H5和H9亚型禽流感病毒联合RT-PCR检测方法的建立

目的建立H5和H9亚型禽流感病毒(AIV)联合RT-PCR检测方法。方法针对H5和H9亚型AIV血凝素(HA)基因,在其裂解位点两侧设计引物。在确定单项RT-PCR检测方法反应条件基础上,建立、优化联合RT-PCR反应体系和反应条件,并通过相关病毒检测试验,验证其敏感性和特异性。结果所建立的H5和H9亚型联合RT-PCR检测方法具有特异、敏感、简便、快捷等特点。结论该方法可用于AIV的检测及致病性分析。     

新城疫病毒一步法RT-PCR检测方法的建立及初步应用

目的建立新城疫病毒(Newcastle disease virus,NDV)一步法RT-PCR检测方法,并进行验证及初步应用。方法根据NDV F基因序列,设计合成了1对特异性引物,建立一步法RT-PCR检测方法,并验证其特异性及敏感性;应用建立的方法对23份疑似新城疫(Newcastle disease,ND)病料进行检测,并与血凝及血凝抑制试验检测结果进行比对。结果建立的一步法RT-PCR同时检测NDV、传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)和禽流感病毒(Avian influenza virus,AIV)H9亚型,仅NDV为阳性,IBDV和AIV H9均为阴性;该方法最低可检出约4 pg的NDV RNA;23份疑似ND病料,一步法RT-PCR检出11份阳性,血凝及血凝抑制试验检出13份阳性,两种方法的阳性符合率为85%。结论建立了NDV一步法RT-PCR检测方法,该方法特异性良好,敏感度较高,用时较短,可从分子水平上对NDV进行早期快速诊断和流行病学调查。     

甲型H1N1流感病毒实时荧光PCR诊断试剂盒的制备

目的制备甲型H1N1流感病毒实时荧光PCR诊断试剂盒,并进行验证。方法采用磁珠法从甲型H1N1流感疑似患者咽拭子样品中提取病毒RNA,逆转录合成cDNA。根据NCBI最新公布的大流行甲型H1N1流感病毒(2009)基因序列,设计针对编码基质蛋白M基因的引物和探针,检测甲型流感病毒;设计针对血凝素(HA)基因和神经氨酸酶(NA)基因特异性的引物和探针,检测甲型H1N1流感病毒;同时针对人的RNaseP基因设计用于内部控制的引物和探针。所有探针均为Taqman探针,5'端标记FAM,3'端标记BHQ1。对最佳荧光PCR反应条件进行优化,在此基础上组装成甲型H1N1流感病毒实时荧光PCR诊断试剂盒,对其特异性、灵敏度、精密性和稳定性进行验证。与市售试剂盒的检测结果进行对比,并对63份临床甲型H1N1流感疑似患者咽拭子样品进行检测。结果设计的PCR引物及探针能对甲型H1N1流感病毒进行准确检测,与流感病毒的其他型和亚型无交叉反应;试剂盒的灵敏度为0.004个血凝素单位;试验内变异系数小于2.5%,批间变异系数小于5%;试剂盒放置-20℃保存,稳定性良好;检测20份甲型H1N1流感疑似患者咽拭子样品的结果与市售试剂盒一致;检测63份流感疑似患者咽拭子样品,其中大流行甲型H1N1流感病毒阳性36份,普通甲型流感病毒阳性5份。结论所制备的甲型H1N1流感病毒实时荧光PCR诊断试剂盒具有较高的灵敏度、特异性、精密性和稳定性,可用于目前流行的甲型H1N1流感病毒的快速检测。     

H5N1禽流感病毒感染豚鼠体内线粒体抗病毒信号蛋白SYBR GreenⅠ相对荧光定量PCR检测方法的建立及应用

目的建立H5N1禽流感病毒(avian influenza virus,AIV)感染豚鼠体内线粒体抗病毒信号蛋白(mitochondrial antiviral signaling protein,MAVS)SYBR GreenⅠ相对荧光定量PCR检测方法,并检测H5N1 AIV感染前、后豚鼠肺脏组织中MAVS的表达水平。方法提取豚鼠肺脏组织RNA,反转录合成cDNA,将cDNA模板按10倍系列稀释为9个浓度(1.00 E+10~1.00 E+02 copies/μl),进行PCR扩增。以β-actin为内参,建立标准曲线,比较两基因的扩增效率,验证该方法的引物特异性、重复性及灵敏度。同时用该方法检测H5N1 AIV攻毒前、后豚鼠肺脏组织中MAVS的表达水平。结果 c DNA模板最佳稀释度范围为1.00 E+09~1.00 E+04。豚鼠MAVS和β-actin基因的标准曲线斜率差值0.1,R~2=0.999,扩增效率分别为100.2%和100.3%;两基因扩增溶解曲线峰单一,可扩增出清晰的目的条带,且无非特异性扩增;两基因Ct值的变异系数(CV)均10%;灵敏度为1.00 E+03 copies/μl。感染H5N1 AIV的豚鼠肺脏组织中MAVS的表达水平下调。结论成功建立了H5N1 AIV感染豚鼠体内MAVS的SYBR GreenⅠ相对荧光定量PCR检测方法,并发现H5N1 AIV感染可导致豚鼠体内MAVS表达水平下调。     

5种病毒性脑炎相关RNA病毒多重RT-PCR检测方法的初步建立

目的建立肠道病毒、乙型脑炎病毒、麻疹病毒、风疹病毒和腮腺炎病毒的多重RT-PCR检测方法。方法根据GenBank中登录的肠道病毒、乙型脑炎病毒、麻疹病毒、风疹病毒和腮腺炎病毒基因组序列,分别设计肠道病毒5′UTR基因、乙型脑炎病毒E基因、麻疹病毒M基因、风疹病毒E基因和腮腺炎病毒M基因的特异性引物,建立多重RT-PCR检测方法,并进行敏感性验证。结果应用5种引物可分别扩增脊髓灰质炎病毒、乙型脑炎病毒、麻疹病毒、风疹病毒和腮腺炎病毒的152、429、111、352和274 bp特异基因条带。对脊髓灰质炎病毒、乙型脑炎病毒、麻疹病毒、风疹病毒和腮腺炎病毒的的敏感度分别为62.5 CCID50/ml、250 PFU/ml、125 CCID50/ml、125 CCID50/ml和125 CCID50/ml。结论初步建立了5种脑炎RNA病毒多重RT-PCR检测方法,该法敏感性良好,可用于5种脑炎相关RNA病毒的快速检测。     

中蜂囊状幼虫病毒RT-PCR检测方法的建立

目的建立中蜂囊状幼虫病毒RT-PCR检测方法。方法参照已发表的中蜂囊状幼虫病毒(CSBV)基因序列,针对其结构蛋白基因设计1对特异性引物,提取感染CSBV的囊状幼虫RNA,反转录为cDNA,以其为模板,进行PCR扩增,对检测方法进行优化,验证该方法的特异性及敏感性,并对31份临床样品进行检测。结果所建立的CSBVRT-PCR检测方法特异性和敏感性良好,以含CSBV结构蛋白基因的重组质粒为模板,最低可检出10pgDNA。临床症状诊断和电镜观察均为阴性的22份样品经RT-PCR检测,有2份扩增出目的条带。结论已建立了中蜂囊状幼虫病毒RT-PCR检测方法,为CSBV检测、流行病学调查及动物模型的建立奠定了基础。     

新疆伊犁地区马脑组织尼帕病毒的感染情况

目的采用一步法荧光定量RT-PCR检测新疆伊犁地区天然牧场放养马群脑组织尼帕病毒(NipahVirus,NiV)核蛋白N基因的表达,调查该地区马群中枢神经系统NiV感染的流行状况。方法针对NiV高度保守区N基因设计特异性引物和探针,建立检测马脑组织样本中低浓度NiV RNA的一步法荧光定量RT-PCR方法,对该方法的敏感性及特异性进行验证,并对新疆伊犁地区天然牧场放养且未接种NiV疫苗的183匹马脑组织进行检测。结果一步法荧光定量RT-PCR的最低检出限为1.1×102 copies/μl;与NiV同为亨尼帕病毒属,且高度同源的亨德拉病毒(Hendra Virus,HeV)及同为单股负链的博尔纳病病毒(Borna Disease Virus,BDV)均无交叉反应;183份马脑组织样本未检出阳性样本。结论新疆伊犁地区天然牧场放养马匹中未发现NiV感染,该地区短时间内爆发NiV的可能性较小。     

猪瘟和猪蓝耳病病毒多重RT-PCR检测方法的建立及初步应用

目的建立猪瘟病毒(Classical swine fever virus,CSFV)和猪蓝耳病病毒(Porcine respiratory and reproductivesyndrome virus,PRRSV)多重RT-PCR检测方法,并进行验证及初步应用。方法根据GenBank中登录的CSFV和PRRSV疫苗株基因组序列,分别设计针对CSFV和PRRSV的两对引物,建立多重RT-PCR检测方法,并进行敏感性及特异性验证。用建立的多重RT-PCR法检测20份疑似猪瘟、猪蓝耳病或混合感染病料,并与市售RT-PCR试剂盒的检测结果进行比较;检测病料的部分PCR产物测序后,与9株具有代表性的CSFV毒株和10株具有代表性的PRRSV毒株进行核苷酸序列同源性比对。结果以CSFV和PRRSV混合引物扩增,分别可扩增出286和664 bp的特异性条带。建立的多重RT-PCR检测方法可检出100倍稀释的病毒RNA;该方法可检出CSFV、PRRSV及CSFV与PRRSV混合病毒,而猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)、猪传染性胃肠炎病毒(Trans-missible gastro-enteritis virus,TGEV)、猪圆环病毒(Porcine circovirus,PCV)、猪细小病毒(Porcine parvovirus,PPV)和猪伪狂犬病病毒(Porcine pseudorabies virus,PRV)的检测结果均为阴性;该方法检测20份疑似猪瘟、猪蓝耳病或混合感染病料的结果与市售RT-PCR试剂盒比较,灵敏度达100%;选取的两份CSFV扩增片段与9株具有代表性的CSFV毒株的核苷酸序列同源性在92.3%⁹8.2%之间,扩增的PRRSV与10株具有代表性的PRRSV毒株的核苷酸序列同源性在94.1%98.2%之间,扩增的PRRSV与10株具有代表性的PRRSV毒株的核苷酸序列同源性在94.1%⁹7.9%之间。结论成功建立了CSFV和PRRSV多重RT-PCR检测方法,为猪瘟和猪蓝耳病的早期诊断及流行病学调查奠定了基础。     

鸡传染性支气管炎病毒S1蛋白单克隆抗体的制备及鉴定

目的制备鸡传染性支气管炎病毒(infectious bronchitis virus,IBV)S1蛋白单克隆抗体,并进行鉴定。方法用差速离心纯化的IBV四川分离株Sczy3免疫BALB/c小鼠,取脾细胞与骨髓瘤细胞SP2/0融合。利用IBV鼠高免血清建立间接ELISA,筛选杂交瘤细胞。强阳性孔经有限稀释法亚克隆,获得分泌单克隆抗体的杂交瘤细胞株,并制备腹水。用鼠单克隆抗体亚型鉴定试剂盒鉴定单抗的亚型,Western blot法分析单抗的反应原性,ELISA法分析单抗的特异性及其与不同IBV毒株的交叉反应性。结果经连续4次亚克隆,获得2株能稳定传代并分泌抗IBV Sczy3株单克隆抗体的杂交瘤细胞株1C8C1和2C10E7,其腹水效价分别为1∶12 800和1∶25 600。2株杂交瘤细胞分泌的单抗均为IgM亚型;均可特异性识别S1蛋白;2株单抗只与IBV发生特异性反应,与新城疫病毒(Newcastle disease virus,NDV)、禽流感病毒(avian influenza virus,AIV)H9亚型和AIV H5抗原均不反应,与其他5种基因型的10株IBV毒株均可产生交叉反应。结论成功制备了2株IBV S1蛋白单克隆抗体,为IBV的检测、抗原表位筛选及致病机理的研究等提供了材料。     

鸡传染性支气管炎病毒M蛋白单克隆抗体的制备及鉴定

目的制备鸡传染性支气管炎病毒(infectious bronchitis virus,IBV)M蛋白单克隆抗体,并进行鉴定。方法利用IBV Sczy3株免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,间接ELISA法筛选杂交瘤细胞,强阳性孔经有限稀释法亚克隆,制备抗IBV M蛋白单克隆抗体。对制备的单抗进行单抗亚型、腹水ELISA效价、特异性、交叉反应性鉴定及Western blot分析。结果获得2株能稳定分泌抗IBV M蛋白单克隆抗体的杂交瘤细胞株,命名为2C10和7H6,单抗亚型分别为IgM和IgG1;腹水ELISA效价分别为1∶25 600和1∶12 800;2株单抗均只与IBV发生特异性反应,而与新城疫病毒(Newcastle disease virus,NDV)、禽流感病毒(avian influenza virus,AIV)H9亚型、AIV H5抗原和鸡传染性法氏囊病毒(infectious bursal disease virus,IBDV)均不反应;2株单抗与5种基因型的其他10株IBV均有良好的反应性。结论制备出2株针对IBV M蛋白的单抗,为今后更深入地研究该蛋白的分子生物学功能、抗原表位的鉴定及抗原捕获ELISA方法的建立等提供了有力的工具。     

H_5N_1亚型禽流感病毒血凝素基因的克隆及分子进化分析

目的　克隆H5N1 亚型禽流感病毒HA基因并进行分子进化分析。方法　应用RT PCR方法 ,以 2株H5N1 亚型禽流感病毒RNA为模板 ,扩增了HA全基因cDNA。将HAcDNA基因克隆于pUCm T载体 ,并对其进行了测序。结果　所克隆的HA基因全长为 1731bp ,共编码 5 6 8个氨基酸。将该毒株与其它 10株H5亚型禽流感病毒HA基因序列核苷酸及氨基酸进行比较 ,其核苷酸同源性在 80 5 %～ 99 2 %之间 ,氨基酸序列同源性在 89 4 %～98 6 %之间。高致病性毒株HA基因裂解位点存在多个碱性氨基酸插入。结论　为禽流感基因工程疫苗的研制奠定了基础 ,同时通过分子进化分析也揭示了各毒株间的亲缘关系。     

Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode

A sensitive electrochemical method for the detection of avian influenza virus (AIV) H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode was developed. To enhance the selectivity and sensitivity, the modified electrode was assembled with multi-wall carbon nanotubes (MWNT), polypyrrole nanowires (PPNWs) and gold nanoparticles (GNPs). This electrode offered a porous structure with a large effective surface area, highly electrocatalytic activities and electronic conductivity. Therefore, the amount of DNA aptamer immobilized onto the electrode was increased while the accessibility of the detection target was maintained. The biosensor is based on the hybridization and preferred orientation of a DNA aptamer immobilized onto a modified electrode surface with its target (H5N1 specific sequence) present in solution. It is selective for the H5N1 specific sequence, and the signal of the indicator was approximately linear to log(concentration) of the H5N1 specific sequence from 5.0 × 10⁻¹² to 1.0 × 10⁻⁹ M (R = 0.9863) with a detection limit of 4.3 × 10⁻¹³ M. These studies showed that the new hybrid nanomaterial (MWNT/PPNWs/GNPs) and the DNA aptamer could be used to fabricate an electrochemical biosensor for gene sequence detection. Furthermore, this design strategy is expected to have extensive applications in other biosensors.     

Development of a Rapid Fluorescent Diagnostic System for Early Detection of the Highly Pathogenic Avian Influenza H5 Clade 2.3.4.4 Viruses in Chicken Stool

Rapid diagnosis is essential for the control and prevention of H5 highly pathogenic avian influenza viruses (HPAIVs). However, highly sensitive and rapid diagnostic systems have shown limited performance due to specific antibody scarcity. In this study, two novel specific monoclonal antibodies (mAbs) for clade 2.3.4.4 H5Nx viruses were developed by using an immunogen from a reversed genetic influenza virus (RGV). These mAbs were combined with fluorescence europium nanoparticles and an optimized lysis buffer, which were further used for developing a fluorescent immunochromatographic rapid strip test (FICT) for early detection of H5Nx influenza viruses on chicken stool samples. The result indicates that the limit of detection (LoD) of the developed FICT was 40 HAU/mL for detection of HPAIV H5 clade 2.3.4.4b in spiked chicken stool samples, which corresponded to 4.78 × 10⁴ RNA copies as obtained from real-time polymerase chain reaction (RT-PCR). An experimental challenge of chicken with H5N6 HPAIV is lethal for chicken three days post-infection (DPI). Interestingly, our FICT could detect H5N6 in stool samples at 2 DPI earlier, with 100% relative sensitivity in comparison with RT-PCR, and it showed 50% higher sensitivity than the traditional colloidal gold-based rapid diagnostic test using the same mAbs pair. In conclusion, our rapid diagnostic method can be utilized for the early detection of H5Nx 2.3.4.4 HPAIVs in avian fecal samples from poultry farms or for influenza surveillance in wild migratory birds.     

H7N7亚型马流感病毒单克隆抗体的制备

目的 制备抗H7N7亚型马流感病毒(Equine influenza virus,EIV)的单克隆抗体,以建立特异、灵敏、简便的H7N7亚型流感病毒金标试纸检测方法.方法 以H7N7亚型EIV为抗原免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞SP2/0进行融合,通过血凝抑制(HI)试验和间接ELISA筛选能稳定分泌抗H...     

国内不同基因型Asia1型口蹄疫病毒RT-PCR检测方法的建立

目的建立口蹄疫病毒(FMDV)Asia1型江苏/05谱系及新疆/03谱系毒株的二联RT-PCR检测方法。方法在比较大量国内外FMDV流行毒株序列的基础上,设计检测不同基因型Asia1型FMDV江苏/05谱系及新疆/03谱系毒株的二联PCR引物,优化单项和二联RT-PCR反应条件,并进行特异性及相关病毒检测。结果优化的单项和二联RT-PCR特异性均良好,检测9份FMDV,病料的结果与其基因序列测定结果完全一致。结论已建立了FMDVAsia1型江苏/05谱系和新疆/03谱系毒株的二联RT-PCR检测方法,为FMDV的诊断、流行病学调查及疫苗应用奠定了基础。     

Glycine Nano-Selenium Enhances Immunoglobulin and Cytokine Production in Mice Immunized with H9N2 Avian Influenza Virus Vaccine

This study was performed to investigate the immune enhancement effect of glycine nano-selenium, a microelement on H9N2 avian influenza virus vaccine (H9N2 AIV vaccine) in mice. Fifty (50) Specific Pathogen Free Kunming mice aged 4–6 weeks (18–20 g Body weight) were randomly divided into five groups: control normal group, which received no immunization + 0.5 mL 0.9% normal saline, positive control group, which received H9N2 AIV vaccine + 0.5 mL 0.9% normal saline, 0.25 mg/kg selenium group, which received H9N2 AIV vaccine + 0.5 mL 0.25 mg/kg selenium solution, 0.5 mg/kg selenium group, which received H9N2 AIV vaccine + 0.5 mL 0.5 mg/kg selenium solution, and 1 mg/kg selenium group, which received H9N2 AIV vaccine + 0.5 mL 1 mg/kg selenium solution. Hematoxylin and eosin staining, enzyme linked immunosorbent assay (ELISA), and quantitative real time polymerase chain reaction (qRT-PCR) methods were used to investigate the pathological changes, immunoglobulin levels, and cytokine gene expressions in this study. The results showed that all tested doses (0.25 mg/kg, 0.5 mg/kg and 1.00 mg/kg) of glycine nano-selenium did not lead to poisoning in mice. In addition, when compared to the positive control group, glycine nano-selenium increased the immunoglobin indexes (IgA, IgG, IgM and AIV-H9 IgG in serum) as well as the mRNA levels of IL-1β, IL-6 and INF-γ in the liver, lungs, and spleen (p < 0.05). In summary, glycine nano-selenium could enhance the efficacy of avian influenza vaccine.