为了从细胞水平探讨PRRSV GD强毒株和PRRSV弱毒疫苗接种仔猪后对其外周血淋巴细胞亚群的影响,运用血常规技术和流式细胞技术分析了猪体外周血淋巴细胞亚群的动态变化.将30日龄20头SPF猪分成3组,分别感染HP-PRRSV GD第5代强毒株、GDr180弱毒疫苗株及留作对照.于感染后第0、3、7、10、14、21、28及35天采血进行外周血淋巴细胞亚群测定,分析淋巴细胞亚群的变化.结果表明:HP-PRRSV GD强毒株感染可导致白细胞、淋巴细胞数量、单核细胞、粒细胞、B细胞、Tc细胞、Th细胞、Tm细胞、和γ6T细胞数量的下降;而弱毒疫苗GDr180株免疫则表现为白细胞、淋巴细胞、单核细胞、引起粒细胞、B细胞、Tc细胞、Th细胞和Tm细胞的上升,对γ6T细胞的影响不大;阴性对照组在整个检测期间各种细胞基本上保持稳定状态.从实验结果可以看出:与HP-PRRSV GD强毒株破坏免疫细胞不同,PRRSV弱毒疫苗GDr180株免疫接种能刺激猪免疫细胞增殖,给试验猪提供良好的免疫反应.

为完善兽用疫苗中支原体污染的检测技术,本研究对污染生物制品的14种常见支原体基因组进行分析,设计一对可以同时检测这14种支原体的通用引物,建立兽用疫苗中支原体污染的PCR检测方法.应用该方法对10种兽用活疫苗的61批次和10种细胞系的21份传代细胞样品进行支原体污染的检测,并与培养法、DNA荧光染色法及成品的支原体PCR检测试剂盒相比较,除成品的支原体PCR检测试剂盒的检测结果不同之外,其他3种方法的检测结果均相同.但培养法耗时长,DNA荧光染色法操作复杂,成品的支原体PCR检测试剂盒只能检测5种污染细胞的常见支原体,出现漏检情况,而本研究建立的PCR检测方法快速、准确,可以为今后兽用疫苗的支原体污染检测工作提供参考.

Due to the urgent need of differentiation of infected from vaccinated animals in control and eradication of classical swine fever (CSF) and the shortcomings of current differential diagnostic tools, this study is aiming to establish a RT-nestPCR assay for differential detection of wild-type viruses and lapinized Chinese vaccine strain (C-strain) of classical swine fever virus (CSFV) of high sensitivity. Two pairs of CSFV-specific primers were designed in the conservative regions of NS5B (a non-structural protein encoded by the CSFV genome, which performs the RNA dependent RNA polymerase activity) and 3? un-translated regions (3?-UTR) to encompass the T-rich insertion uniquely existing in the 3?-UTR of C-strain genome. Thus the amplification fragment of C-strain is longer than that of the wild-type viruses for it contains the T-rich insertion region. Two pairs of primers were used in combination and the wild-type viruses and C-strain of CSFV could be detected and accurately distinguished with a high sensitivity through super fine resolution (SFR) argarose gel electrophoresis that displays the different lengths of the amplicons. The detection limit of the C-strain and Shimen strain were respectively 4.5×10-2 pg and 3.2×10-2 pg of viral RNA. The results of the specificity test showed that this method can detect different strains of CSFV without amplifying other non-CSFV pathogens. The results of the detection of 400 clinical samples indicated that 16 samples were CSFV positive in total; in which 4 samples were C-Strain positive and 12 were wild-type CSFV positive. The total CSFV positive rate was 4%. The detection results of the 14 batches of C-Strain vaccines showed that all samples displayed bands of C-Strain amplicons in the SFR argarose gel electrophoresis and all vaccines were free of wild-type virus contamination. In conclusion, the RT-nestPCR assay established in the present study could supply a sensitive and specific test method for distinguishing wild-type CSFV infected animals from those vaccinated with C-strain vaccines in the field.

自1956年国外学者第一次检测到细胞培养物中存在支原体污染以来,这个世界性问题持续了半个多世纪.对于生物制品中支原体污染的检测方法多种多样,但尚无统一的内控标准,而建立一种快速、准确的方法用于支原体污染的检测,可以有效地减少和预防支原体污染,从而提高生物制品的纯净性.本文对支原体污染的几种常见检测及鉴定方法进行综述,包括DNA荧光染色法、PCR、IFA与ELISA等,以期为支原体污染的检测及鉴定研究提供参者.

Primers and probe were designed with PrimerExpress 3.0 and synthesized according to the conserved gene sequences of 24 highly pathogenic porcine reproductive and respiratory syndrome(HP-PRRSV) strains and 5 classical PRRSV strains available in GenBank.A real-time fluorescent quantitative PCR assay was established for detection,differentiation and quantitation of HP-PRRSV and classical PRRSV.A known concentration of 10-fold series of dilutions of pGET-258 plasmid DNA were detected by using the established real-time FQ-PCR assay,then compared to the detection result of routine PCR.The developed real-time FQ-PCR assay could detect 1.5 copies with plasmid DNA and its sensitivity was 100 times higher than that of the routine PCR.Three plasmid standards were examined using the real-time FQ-PCR repeatedly at three different time,the coefficient of variations were below 2%,and the results indicated that the real-time FQ-PCR assay was reproducible and could be used for the diagnosis and differentiation of HP-PRRSV infection.

为建立一种能够快速鉴别检测猪繁殖与呼吸综合征病毒（PRRSV）GDr180疫苗株与GD野毒株的双重荧光定量RT-PCR方法,本研究在PPRSV的Nsp2基因和Orf7基因区域分别设计不同荧光标记的MGB探针及特异性引物。这两种MGB探针能够分别特异结合GD野毒株和GDr180疫苗株。通过对PRRSV培养液、感染猪血清和组织样品检测证明了该方法的可行性;采用双重实时荧光定量RT-PCR方法检测PRRSV GDr180疫苗株和GD强毒株时敏感性分别达到19.4拷贝/μL和34.2拷贝/μL;该方法与PRRSV其他8个病毒株和猪瘟病毒、猪伪狂犬病毒、猪圆环病毒2型和猪细小病毒不发生交叉反应,表明该方法具有良好的特异性;因此可以用于PRRSV GDr180疫苗株与野毒株的鉴别检测。