不同厂家猪瘟疫苗剂量快速检测方法建立及临床免疫效果比较
发布时间：2018-08-18 17:53
【摘要】：猪瘟是由猪瘟病毒引起的一种严重危害养猪业的烈性传染病,疫苗接种是预防猪瘟的有效措施。生产猪瘟疫苗的厂家众多,但不同厂家猪瘟疫苗的剂量存在明显差异,这种差异可能会影响临床免疫效果。本研究通过建立一种快速检测猪瘟疫苗剂量的荧光定量PCR方法来检测京津冀地区常用的6个厂家猪瘟疫苗的病毒载量,检测了较高与较低剂量的2个厂家的猪瘟疫苗接种后猪群血清中猪瘟病毒抗体的动态变化,比较分析了2种猪瘟疫苗的临床免疫效果。用RT-PCR方法扩增猪瘟疫苗病毒(HCLV)基因200 bp片段并克隆到T载体,构建含有该200 bp片段的重组质粒,对此重组质粒进行系列稀释后作为SYBR Green I荧光定量PCR模板,来绘制定量检测HCLV的标准曲线。结果显示,在(6.4×10~7~6.4×10~2)copies/μL模板浓度范围内,荧光定量PCR的扩增效率为92.2%,标准曲线的决定系数为0.9997。该方法的精确灵敏度为6.4×10~2 copies/μL,重复性试验的变异系数小于2%,对牛病毒性腹泻病毒、猪繁殖与呼吸综合征病毒等的检测结果均为阴性,表明建立了1种具有良好特异性、敏感性与可重复性的HCLV荧光定量PCR检测方法。用该方法检测了京津冀地区常用的6个厂家猪瘟疫苗中每头份剂量的病毒载量,结果显示不同厂家猪瘟疫苗的HCLV载量存在明显差异。选取较高剂量猪瘟疫苗E和较低剂量猪瘟疫苗A,在一个基础母猪为900头的猪场进行临床免疫试验。将60头断奶空怀母猪随机平均分为2组,1组接种疫苗E(E组),另1组接种疫苗A(A组),6个月后再分别接种1次;当母猪产仔后,所产仔猪在20 d与60 d时分别免疫1次;采集空怀期、妊娠55 d、哺乳14 d的母猪血清和14 d、35 d、56 d的仔猪血清及77 d、98 d、119d、140 d的生长猪血清,用阻断ELISA方法检测猪瘟病毒抗体。结果显示,在空怀母猪阶段两组无差异。在其他试验阶段,E组猪瘟病毒抗体阻断率一直高于A组,各个阶段均达到显著(P0.05)水平;除35 d与56 d仔猪外,E组猪瘟病毒抗体阻断率变异系数都小于A组;E组猪瘟病毒抗体阳性率都高于A组,其中在35 d仔猪阶段达到显著水平。抗体检测结果显示,E组猪瘟免疫抗体数据优于A组,表明猪瘟疫苗E的临床免疫效果优于疫苗A,提示较高剂量猪瘟疫苗的临床免疫效果好于较低剂量疫苗。综合分析表明,建立了1种快速检测猪瘟疫苗剂量的荧光定量PCR方法,不同厂家猪瘟疫苗的剂量存在明显差异,较高剂量猪瘟疫苗的临床免疫效果优于较低剂量疫苗,为剂量存在差异的不同厂家猪瘟疫苗的临床选用提供了实验依据。
[Abstract]:Swine fever is a severe infectious disease caused by swine fever virus. Vaccination is an effective measure to prevent swine fever. There are many manufacturers producing CSFV vaccine, but the dose of CSFV vaccine is different, which may affect the clinical immune effect. In this study, a fluorescent quantitative PCR method was established to detect the viral load of classical swine fever vaccine from six manufacturers in Beijing, Tianjin and Hebei. The dynamic changes of antibody to swine fever virus in serum of swine swarm were detected after inoculation of high and low doses of swine fever vaccine, and the clinical immune effects of two kinds of classical swine fever vaccine were compared and analyzed. The 200bp fragment of (HCLV) gene of CSV was amplified by RT-PCR method and cloned into T vector. The recombinant plasmid containing 200bp fragment was constructed. The recombinant plasmid was diluted by serial dilution and used as SYBR Green I fluorescent quantitative PCR template. To draw the standard curve for quantitative detection of HCLV. The results showed that in the range of (6.4 脳 10 ~ (7) 脳 10 ~ (2) copies/ 渭 L template concentration, the amplification efficiency of fluorescent quantitative PCR was 92.2, and the determination coefficient of the standard curve was 0.9997. The accurate sensitivity of this method is 6.4 脳 10 ~ (2) copies/ 渭 L, the coefficient of variation of repeatability test is less than 2, and the detection results of bovine viral diarrhea virus and porcine reproductive and respiratory syndrome virus are all negative. Sensitivity and reproducibility of HCLV fluorescence quantitative PCR assay. This method was used to detect the viral load of each dose of swine fever vaccine from six manufacturers in Beijing, Tianjin and Hebei. The results showed that the HCLV load of swine fever vaccine from different manufacturers had obvious difference. High dose swine fever vaccine E and low dose swine fever vaccine A were selected for clinical immunological test in a 900 pig farm with base sows. 60 weanling empty pregnant sows were randomly divided into two groups, one group was vaccinated with E (E group, the other group was vaccinated with A (A group (6 months later), and the piglets were immunized once at 20 days and 60 days after birth. Serum samples were collected from sows on day 55, day 14, day 14 at day 14, piglet serum from day 35 to day 56 and serum from growing pig from day 77 at day 98 to day 119 at day 140. The antibody against swine fever virus was detected by blocking ELISA method. The results showed that there was no difference between the two groups in the stage of empty pregnant sows. The blocking rate of CSFV antibody in group E was significantly higher than that in group A in other test stages (P0.05). Except for 35 d and 56 d of piglets, the coefficient of variation of antibody blocking of swine fever virus in group E was lower than that in group A, and the positive rate of antibody in group E was higher than that in group A, and reached a significant level at the stage of 35 d. The results of antibody detection showed that the data of CSFV antibody in group E was better than that in group A, which indicated that the clinical immune effect of CSFV vaccine E was better than that of vaccine A, indicating that the clinical immune effect of high dose CSFV vaccine was better than that of low dose vaccine. Comprehensive analysis showed that a fluorescent quantitative PCR method was established for rapid detection of CSFV doses. The dose of CSFV vaccine from different manufacturers was significantly different, and the clinical immunization effect of high dose CSFV vaccine was better than that of low dose CSFV vaccine. It provides experimental basis for clinical selection of swine fever vaccine from different manufacturers with different doses.
【学位授予单位】：北京农学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.28
本文编号：2190235
[Abstract]:Swine fever is a severe infectious disease caused by swine fever virus. Vaccination is an effective measure to prevent swine fever. There are many manufacturers producing CSFV vaccine, but the dose of CSFV vaccine is different, which may affect the clinical immune effect. In this study, a fluorescent quantitative PCR method was established to detect the viral load of classical swine fever vaccine from six manufacturers in Beijing, Tianjin and Hebei. The dynamic changes of antibody to swine fever virus in serum of swine swarm were detected after inoculation of high and low doses of swine fever vaccine, and the clinical immune effects of two kinds of classical swine fever vaccine were compared and analyzed. The 200bp fragment of (HCLV) gene of CSV was amplified by RT-PCR method and cloned into T vector. The recombinant plasmid containing 200bp fragment was constructed. The recombinant plasmid was diluted by serial dilution and used as SYBR Green I fluorescent quantitative PCR template. To draw the standard curve for quantitative detection of HCLV. The results showed that in the range of (6.4 脳 10 ~ (7) 脳 10 ~ (2) copies/ 渭 L template concentration, the amplification efficiency of fluorescent quantitative PCR was 92.2, and the determination coefficient of the standard curve was 0.9997. The accurate sensitivity of this method is 6.4 脳 10 ~ (2) copies/ 渭 L, the coefficient of variation of repeatability test is less than 2, and the detection results of bovine viral diarrhea virus and porcine reproductive and respiratory syndrome virus are all negative. Sensitivity and reproducibility of HCLV fluorescence quantitative PCR assay. This method was used to detect the viral load of each dose of swine fever vaccine from six manufacturers in Beijing, Tianjin and Hebei. The results showed that the HCLV load of swine fever vaccine from different manufacturers had obvious difference. High dose swine fever vaccine E and low dose swine fever vaccine A were selected for clinical immunological test in a 900 pig farm with base sows. 60 weanling empty pregnant sows were randomly divided into two groups, one group was vaccinated with E (E group, the other group was vaccinated with A (A group (6 months later), and the piglets were immunized once at 20 days and 60 days after birth. Serum samples were collected from sows on day 55, day 14, day 14 at day 14, piglet serum from day 35 to day 56 and serum from growing pig from day 77 at day 98 to day 119 at day 140. The antibody against swine fever virus was detected by blocking ELISA method. The results showed that there was no difference between the two groups in the stage of empty pregnant sows. The blocking rate of CSFV antibody in group E was significantly higher than that in group A in other test stages (P0.05). Except for 35 d and 56 d of piglets, the coefficient of variation of antibody blocking of swine fever virus in group E was lower than that in group A, and the positive rate of antibody in group E was higher than that in group A, and reached a significant level at the stage of 35 d. The results of antibody detection showed that the data of CSFV antibody in group E was better than that in group A, which indicated that the clinical immune effect of CSFV vaccine E was better than that of vaccine A, indicating that the clinical immune effect of high dose CSFV vaccine was better than that of low dose vaccine. Comprehensive analysis showed that a fluorescent quantitative PCR method was established for rapid detection of CSFV doses. The dose of CSFV vaccine from different manufacturers was significantly different, and the clinical immunization effect of high dose CSFV vaccine was better than that of low dose CSFV vaccine. It provides experimental basis for clinical selection of swine fever vaccine from different manufacturers with different doses.
【学位授予单位】：北京农学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.28
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