高致病性猪繁殖与呼吸综合征病毒及其疫苗毒株JXA1-R荧光RT-PCR鉴别检测方法的建立

发布时间：2018-03-11 02:02

本文选题：HP-PRRSV　切入点：JXA1-R　出处：《南京农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：猪繁殖与呼吸综合征(PRRS)是以母猪生殖障碍和各种日龄猪(尤其仔猪)的呼吸道症状为主要特征的急性、高度接触性传染病,该病病原为猪繁殖与呼吸综合征病毒(PRRSV)。2006年之前我国主要的流行毒株是经典PRRSV,2006年出现了高致病性PRRSV(HP-PRRSV)感染和流行。目前,预防HP-PRRSV感染的主要措施是接种疫苗,疫苗的种类有JXA1-R、HuN4-F112及TJM-F92,其中,JXA1-R株应用最为广泛。本研究成功建立了 HP-PRRSV野毒及其疫苗毒株JXA1-R双重荧光RT-PCR鉴别检测方法,其主要研究内容如下:1.HP-PRRSV野毒荧光RT-PCR检测方法的建立根据GenBank中的经典PRRSV及HP-PRRSV野毒株全基因序列的比较结果,在HP-PRRSV nsp2基因设计了一对引物和一个探针,建立了 HP-PRRSV的荧光RT-PCR检测方法。该方法敏感性达4.43拷贝/μL,标准曲线相关系数为0.998;用三份不同稀释浓度的标准品对该方法的重复性和稳定性进行评估,该方法具有良好的重复性,组内变异系数为0.39%-0.96%,组间变异系数为1.1%-2.6%;用建立的方法同时检测经典 PRRSV、HP-PRRSV 野毒、PCV、PEDV、PRV、TGEV,只有 HP-PRRSV 野毒有特异性的荧光曲线,表明该方法的特异性好;该方法的检测灵敏度为普通RT-PCR的1000 倍。2.JXA1-R疫苗毒株荧光RT-PCR检测方法的建立根据HP-PRRSV与JXA1-R株的基因序列比较结果,设计一对引物和一个探针,建立了 JXA1-R株荧光RT-PCR检测方法。该方法灵敏度达8.25拷贝/μL,标准曲线相关系数为0.999;该方法的组内变异系数为0.42%-1.9%,组间变异系数为0.68%-2.74%,表明具有良好的重复性;用此方法对HP-PRRSV、经典PRRSV、PCV、PEDV、PRV、TGEV进行检测,均没有交叉反应,表明该方法的特异性好;并与常规RT-PCR做对比,灵敏度是其的1000倍。3.HP-PRRSV野毒及其疫苗毒株JXA1-R双重荧光RT-PCR鉴别检测方法的建立及应用在建立单一荧光RT-PCR检测方法的基础上,经过条件的优化,建立了鉴别检测HP-PRRSV和JXA1-R的双重荧光RT-PCR。该方法对HP-PRRSV和JXA1-R的检测灵敏度分别为4.43拷贝/μL和4.12拷贝/μL,对HP-PRRSV检测的批内重复性变异系数CV%最大值为0.70%,批间重复性变异系数CV%最大值为2.8%,对JXA1-R检测的批内重复性变异系数CV%最大值为0.62%,批间重复性变异系数CV%最大值为3.0%,均不超过5%,表明建立的方法重复性好;用此方法对经典PRRSV、PCV、PEDV、PRV、TGEV等进行检测,均没有交叉反应,表明该方法的特异性好。用建立的方法对临床200份血液样品进行了检测,HP-PRRSV荧光RT-PCR方法检测出32份阳性,阳性率为16%(32/200);JXA1-R疫苗毒株荧光RT-PCR方法检测出9份阳性,阳性率为4.5%(9/200);用双重荧光RT-PCR方法对这200份样品进行检测,结果与单一荧光RT-PCR方法检测一致,表明建立的方法具有很好的临床应用价值。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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