基于TaqMan探针的荧光定量PCR技术在PCV2检测中应用研究

目的 针对猪圆环病毒(PCV2)特点,利用TaqMan探针建立一种能够快速、准确并具有临床应用价值的PCV2检测方法.方法 参照GenBank已收录PCV20RF2,利用软件设计合成1对特异引物以及与之相匹配的特异性TapMan探针,采用PCR对PCV2衣壳蛋白部分基因进行克隆并鉴定,将纯化回收产物连接至pMT19-T载体,然后转化至大肠杆菌DH5a感受态细胞,涂布至Amp+琼脂平板,筛选阳性克隆并扩大培养,进行2％琼脂糖凝胶电泳鉴定并测序.采用矩阵法确定引物和探针佳浓度.对基于TapMan探针的实时荧光定量PCR循环进行优化,筛选佳退火温度.以100～109倍稀释的标准品为模板,进行敏感性试验.提取CSFV、PRRSV、PRV和PCV1的DNA(CSFV和CSFV在提取RNA后反转录成cDNA)作为模板,进行TapMan荧光定量PCR,检测其特异性.采用105～108倍稀释的标准品为模板进行重复性实验,其中批内和批间均重复4次,根据所得值计算变异系数.结果 PCR产物经20 g/L琼脂糖凝胶电泳鉴定后大小为114 bp,与预期相符;测序分析PCR产物序列与参考序列同源性为100％.优化的PCR条件为:引物和探针浓度为10 μmol/L,退火温度60℃.建立了PCV2 TaqMan实时荧光定量PCR检测标准曲线,起始模板拷贝数为38.403,斜率为-3.69,相关系数R2为0.998,曲线呈线性.检测方法的敏感性为10 2拷贝/μl质粒标准品,是常规PCR检测敏感性的100倍.特异性检测显示,CSFV、PRRSV、PRV和PCV1扩增反应均阴性.重复性检测显示,PCR扩增循环阈值平均值基本一致,其变异系数均小于2％.采用TaqMan荧光定量PCR方法检测40份样品14份阳性,阳性率35.0％;普通PCR检测11份阳性,阳性率27.5％,差异无统计学意义(P＞0.05).结论 建立的PCV2TaqMan实时荧光定量PCR检测方法具有以下优点:1)敏感性高,能够用于精确计算PCV2病毒载量;2)特异性强,适用于检测PCV2与其他病原混合感染;3)用时短且可靠性强,可用于PCV2感染检测.

欧洲型PRRSV、PCV2重组痘病毒疫苗灌注式生物反应器培养工艺的建立

目的 利用微载体-生物反应器技术培养乳仓鼠肾细胞,接种重组痘病毒rVTT-ORF2-EUO3O5,建立规模化制备欧洲型PRRSV、PCV2重组痘病毒疫苗的灌注式生产工艺. 方法 利用2L体积灌注式生物反应器培养BHK细胞,设定培养参数为温度37℃、溶氧度(DO)为60％,转速为80 r/min,pH值为7.3.当90％微载体表面贴满BHK细胞时以0.1 MOI的感染剂量接种重组痘病毒rVTT-ORF2-EUO3O5,设定感染参数为温度35℃、DO为50％、转速为50 r/min,pH值为7.1,继续培养观察. 结果 在采用灌注式生物反应器技术制备重组痘病毒过程中的细胞培养阶段,细胞培养96 h可获得1.587×1010个细胞数,细胞增殖39.67倍;在病毒增殖阶段,微载体上细胞完全脱落时的病毒滴度为2.12×109 PFU/ml. 结论 成功建立了欧洲型PRRSV、PCV2重组痘苗病毒灌注式生产工艺,为规模化制备重组痘病毒疫苗提供了技术参数.

VC2002, isolated from postweaning multisystemic wasting syndrome (PMWS)-affected pig, is a mixture of two porcine circovirus genotype 2b (PCV2b) viruses, K2 and K39. Preliminary experiments disclosed short-term adverse effects of K39, but not K2, on porcine foetuses. These findings led to the hypothesis that infection of immuno-incompetent foetuses with K2 confers a status of immunotolerance, and postnatal super-infection with K39 triggers PMWS. To explore this hypothesis, nine 55-day-old foetuses were inoculated in utero (three with K2-104.3TCID50, three with K39-104.3TCID50 and three with medium), and foeto-pathogenicity examined. At 21 days post-inoculation (dpi), K2 did not induce pathology, whereas pathological effects of K39 were evident. Twenty-four 45-day-old foetuses were subsequently inoculated to examine the long-term effect of K2, including six with K2-high dose-104.3TCID50, six with K2-low dose-102.3TCID50 and 12 mock-inoculated controls. Both doses resulted in ifve mummiifed foetuses and one live-born piglet each (69dpi). K2 was recovered from all mummies. K2 and K2-speciifc antibodies were not detected in serum of the two live-born piglets at birth, indicating full control of K2 infection. The K2-low dose-infected piglet was immunostimulated at day 2, but not the K2-high dose-infected piglet. Both non-stimulated and stimulated K2-infected piglets were super-inoculated with K39 at day 6 or 8 (taken as 0 days post super-inoculation). Low viral replication was observed in the non-stimulated K2-K39 piglet (up to 103.3 TCID50/g;identiifed as K39). In contrast, viral replication was extremely high in the stimulated K2-K39 piglet (up to 105.6TCID50/g) and identiifed as K2, indicating that K2 infection is controlled during foetal life, but emerges after birth upon immunostimulation. However, none of the piglets showed any signs of PMWS.