發(fā)布時(shí)間：2018-03-11 02:02

  本文選題：HP-PRRSV　切入點(diǎn)：JXA1-R　出處：《南京農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：豬繁殖與呼吸綜合征(PRRS)是以母豬生殖障礙和各種日齡豬(尤其仔豬)的呼吸道癥狀為主要特征的急性、高度接觸性傳染病,該病病原為豬繁殖與呼吸綜合征病毒(PRRSV)。2006年之前我國主要的流行毒株是經(jīng)典PRRSV,2006年出現(xiàn)了高致病性PRRSV(HP-PRRSV)感染和流行。目前,預(yù)防HP-PRRSV感染的主要措施是接種疫苗,疫苗的種類有JXA1-R、HuN4-F112及TJM-F92,其中,JXA1-R株應(yīng)用最為廣泛。本研究成功建立了 HP-PRRSV野毒及其疫苗毒株JXA1-R雙重?zé)晒釸T-PCR鑒別檢測方法,其主要研究內(nèi)容如下:1.HP-PRRSV野毒熒光RT-PCR檢測方法的建立根據(jù)GenBank中的經(jīng)典PRRSV及HP-PRRSV野毒株全基因序列的比較結(jié)果,在HP-PRRSV nsp2基因設(shè)計(jì)了一對(duì)引物和一個(gè)探針,建立了 HP-PRRSV的熒光RT-PCR檢測方法。該方法敏感性達(dá)4.43拷貝/μL,標(biāo)準(zhǔn)曲線相關(guān)系數(shù)為0.998;用三份不同稀釋濃度的標(biāo)準(zhǔn)品對(duì)該方法的重復(fù)性和穩(wěn)定性進(jìn)行評(píng)估,該方法具有良好的重復(fù)性,組內(nèi)變異系數(shù)為0.39%-0.96%,組間變異系數(shù)為1.1%-2.6%;用建立的方法同時(shí)檢測經(jīng)典 PRRSV、HP-PRRSV 野毒、PCV、PEDV、PRV、TGEV,只有 HP-PRRSV 野毒有特異性的熒光曲線,表明該方法的特異性好;該方法的檢測靈敏度為普通RT-PCR的1000 倍。2.JXA1-R疫苗毒株熒光RT-PCR檢測方法的建立根據(jù)HP-PRRSV與JXA1-R株的基因序列比較結(jié)果,設(shè)計(jì)一對(duì)引物和一個(gè)探針,建立了 JXA1-R株熒光RT-PCR檢測方法。該方法靈敏度達(dá)8.25拷貝/μL,標(biāo)準(zhǔn)曲線相關(guān)系數(shù)為0.999;該方法的組內(nèi)變異系數(shù)為0.42%-1.9%,組間變異系數(shù)為0.68%-2.74%,表明具有良好的重復(fù)性;用此方法對(duì)HP-PRRSV、經(jīng)典PRRSV、PCV、PEDV、PRV、TGEV進(jìn)行檢測,均沒有交叉反應(yīng),表明該方法的特異性好;并與常規(guī)RT-PCR做對(duì)比,靈敏度是其的1000倍。3.HP-PRRSV野毒及其疫苗毒株JXA1-R雙重?zé)晒釸T-PCR鑒別檢測方法的建立及應(yīng)用在建立單一熒光RT-PCR檢測方法的基礎(chǔ)上,經(jīng)過條件的優(yōu)化,建立了鑒別檢測HP-PRRSV和JXA1-R的雙重?zé)晒釸T-PCR。該方法對(duì)HP-PRRSV和JXA1-R的檢測靈敏度分別為4.43拷貝/μL和4.12拷貝/μL,對(duì)HP-PRRSV檢測的批內(nèi)重復(fù)性變異系數(shù)CV%最大值為0.70%,批間重復(fù)性變異系數(shù)CV%最大值為2.8%,對(duì)JXA1-R檢測的批內(nèi)重復(fù)性變異系數(shù)CV%最大值為0.62%,批間重復(fù)性變異系數(shù)CV%最大值為3.0%,均不超過5%,表明建立的方法重復(fù)性好;用此方法對(duì)經(jīng)典PRRSV、PCV、PEDV、PRV、TGEV等進(jìn)行檢測,均沒有交叉反應(yīng),表明該方法的特異性好。用建立的方法對(duì)臨床200份血液樣品進(jìn)行了檢測,HP-PRRSV熒光RT-PCR方法檢測出32份陽性,陽性率為16%(32/200);JXA1-R疫苗毒株熒光RT-PCR方法檢測出9份陽性,陽性率為4.5%(9/200);用雙重?zé)晒釸T-PCR方法對(duì)這200份樣品進(jìn)行檢測,結(jié)果與單一熒光RT-PCR方法檢測一致,表明建立的方法具有很好的臨床應(yīng)用價(jià)值。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRSs) is an acute, highly contagious disease characterized by reproductive disorders in sows and respiratory symptoms in various day-old pigs (especially piglets). The pathogen of the disease is porcine reproductive and respiratory syndrome virus (PRRSVV). Before 2006, the main epidemic strain in China was the classic PRRSVV.The infection and prevalence of high pathogenicity PRRSVV HP-PRRSVS appeared in 2006. At present, vaccination is the main measure to prevent HP-PRRSV infection. The kinds of vaccines are JXA1-RN4-F112 and TJM-F92.In this study, the identification and detection of HP-PRRSV wild virus and its vaccine strain JXA1-R by double fluorescence RT-PCR were successfully established, among which JXA1-R strain was the most widely used. The main contents of this study are as follows: 1. The establishment of fluorescence RT-PCR method for detecting wild virus of HP-PRRSv. According to the results of comparison of the whole gene sequences of classical PRRSV and HP-PRRSV wild strain in GenBank, a pair of primers and a probe were designed in the HP-PRRSV nsp2 gene. A fluorescence RT-PCR method for the detection of HP-PRRSV was established. The sensitivity of the method was 4.43 copies / 渭 L and the correlation coefficient of the standard curve was 0.998.The repeatability and stability of the method were evaluated with three standard samples with different dilution concentrations. The coefficient of variation within the group was 0.39-0.96, and the coefficient of variation between the groups was 1.1-2.6. The established method was used to simultaneously detect the classical PRRSVHP-PRRSV wild virus, PCV-PEDVV and TGEV. Only the HP-PRRSV wild virus had specific fluorescence curve, which indicated that the method was specific. The sensitivity of this method is 1000 times of that of ordinary RT-PCR. 2. The method of fluorescent RT-PCR detection of JXA1-R vaccine strain was established. According to the results of gene sequence comparison between HP-PRRSV and JXA1-R strain, a pair of primers and a probe were designed. The sensitivity of the method was 8.25 copies / 渭 L, the correlation coefficient of standard curve was 0.999 9, the coefficient of variation within group was 0.42% -1.9%, and the coefficient of variation between groups was 0.68 -2.74, which indicated that the method had good repeatability. The method was used to detect HP-PRRSVand classical PRRSVP PEVV RT-PCR without cross reaction, which indicated that the method had good specificity, and was compared with conventional RT-PCR. The sensitivity is 1000 times as high as that of HP-PRRSv and its vaccine strain JXA1-R. The establishment and application of double fluorescent RT-PCR detection method based on the establishment of a single fluorescent RT-PCR detection method, through the optimization of the conditions. A dual fluorescence RT-PCR was established for the identification and detection of HP-PRRSV and JXA1-R. The sensitivity of this method for HP-PRRSV and JXA1-R was 4.43 copies / 渭 L and 4.12 copies / 渭 L, respectively. The maximum coefficient of variation for intra-assay reproducibility of HP-PRRSV was 0.70%, and the coefficient of variation for inter-assay repeatability was 0.70. The maximum value of CV% was 2.8, the maximum coefficient of variation of intra-assay repeatability was 0.62% for JXA1-R, and the maximum value of CV% for inter-assay was 3.0%, which was less than 5, which indicated that the established method had good reproducibility. This method was used to detect the classical PRRSVV PCVV PEVV and PRVV TGEV, all of which showed no cross reaction, which showed that the method was specific. The established method was used to detect 32 HP-PRRSV positive samples in 200 clinical blood samples. The positive rate of JXA1-R vaccine strain was 16 / 32 / 200. Nine samples were positive by fluorescent RT-PCR method, and the positive rate was 4.50.The results of double fluorescence RT-PCR method were consistent with that of single fluorescent RT-PCR method. The results show that the established method has good clinical application value.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.28

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李學(xué)伍,肖治軍,張改平,鄧瑞廣,楊艷艷,趙東,柴書軍,邢廣旭;雞傳染性法氏囊病疫苗毒株的生物學(xué)特性研究[J];河南農(nóng)業(yè)科學(xué);2003年01期

2 陳昌如;陳曉琴;韋建林;劉克華;;提高家禽免疫效果的綜合措施[J];中國家禽;2009年06期

3 沈明君;;疫苗免疫需注意的幾個(gè)方面[J];中國禽業(yè)導(dǎo)刊;2009年12期

4 蔡汝健;蔣智勇;劉燕玲;張樂宜;宋長緒;;我國豬繁殖與呼吸綜合征病毒的分子演化及與疫苗毒株的比較分析[J];廣東農(nóng)業(yè)科學(xué);2013年08期

5 李本勝;;雞群產(chǎn)生有效抗體,必須控制的五個(gè)關(guān)鍵環(huán)節(jié)[J];北方牧業(yè);2007年12期

6 池躍田;;疫苗免疫失敗的因素[J];養(yǎng)殖技術(shù)顧問;2013年09期

7 胡少昶;讓疫苗接種產(chǎn)生最大效力[J];國外畜牧學(xué)(豬與禽);1999年05期

8 冉廣忠;;提高動(dòng)物免疫效果的主要措施[J];今日畜牧獸醫(yī);2013年06期

9 吳國彬;;新城疫緊急免疫接種疫苗毒株和接種方式的選擇探討[J];北方牧業(yè);2007年21期

10 ;大華農(nóng)獲得廣東省科學(xué)技術(shù)一等獎(jiǎng)[J];飼料廣角;2012年13期

相關(guān)會(huì)議論文 前1條

1 丁旭娜;呂茂杰;楊曉紅;何召慶;楊保收;王宏偉;;五種不同豬偽狂犬病滅活疫苗免疫效果評(píng)價(jià)[A];中國畜牧獸醫(yī)學(xué)會(huì)家畜傳染病學(xué)分會(huì)第八屆全國會(huì)員代表大會(huì)暨第十五次學(xué)術(shù)研討會(huì)論文集[C];2013年

相關(guān)重要報(bào)紙文章 前2條

1 記者 李穎;北京科興：正等待大批量生產(chǎn)用疫苗毒株[N];科技日?qǐng)?bào);2009年

2 記者 張錦芳;韓國初步研制出禽流感疫苗毒株[N];新華每日電訊;2004年

相關(guān)博士學(xué)位論文 前1條

1 王祥;豬乙型腦炎減毒活疫苗與基因免疫研究[D];華中農(nóng)業(yè)大學(xué);2001年

相關(guān)碩士學(xué)位論文 前4條

1 楊雯慧;河南省PRV血清學(xué)調(diào)查及鑒別野毒/疫苗毒株二重FQ-PCR方法的建立[D];河南科技大學(xué);2015年

2 鄒玖零;高致病性豬繁殖與呼吸綜合征病毒及其疫苗毒株JXA1-R熒光RT-PCR鑒別檢測方法的建立[D];南京農(nóng)業(yè)大學(xué);2015年

3 于靜;雞傳染性支氣管炎病毒生產(chǎn)用種毒的分子生物學(xué)鑒定方法的研究[D];中國獸醫(yī)藥品監(jiān)察所;2012年

4 龔國華;口蹄疫O型、A型雞胚化活疫苗毒株VP1基因的克隆與序列分析[D];新疆農(nóng)業(yè)大學(xué);2006年

，

本文編號(hào)：1596064