according to the sequence of pseudorabies virus(PRV) gB gene in GenBank(accession No.:M17321.1),a Real-time quantitative PCR method was established,and the virus content of live PR vaccine from 6 different manufacturers was analyzed.The results showed that a Real-time quantitative PCR method was successfully established for detection of PRV.There was a good linear relationship between the number of plasmid copies diluted in the standard curve and Ct value,the standard curve equation was Y=-0.97 X+33.66(R~2=0.999).The method had a minimum detection virus content of 3.19×10~1 copies/μL,high sensitivity,good repeatability and specificity.The Real-time quantitative PCR method was applied to detect the viral content of live PR vaccine produced by 6 manufacturers in Guizhou province.The results showed that the virus content of 6 live PR vaccine(A-F) sold in 6 markets were 1.67×10~7,4.83×10~5,2.64×10~6,4.27×10~7,3.39×10~6 and 3.68×10~5 copies/μL,the viral content of the vaccine from different manufacturers was significantly different,the biggest difference could be 116 times.Two kinds of live PR vaccine from A and D manufacturers had higher viral content.The Real-time quantitative PCR method established in this experiment could be used for rapid detection and evaluation of the viral content of live vaccine against porcine PR,it had a certain guiding significance for the quality control of the live vaccine of PR and the selection of clinical vaccine.
引文
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[7] 王继春,曾容愚,Torrents D,等.猪伪狂犬病活疫苗(Bartha K61株)对变异株的保护效力[J].畜牧与兽医,2015,47(12):1-4. WANG J C,ZENG R Y,TORRENTS D,et al.Protection of pseudorabies vaccine (Bartha K61 strain) against pseudorabies virus variant in pigs[J].Animal Husbandry & Veterinary Medicine,2015,47(12):1-4.(in Chinese)
[8] LIU Y,ZHANG S,XU Q,et al.Investigation on pseudorabies prevalence in Chinese swine breeding farms in 2013-2016[J].Tropical Animal Health & Production,2018,50(6):1279-1285.
[9] 曹玉美,孙龙,兰云,等.Cpg DNA佐剂对猪伪狂犬病疫苗免疫效果的影响[J].广西畜牧兽医,2017,33(4):171-172. CAO Y M,SUN L,LAN Y,et al.Effect of Cpg DNA adjuvant on the immune efficacy of porcine pseudorabies vaccine[J].Guangxi Animal Husbandry and Veterinary,2017,33(4):171-172.(in Chinese)
[10] 李萌,李雪,郑志强.实时荧光定量PCR技术的应用及研究进展[J].饲料博览,2018,7:94. LI M,LI X,ZHENG Z Q.Application and research progress of Real-time fluorescence quantitative PCR technology[J].Feed Review,2018,7:94.(in Chinese)
[11] 张志,郝占武,张美晶,等.猪圆环病毒3型实时荧光定量PCR方法的建立和应用[J].中国兽医科学,2018,48(6):686-691. ZHANG Z,HAO Z W,ZHANG M J,et al.Development and application of a Real-time PCR for the detection of porcine circovirus 3[J].Chinese Veterinary Science,2018,48(6):686-691.(in Chinese)
[12] 王香君,李梦茹,段杉.实时荧光定量PCR法定量微生物的条件及在鱼露和虾油微生物检测中的应用研究[J].食品与发酵工业,2018,44(2):194-201. WANG X J,LI M R,DUAN S.Determination of the conditions for the counting of microbes by Real-time fluorescence quantitative PCR and research on the detection of microbial populations in fish sauce and shrimp sauce[J].Food and Fermentation Industry,2018,44(2):194-201.(in Chinese)
[13] 李秋阳,姚瑶.实时荧光定量PCR技术在食品检验中的应用[J].食品安全导刊,2018,15:130. LI Q Y,YAO Y.Application of Real-time fluorescence quantitative PCR in food inspection[J].Food Safety Guide,2018,15:130.(in Chinese)
[14] 马颖颖.实时荧光定量PCR技术在粮油转基因成分检测中的应用[J].信息记录材料,2018,19(3):62-63. MA Y Y.Application of Real-time fluorescence quantitative PCR in the detection of transgenic components in grain and oil[J].Information Recording Materials,2018,19(3):62-63.(in Chinese)
[15] WANG Y,QIAO S,LI X,et al.Molecular epidemiology of outbreak-associated pseudorabies virus (PRV) strains in central China[J].Virus Genes,2015,50(3):401-409.
[16] SUN Y,LUO Y,WANG C H,et al.Control of swine pseudorabies in China:Opportunities and limitations[J].Veterinary Microbiology,2016,183:119-124.
[17] 牛晓芸,赖月辉,黄秋雪,等.猪伪狂犬病疫苗的研究进展[J].中国猪业,2017,12(8):71-75. NIU X Y,LAI Y H,HUANG Q X,et al.Research progress on porcine pseudorabies vaccine[J].China Pig Industry,2017,12(8):71-75.(in Chinese)
[18] 宁慧波,陈同煜,李聪.猪伪狂犬病的诊治实例及免疫体会[J].养猪,2018,1:93-96. NING H B,CHEN T Y,LI C.Diagnosis and treatment of porcine pseudorabies and immunization experience[J].Pig Raising,2018,1:93-96.(in Chinese)
[19] 刘扶摇,王梦君,李雨轩,等.Montanide(TM)Gel 01 ST水性佐剂对猪乙型脑炎活疫苗免疫小鼠后的早期免疫保护效果的影响[J].中国预防兽医学报,2018,40(1):40-44. LIU F Y,WANG M J,LI Y X,et al.The effect of aqueous adjuvant Montanide(TM) Gel 01 ST on early immune protection of swine Japanese encephalitis live vaccine in mice[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(1):40-44.(in Chinese)
[20] 任卫科,池晶晶,李秀丽,等.猪伪狂犬病疫苗研究及应用现状[J].天津农业科学,2017,23(8):37-41. REN W K,CHI J J,LI X L,et al.Research and application of porcine pseudorabies vaccine[J].Tianjin Agricultural Science,2017,23(8):37-41.(in Chinese)
[21] 周志军,孙文,施金荣,等.检测狂犬病疫苗抗原含量的竞争ELISA的建立及初步应用[J].微生物学免疫学进展,2007,4:28-31. ZHOU Z J,SUN W,SHI J R,et al.Establishment and application of competitive-ELISA to screening virus antigen content in rabies vaccine production[J].Progress in Microbiology and Immunology,2007,4:28-31.(in Chinese)
[22] 雷宁,张远,肖海波,等.邻苯二甲醛(OPA)法检测铝吸附疫苗中抗原含量[J].中国生物工程杂志,2011,31(7):109-113. LEI N,ZHANG Y,XIAO H B,et al.Determination of protein content of alhydrogel-absorbed vaccine using O-phthalaldehyde (OPA) fluorometric method[J].Chinese Biotechnology,2011,31(7):109-113.(in Chinese)
[23] 马志亮,孙亭亭,杨洁,等.猪繁殖与呼吸综合征活疫苗中病毒含量荧光定量PCR测定方法的建立及初步应用[J].中国畜牧兽医,2014,41(5):71-76. MA Z L,SUN T T,YANG J,et al.Development and preliminary application of Real-time PCR assay for detecting quantitation of PRRSV in vaccine[J].China Animal Husbandry & Veterinary Medicine,2014,41(5):71-76.(in Chinese)
[24] RISATTI G,HOLINKA L,LU Z,et al.Diagnostic evaluation of a Real-time reverse transcriptase PCR assay for detection of classical swine fever virus[J].Journal of Clinical Microbiology,2005,43(1):468-471.
[2] 洪天旗,陈培华,陈彬,等.猪伪狂犬病的诊断、防控及净化[J].畜牧与兽医,2018,50(3):131-136. HONG T Q,CHEN P H,CHEN B,et al.Diagnosis,control and eradication of pseudorabies in pigs[J].Animal Husbandry & Veterinary Medicine,2018,50(3):131-136.(in Chinese)
[3] 林鸷,何锡忠,潘洁,等.猪伪狂犬病研究进展[J].国外畜牧学(猪与禽),2018,38(8):18-20. LIN Z,HE X Z,PAN J,et al.Progress on porcine pseudorabies[J].Overseas Animal Science (Pig and Poultry),2018,38(8):18-20.(in Chinese)
[4] 刘曜综,芮萍,马芮,等.伪狂犬病毒固有免疫逃避机制[J].病毒学报,2015,31(6):698-703. LIU Y Z,RUI P,MA R,et al.Innate immune evasion mechanisms of pseudorabies virus[J].Journal of Virus,2015,31(6):698-703.(in Chinese)
[5] 雷有玲,李国泰,鲍玉林,等.猪伪狂犬病毒gE蛋白单克隆抗体制备与鉴定[J].畜牧与兽医,2018,50(1):109-112. LEI Y L,LI G T,BAO Y L,et al.Preparation and identification of monoclonal antibodies against porcine pseudorabies virus gE protein[J].Animal Husbandry & Veterinary Medicine,2018,50 (1):109-112.(in Chinese)
[6] 陈娟,喻正军.不同伪狂犬病活疫苗免疫效力比较[J].中国兽药杂志,2017,51(1):15-18. CHEN J,YU Z J.Comparison of immune efficacy of pseudorabies vaccines from different bio-factories[J].Chinese Journal of Veterinary Medicine,2017,51(1):15-18.(in Chinese)
[7] 王继春,曾容愚,Torrents D,等.猪伪狂犬病活疫苗(Bartha K61株)对变异株的保护效力[J].畜牧与兽医,2015,47(12):1-4. WANG J C,ZENG R Y,TORRENTS D,et al.Protection of pseudorabies vaccine (Bartha K61 strain) against pseudorabies virus variant in pigs[J].Animal Husbandry & Veterinary Medicine,2015,47(12):1-4.(in Chinese)
[8] LIU Y,ZHANG S,XU Q,et al.Investigation on pseudorabies prevalence in Chinese swine breeding farms in 2013-2016[J].Tropical Animal Health & Production,2018,50(6):1279-1285.
[9] 曹玉美,孙龙,兰云,等.Cpg DNA佐剂对猪伪狂犬病疫苗免疫效果的影响[J].广西畜牧兽医,2017,33(4):171-172. CAO Y M,SUN L,LAN Y,et al.Effect of Cpg DNA adjuvant on the immune efficacy of porcine pseudorabies vaccine[J].Guangxi Animal Husbandry and Veterinary,2017,33(4):171-172.(in Chinese)
[10] 李萌,李雪,郑志强.实时荧光定量PCR技术的应用及研究进展[J].饲料博览,2018,7:94. LI M,LI X,ZHENG Z Q.Application and research progress of Real-time fluorescence quantitative PCR technology[J].Feed Review,2018,7:94.(in Chinese)
[11] 张志,郝占武,张美晶,等.猪圆环病毒3型实时荧光定量PCR方法的建立和应用[J].中国兽医科学,2018,48(6):686-691. ZHANG Z,HAO Z W,ZHANG M J,et al.Development and application of a Real-time PCR for the detection of porcine circovirus 3[J].Chinese Veterinary Science,2018,48(6):686-691.(in Chinese)
[12] 王香君,李梦茹,段杉.实时荧光定量PCR法定量微生物的条件及在鱼露和虾油微生物检测中的应用研究[J].食品与发酵工业,2018,44(2):194-201. WANG X J,LI M R,DUAN S.Determination of the conditions for the counting of microbes by Real-time fluorescence quantitative PCR and research on the detection of microbial populations in fish sauce and shrimp sauce[J].Food and Fermentation Industry,2018,44(2):194-201.(in Chinese)
[13] 李秋阳,姚瑶.实时荧光定量PCR技术在食品检验中的应用[J].食品安全导刊,2018,15:130. LI Q Y,YAO Y.Application of Real-time fluorescence quantitative PCR in food inspection[J].Food Safety Guide,2018,15:130.(in Chinese)
[14] 马颖颖.实时荧光定量PCR技术在粮油转基因成分检测中的应用[J].信息记录材料,2018,19(3):62-63. MA Y Y.Application of Real-time fluorescence quantitative PCR in the detection of transgenic components in grain and oil[J].Information Recording Materials,2018,19(3):62-63.(in Chinese)
[15] WANG Y,QIAO S,LI X,et al.Molecular epidemiology of outbreak-associated pseudorabies virus (PRV) strains in central China[J].Virus Genes,2015,50(3):401-409.
[16] SUN Y,LUO Y,WANG C H,et al.Control of swine pseudorabies in China:Opportunities and limitations[J].Veterinary Microbiology,2016,183:119-124.
[17] 牛晓芸,赖月辉,黄秋雪,等.猪伪狂犬病疫苗的研究进展[J].中国猪业,2017,12(8):71-75. NIU X Y,LAI Y H,HUANG Q X,et al.Research progress on porcine pseudorabies vaccine[J].China Pig Industry,2017,12(8):71-75.(in Chinese)
[18] 宁慧波,陈同煜,李聪.猪伪狂犬病的诊治实例及免疫体会[J].养猪,2018,1:93-96. NING H B,CHEN T Y,LI C.Diagnosis and treatment of porcine pseudorabies and immunization experience[J].Pig Raising,2018,1:93-96.(in Chinese)
[19] 刘扶摇,王梦君,李雨轩,等.Montanide(TM)Gel 01 ST水性佐剂对猪乙型脑炎活疫苗免疫小鼠后的早期免疫保护效果的影响[J].中国预防兽医学报,2018,40(1):40-44. LIU F Y,WANG M J,LI Y X,et al.The effect of aqueous adjuvant Montanide(TM) Gel 01 ST on early immune protection of swine Japanese encephalitis live vaccine in mice[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(1):40-44.(in Chinese)
[20] 任卫科,池晶晶,李秀丽,等.猪伪狂犬病疫苗研究及应用现状[J].天津农业科学,2017,23(8):37-41. REN W K,CHI J J,LI X L,et al.Research and application of porcine pseudorabies vaccine[J].Tianjin Agricultural Science,2017,23(8):37-41.(in Chinese)
[21] 周志军,孙文,施金荣,等.检测狂犬病疫苗抗原含量的竞争ELISA的建立及初步应用[J].微生物学免疫学进展,2007,4:28-31. ZHOU Z J,SUN W,SHI J R,et al.Establishment and application of competitive-ELISA to screening virus antigen content in rabies vaccine production[J].Progress in Microbiology and Immunology,2007,4:28-31.(in Chinese)
[22] 雷宁,张远,肖海波,等.邻苯二甲醛(OPA)法检测铝吸附疫苗中抗原含量[J].中国生物工程杂志,2011,31(7):109-113. LEI N,ZHANG Y,XIAO H B,et al.Determination of protein content of alhydrogel-absorbed vaccine using O-phthalaldehyde (OPA) fluorometric method[J].Chinese Biotechnology,2011,31(7):109-113.(in Chinese)
[23] 马志亮,孙亭亭,杨洁,等.猪繁殖与呼吸综合征活疫苗中病毒含量荧光定量PCR测定方法的建立及初步应用[J].中国畜牧兽医,2014,41(5):71-76. MA Z L,SUN T T,YANG J,et al.Development and preliminary application of Real-time PCR assay for detecting quantitation of PRRSV in vaccine[J].China Animal Husbandry & Veterinary Medicine,2014,41(5):71-76.(in Chinese)
[24] RISATTI G,HOLINKA L,LU Z,et al.Diagnostic evaluation of a Real-time reverse transcriptase PCR assay for detection of classical swine fever virus[J].Journal of Clinical Microbiology,2005,43(1):468-471.