不同廠家豬瘟疫苗劑量快速檢測方法建立及臨床免疫效果比較
發(fā)布時間：2018-08-18 17:53
【摘要】：豬瘟是由豬瘟病毒引起的一種嚴(yán)重危害養(yǎng)豬業(yè)的烈性傳染病,疫苗接種是預(yù)防豬瘟的有效措施。生產(chǎn)豬瘟疫苗的廠家眾多,但不同廠家豬瘟疫苗的劑量存在明顯差異,這種差異可能會影響臨床免疫效果。本研究通過建立一種快速檢測豬瘟疫苗劑量的熒光定量PCR方法來檢測京津冀地區(qū)常用的6個廠家豬瘟疫苗的病毒載量,檢測了較高與較低劑量的2個廠家的豬瘟疫苗接種后豬群血清中豬瘟病毒抗體的動態(tài)變化,比較分析了2種豬瘟疫苗的臨床免疫效果。用RT-PCR方法擴增豬瘟疫苗病毒(HCLV)基因200 bp片段并克隆到T載體,構(gòu)建含有該200 bp片段的重組質(zhì)粒,對此重組質(zhì)粒進行系列稀釋后作為SYBR Green I熒光定量PCR模板,來繪制定量檢測HCLV的標(biāo)準(zhǔn)曲線。結(jié)果顯示,在(6.4×10~7~6.4×10~2)copies/μL模板濃度范圍內(nèi),熒光定量PCR的擴增效率為92.2%,標(biāo)準(zhǔn)曲線的決定系數(shù)為0.9997。該方法的精確靈敏度為6.4×10~2 copies/μL,重復(fù)性試驗的變異系數(shù)小于2%,對牛病毒性腹瀉病毒、豬繁殖與呼吸綜合征病毒等的檢測結(jié)果均為陰性,表明建立了1種具有良好特異性、敏感性與可重復(fù)性的HCLV熒光定量PCR檢測方法。用該方法檢測了京津冀地區(qū)常用的6個廠家豬瘟疫苗中每頭份劑量的病毒載量,結(jié)果顯示不同廠家豬瘟疫苗的HCLV載量存在明顯差異。選取較高劑量豬瘟疫苗E和較低劑量豬瘟疫苗A,在一個基礎(chǔ)母豬為900頭的豬場進行臨床免疫試驗。將60頭斷奶空懷母豬隨機平均分為2組,1組接種疫苗E(E組),另1組接種疫苗A(A組),6個月后再分別接種1次;當(dāng)母豬產(chǎn)仔后,所產(chǎn)仔豬在20 d與60 d時分別免疫1次;采集空懷期、妊娠55 d、哺乳14 d的母豬血清和14 d、35 d、56 d的仔豬血清及77 d、98 d、119d、140 d的生長豬血清,用阻斷ELISA方法檢測豬瘟病毒抗體。結(jié)果顯示,在空懷母豬階段兩組無差異。在其他試驗階段,E組豬瘟病毒抗體阻斷率一直高于A組,各個階段均達(dá)到顯著(P0.05)水平;除35 d與56 d仔豬外,E組豬瘟病毒抗體阻斷率變異系數(shù)都小于A組;E組豬瘟病毒抗體陽性率都高于A組,其中在35 d仔豬階段達(dá)到顯著水平�？贵w檢測結(jié)果顯示,E組豬瘟免疫抗體數(shù)據(jù)優(yōu)于A組,表明豬瘟疫苗E的臨床免疫效果優(yōu)于疫苗A,提示較高劑量豬瘟疫苗的臨床免疫效果好于較低劑量疫苗。綜合分析表明,建立了1種快速檢測豬瘟疫苗劑量的熒光定量PCR方法,不同廠家豬瘟疫苗的劑量存在明顯差異,較高劑量豬瘟疫苗的臨床免疫效果優(yōu)于較低劑量疫苗,為劑量存在差異的不同廠家豬瘟疫苗的臨床選用提供了實驗依據(jù)。
[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.
【學(xué)位授予單位】：北京農(nóng)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.28
本文編號：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.
【學(xué)位授予單位】：北京農(nóng)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.28
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