共表达H5、H7亚型AIV HA基因与鸡IL-18基因的重组鸡痘病毒构建

以鸡痘病毒(FPV)疫苗株为载体,将H5和H7亚型禽流感病毒血凝素(AIV HA)基因串联后(拥有同一个阅读框)和鸡白细胞介素-18(IL-18)基因分别插入到鸡痘病毒表达载体pUTA-16-LacZ复合启动子(ATI-P7.5×20)和单一启动子1(P7.5)下游,构建了携带AIV HA基因和鸡白细胞介素-18基因的重组鸡痘病毒转移载体质粒pUTAL-H5-H7-IL18;用相同的方法构建重组鸡痘病毒转移载体质粒pUTAL-H5-IL18;将H5亚型AIV HA基因插入到鸡痘病毒表达载体pUTA2复合启动子(ATI-P7.5×20)下游,构建了携带H5亚型AIV HA基因的重组鸡痘病毒转移载体质粒pUTA2-H5.应用脂质体转染法,将重组鸡痘病毒转移载体质粒与282E4株鸡痘病毒共转染鸡胚成纤维细胞(CEF),经BrdU进行三次加压蚀斑筛选后,以不同代次的细胞mRNA为模板,利用H5亚型AIV HA基因、H7亚型AIV HA基因和鸡IL-18基因特异引物进行RT-PCR和蛋白印迹检测,筛选出H5HA-H7HA融合蛋白基因和鸡IL-18基因共表达的重组鸡痘病毒rFPV-H5HA-H7HA-IL18,H5亚型AIV HA基因和鸡IL-18基因共表达的重组鸡痘病毒rFPV-H5HA-IL18以及单独表达H5亚型AIV HA基因的重组鸡痘病毒rFPV-H5HA.这些重组鸡痘病毒的构建为AIV活载体疫苗的研制奠定了基础.     

Taqman MGB探针快速定量检测VHSV方法的研究

为建立准确实时地定量检测病毒性出血性败血症病毒(VHSV),在VHSV-N基因保守区设计了Taqman MGB探针与引物对,随后,采用体外转录技术获得了VHSV-N基因RNA,并以此为绝对定量标准品,建立了绝对定量(AQ)检测VHSV的实时荧光RT-PCR法(AQ-RT-PCR方法),并与世界动物卫生组织(OIE)推荐的普通RT-PCR法进行了比较。此荧光RT-PCR法特异性好,与其他鱼类弹状病毒无交叉反应。检测线性范围为10~(10)～10~2拷贝/反应,灵法度达10~2拷贝/反应。此检测灵敏度比OIE推举的RT-PCR法高出5个数量级,比嵌套RT-PCR高出1个数量级。此法是出入境检疫VHSV的有效方法。     

食品中亚利桑那菌环介导等温扩增检测方法的建立

应用环介导等温扩增(loop mediated isothermal amplification,LAMP)技术,研究建立一种快速、敏感、特异的检测亚利桑那菌(Salmonella arizonae)方法,为食品安全检测工作中亚利桑那菌检测提供一种快速准确工具。针对亚利桑那菌spv A基因中39个保守且特异的碱基序列,设计LAMP特异性引物,利用实时浊度仪对引物及反应条件进行优化,并对检测方法的灵敏度和特异性进行验证。该方法在63℃恒温下作用40 min,亚利桑那菌DNA获得高效率的特异性扩增;其检出限量为培养菌液浓度10~6CFU/m L,敏感性高;通过对25份沙门氏菌阳性食品的LAMP检测,检出2例亚利桑那菌。该文所建立的亚利桑那菌环介导等温扩增LAMP检测方法具有特异、灵敏、简便快捷等特点,适用于食品安全检测中亚利桑那菌的快速筛检。     

H7N9禽流感病毒检测取得重大突破

本刊讯近日,由北京市检验检疫局承担的国家质检总局科技计划项目"禽流感病毒H7N9亚型双重荧光RT-PCR定型技术研究"顺利通过专家鉴定。鉴定会上,专家组一致认为:该项目创新性地实现了对禽流感病毒H7N9亚型的一步快速定型,可在一次检测中确认样品中是否存在H7N9亚型禽流感病毒或其他H7亚型和N9亚型的禽流感病毒;在通用性、特异性、灵敏性上,技术优势突出。     

LAMP实时浊度法快速检测大肠杆菌方法的建立与优化研究

根据大肠杆菌的特异性基因（malB）,设计与筛查环介导等温扩增（LAMP）的引物,通过对甜菜碱用量、环引物的浓度、反应温度、dNTPs用量等反应条件的优化,得到较佳的LAMP反应体系,建立了LAMP实时浊度法快速检测大肠杆菌;在较优条件下,先后对9株非大肠杆菌菌株进行特异性检测和5株大肠杆菌的进行同源性检测。此外,结合课题组自行研制的多通道浊度仪,对LAMP扩增产物进行实时浊度监测。结果表明：LAMP实时浊度法检测大肠杆菌的灵敏度达16.010 ng/L,与实时荧光定量PCR的检测限相当。因此,建立与优化LAMP实时浊度法可为食品中大肠杆菌的现场快速检测提供了有力手段。     

小麦印度腥黑穗病菌的分子检测

根据GenBank中登录Tilletia属的20个种的核糖体转录间隔区(ITS)序列差异设计1对引物T1／T2，可以特异地从T.indica和T.wallceri菌株中扩增到一条450bp左右的条带，而从其他供试菌株中不能扩增出条带，表明该对引物可以将这二个种与Tilletia其他种区分开。进一步根据T．indica和T．walked的线粒体DNA序列的差异设计一对引物M1／M2，只能特异地从T．indica DNA中扩增到一条250bp左右的条带，表明该对引物可以将T．indica与T．walked区分开。利用上述2对引物分别与ITS区通用引物ITS1/ITS4．和线粒体引物Ti-1／Ti4组合建立套式PCR检测体系，能够直接将T．indica与其他相似或相关种区分开，且灵敏度可达1个冬孢子。此外，还观察到在反应体系中加入适量的(NH4)2SO4对PCR反应具有增效作用，可以提高检测灵敏度。上述结果提示建立的套式PCR反应体系可望能应用于口岸对T．indica的检疫。     

金华火腿中金黄色葡萄球菌的聚合酶链式反应检测

建立了一种用于金华火腿中金黄色葡萄球菌的聚合酶链式反应(PCR)检测方法.针对金黄色葡萄球菌独有的肠毒素基因(sea)设计了一对特异引物,在PCR体系中对相应片断进行扩增,扩增产物通过电泳技术与阳性对照进行对比来判断阴阳性.结果表明,该方法检出率高,样品中模板DNA含量仅有0.05 pg即可检出金黄色葡萄球菌,24 h即可报告结果.因此,PCR方法可以作为一种高效、敏感、特异性高的检测技术,用于金华火腿中金黄色葡萄球菌的快速检测.     

利用环介导等温扩增技术快速检测沙眼衣原体方法的建立

建立一种利用环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)快速检测沙眼衣原体的方法。针对沙眼衣原体gyr A基因设计LAMP特异引物,并加入一条环引物LB提高反应效率,对反应的特异性、灵敏度和重复性进行评价,同时用沙眼衣原体阳性的临床样本进行LAMP检测验证。实时荧光LAMP结果表明,建立的LAMP方法特异性好,不与10种常见的病原微生物以及生殖道感染相关细菌发生非特异反应,灵敏度可以达到4.13×101 copies/μL;20次重复试验的Cq值变异系数为7.23%,反应稳定性良好;用LAMP方法对6份临床阳性样本进行检测,阳性符合率为100%。因此,该研究建立的沙眼衣原体LAMP检测方法快速、灵敏、稳定性好,能为感染筛查和早期诊断提供参考。     

H7N9禽流感病毒检测质粒DNA标准物质研制成功

正2016年7月,上海市计量测试技术研究院承担的国家质检总局项目《H7N9禽流感病毒检测质粒DNA标准物质的研制》顺利通过专家组验收。项目组以H7N9禽流感病毒为目标,研发了一种含有H7基因、N9基因、M基因及Rnase P基因的质粒DNA标准物质,可适用于H7N9禽流感病毒实时荧光RT-PCR检测方法的量值溯源。该标准物质由九     

PIK3CA基因突变数字PCR参考测量方法研究

PIK3CA突变检测是实现肿瘤个体化治疗的重要预测因子。以PIK3CA为目的基因，选择E542K、E545K、H1047R三个热点突变，建立对其准确定量检测的微滴式数字聚合酶链式反应(ddPCR)方法。优化退火温度、引物探针浓度等条件，对方法特异性、线性范围、重复性等方面进行考察。结果表明，建立的ddPCR检测方法精密度好，在突变丰度(0.05～82.75)%范围内相对标准偏差值介于(0.392～14.031)%,准确性高，与重量法的相关系数达到99.91%、99.98%、99.94%;在突变丰度0.2%以上，方法的重复性可达5%以内；在突变丰度0.2%以下，重复性保持在15%以下。在20μL反应体系中3个热点突变的空白限分别为0.03%、 0.04%、0.04%,检测下限和定量下限均为0.05%。因此，所建立的PIK3CA基因E542K、E545K及H1047R位点突变的ddPCR参考测量灵敏度高，重复性好，在癌症诊断、个体化用药指导和预后方面具有良好的应用。     

Robust and highly sensitive fluorescence approach for point-of-care virus detection based on immunomagnetic separation

In this work, robust approach for a highly sensitive point-of-care virus detection was established based on immunomagnetic nanobeads and fluorescent quantum dots (QDs). Taking advantage of immunomagnetic nanobeads functionalized with the monoclonal antibody (mAb) to the surface protein hemagglutinin (HA) of avian influenza virus (AIV) H9N2 subtype, H9N2 viruses were efficiently captured through antibody affinity binding, without pretreatment of samples. The capture kinetics could be fitted well with a first-order bimolecular reaction with a high capturing rate constant k(f) of 4.25 × 10(9) (mol/L)(-1) s(-1), which suggested that the viruses could be quickly captured by the well-dispersed and comparable-size immunomagnetic nanobeads. In order to improve the sensitivity, high-luminance QDs conjugated with streptavidin (QDs-SA) were introduced to this assay through the high affinity biotin-streptavidin system by using the biotinylated mAb in an immuno sandwich mode. We ensured the selective binding of QDs-SA to the available biotin-sites on biotinylated mAb and optimized the conditions to reduce the nonspecific adsorption of QDs-SA to get a limit of detection low up to 60 copies of viruses in 200 μL. This approach is robust for application at the point-of-care due to its very good specificity, precision, and reproducibility with an intra-assay variability of 1.35% and an interassay variability of 3.0%, as well as its high selectivity also demonstrated by analysis of synthetic biological samples with mashed tissues and feces. Moreover, this method has been validated through a double-blind trial with 30 throat swab samples with a coincidence of 96.7% with the expected results.     

Exploring Sialic Acid Receptors‐Related Infection Behavior of Avian Influenza Virus in Human Bronchial Epithelial Cells by Single‐Particle Tracking

Human respiratory tract epithelial cells are the portals of human infection with influenza viruses. However, the infection pathway of individual avian influenza viruses in human respiratory cells remains poorly reported so far. The single‐particle tracking technique (SPT) is a powerful tool for studying the transport mechanism of biomolecules in live cells. In this work, we use quantum dots to label avian influenza H9N2 virus and elaborate on the infection mechanism of the virus in human bronchial epithelial (HBE) cells using a three‐dimensional SPT technique. We have found that the H9N2 virus can infect HBE cells directly and the virus infection follows an actin filament‐ and microtubule‐dependent process with a three‐stage pattern. The transport behaviors show a high degree of consistency between the sialic acid receptors and the influenza virus. Real‐time SPT provides dynamic evidence of the sialic acid receptors‐related infection behavior of the avian influenza virus in live cells. The study of the influence of sialic acid receptors on virus infection may contribute to a better understanding of the cross‐species transmission of the avian influenza virus.     

Optimizing the early detection of low pathogenic avian influenza H7N9 virus in live bird markets

In Southeast Asia, surveillance at live bird markets (LBMs) has been identified as crucial for detecting avian influenza viruses (AIV) and reducing the risk of human infections. However, the design of effective surveillance systems in LBMs remains complex given the rapid turn-over of poultry. We developed a deterministic transmission model to provide guidance for optimizing AIV surveillance efforts. The model was calibrated to fit one of the largest LBMs in northern Vietnam at high risk of low pathogenic H7N9 virus introduction from China to identify the surveillance strategy that optimizes H7N9 detection. Results show that (i) using a portable diagnostic device would slightly reduce the number of infected birds leaving the LBM before the first detection, as compared to a laboratory-based diagnostic strategy, (ii) H7N9 detection could become more timely by sampling birds staying overnight, just before new susceptible birds are introduced at the beginning of a working day, and (iii) banning birds staying overnight would represent an effective intervention to reduce the risk of H7N9 spread but would decrease the likelihood of virus detection if introduced. These strategies should receive high priority in Vietnam and other Asian countries at risk of H7N9 introduction.     

Quantification of immunoreactive viral influenza proteins by immunoaffinity capture and isotope-dilution liquid chromatography-tandem mass spectrometry

An immunocapture isotope dilution mass spectrometry (IC-IDMS) method was developed to quantify antibody-bound influenza hemagglutinins (HA) in trivalent influenza vaccines (TIV). Currently, regulatory potency requirements for TIV require HA quantification based on the single radial immunodiffusion (SRID) assay, which is time-consuming, laborious, and requires production of large quantities of reagents globally. In IC-IDMS, antiserum to the HA of interest captured viral proteins that were in the correct conformation to be recognized by the antibodies. The captured proteins were digested, and evolutionarily conserved tryptic peptides were quantified using isotope-dilution liquid chromatography-tandem mass spectrometry. IC-IDMS relies on antibody-antigen binding similar to SRID but incorporates the accuracy and precision of IDMS. Polyclonal antibodies (pAb-H3) prepared by injection of sheep with purified H3 HA captured 82.9% (55.26 fmol/μL) of the total H3 HA (66.69 fmol/μL) from the commercial TIV and 93.6% (57.23 fmol/μL) of the total H3 HA (61.14 fmol/μL) in purified virus. While other HA (H1, B), neuraminidase (N1, N2, NB), viral matrix proteins, and nucleoproteins were also captured by this antiserum, our results were not affected due to the specificity of the mass spectrometer. IC-IDMS is an accurate, precise, sensitive, and selective method to measure antibody-bound HA in purified virus and commercial vaccines.     

A Nanostructured Microfluidic Immunoassay Platform for Highly Sensitive Infectious Pathogen Detection

Rapid and simultaneous detection of multiple potential pathogens by portable devices can facilitate early diagnosis of infectious diseases, and allow for rapid and effective implementation of disease prevention and treatment measures. The development of a ZnO nanorod integrated microdevice as a multiplex immunofluorescence platform for highly sensitive and selective detection of avian influenza virus (AIV) is described. The 3D morphology and unique optical property of the ZnO nanorods boost the detection limit of the H5N2 AIV to as low as 3.6 × 10³ EID₅₀ mL^?1 (EID₅₀: 50% embryo infectious dose), which is ≈22 times more sensitive than conventional enzyme‐linked immunosorbent assay. The entire virus capture and detection process could be completed within 1.5 h with excellent selectivity. Moreover, this microfluidic biosensor is capable of detecting multiple viruses simultaneously by spatial encoding of capture antibodies. One prominent feature of the device is that the captured H5N2 AIV can be released by simply dissolving ZnO nanorods under slightly acidic environment for subsequent off‐chip analyses. As a whole, this platform provides a powerful tool for rapid detection of multiple pathogens, which may extent to the other fields for low‐cost and convenient biomarker detection.     

番杏Na+/H+逆向转运蛋白基因的克隆及特性分析

根据同源序列设计筒并引物,通过RT—PCR及RACE的方法从盐生植物番杏中克隆出Na^ ／H^ 逆向运输蛋白基因。序列分析表明,该cDNA全长2199bp,开放读码框为1665bp,可编码一个554个氨基酸的多肽,与其他植物中已经克隆的Na^ ／H^ 逆向转运蛋白具有很高的同源性。系统发育分析表明该蛋白(TtNHX1)与液泡型的Na^ ／H^ 逆向转运蛋白的亲缘较近,与质膜型的Na^ ／H^ 逆向转运蛋白亲缘关系较远。TtNHX1的表达具有组织特异性,在番杏的根、茎和叶中均有表达,而花中没有表达。经NaCl诱导后根、茎和叶中的表达量增加,而花中仍没有表达。     

纳米Cu2+/TiO2抗菌膜对禽流感病毒(H9N2)的灭活效应

利用病毒滴度测定和MDCK细胞电镜观察法,初步探讨了在365nm的黑光灯(UV)照射下,纳米Cu2+/TiO2抗菌膜对禽流感病毒(H9N2)的光催化灭活效应,并分别考察了UV强度、UV照射时间以及H9N2病毒量对H9N2病毒光催化灭活效应的影响.实验结果表明,在365nm黑光灯的照射下,纳米Cu2+/TiO2抗茵膜对H9N2病毒具有显著的灭活效应,在UV强度为0.5mW/cm2、UV照射时间为2.5h、病毒量为0.1ml时,H9N2病毒的灭活率达到了100%.研究结果表明,纳米Cu2+/TiO2抗菌膜在抑制禽流感(H9N2)病毒在环境媒介中的扩散与传播方面有潜在的应用价值.     

长爪沙鼠Tim-1基因部分序列克隆及表达分析

根据美国国立生物技术信息中心(NCBI)中基因库(GenBank)里查询到已登录的小鼠、大鼠的Tim-1mRNA序列,通过同源比较,设计引物,首次对长爪沙鼠Tim-1基因部分编码序列进行分子克隆.经过PCR扩增,获得长爪沙鼠Tim-1基因243bp部分编码序列,经测序后登录GenBank(JN628997),发现该序列与小鼠、大鼠Tim-1相应部分有80%以上的相似性.利用所克隆的序列设计引物,建立荧光定量PCR方法检测长爪沙鼠Tim-1基因在不同组织中表达情况.结果显示,长爪沙鼠Tim-1基因在不同组织中表达差异性较大,其中在肾脏组织中表达水平高于其他组织,在肌肉、心脏、小肠、脾脏、肝脏、肺组织中表达均较低.     

Identification of A/H5N1 influenza viruses using a single gene diagnostic microarray

In previous work, a simple diagnostic DNA microarray that targeted only the matrix gene segment of influenza A (MChip) was developed and evaluated with patient samples. In this work, the analytical utility of the MChip for detection and subtyping of an emerging virus was evaluated with a diverse set of A/H5N1 influenza viruses. A total of 43 different highly pathogenic A/H5N1 viral isolates that were collected from diverse geographic locations, including Vietnam, Nigeria, Indonesia, and Kazakhstan, representing human, feline, and a variety of avian infections spanning the time period 2003-2006 were used in this study. A probabilistic artificial neural network was developed for automated microarray image interpretation through pattern recognition. The microarray assay and subsequent subtype assignment by the artificial neural network resulted in correct identification of 24 "unknown" A/H5N1 positive samples with no false positives. Analysis of a data set composed of A/H5N1, A/H3N2, and A/H1N1 positive samples and negative controls resulted in a clinical sensitivity of 97% and a clinical specificity of 100%.     

Conjugation of membrane-destabilizing peptide onto gelatin–siloxane nanoparticles for efficient gene expression

One of the crucial steps in gene delivery with non-viral vectors is the escape of DNA complexes from the endosome. In order to improve gene transfection efficiency, we designed a novel gene delivery vector gelatin–siloxane nanoparticles (GS NPs) conjugated with two different membrane-destabilizing peptides, octaarginine (R8) and a subunit of influenza virus haemagglutinin HA2. Both R8-GS NPs and HA2-GS NPs had high positive surface charges. They could condense and protect DNA against serum/DNase degradation. Results from flow cytometry and confocal laser scanning microscope respectively indicated that R8-GS NPs had higher uptake efficiency than HA2-GS NPs, whereas HA2-GS had higher endosome escaping efficiency. Furthermore, in vitro transfection displayed a higher gene expression level with HA2-modified GS NPs, which suggested that endosome escaping is the crucial step for nanoparticle mediated gene therapy.