家禽禽流感病毒AGID抗体定性检测及H5、H9亚型HI抗体定量检测能力验证结果分析

通过对全国多个行业的24个实验室禽流感血清学检测技术的能力验证考核，全面了解和掌握这些实验室这方面的技术水平，为实验室认可、国际互认检测结果和领导决策提供了有力的技术数据。     

我国率先攻克并掌握检测食品中是否存在SARS病毒的检测技术

5月8日,由国家质检总局中国进出口商品检验技术研究所自主设计开发并自行实验完成的检测食品、动植物及其产品中是否存在SARS病毒的检测方法在京通过专家鉴定,这在国际上尚属首次,填补了国内外这一领域的空白。该课题是国家质检总局从应对国外技术壁垒提出的,课题组以高度的政治责任感和使命感,夜以继日,连续奋战,仅用了18天时间就完成了课题的研究。该方法是在人工控制的情况下利用病毒分离技术,将人为添加在食品样品中的SARS病毒有效富集、分离,采用RT-PCR(逆转录聚合酶链式反应)方法快速检测食品中的SARS病毒。该方法不仅可用于检…     

反转录病毒介导的HSV-tk/ACV系统基因治疗的安全性研究

研究了反转录病毒介导的ＨＳＶ－ｔｋ／ＡＣＶ系统基因治疗脑肿瘤的安全性。     

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.     

食品中金黄色葡萄球菌PCR检测方法的建立

目的:建立食品中金黄色葡萄球菌的PCR检测方法。方法:采用PCR技术特异性扩增金黄色葡萄球菌的耐热核酸酶编码基因(nuc基因),检测模拟样品中金黄色葡萄球菌。结果:所建立的模拟食品中金黄色葡萄球菌的PCR检测方法,特异性良好,敏感性可达61.76pg/μl。结论:该方法简便、快捷、准确,为食源性金黄色葡萄球菌的检验提供了快速检测的手段,并为今后相关技术的研究积累了资料。     

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。     

基于HCR放大的无标记型荧光传感器的构建及H5N1 DNA检测

对于高致病性H5N1禽流感病毒,构建检测该病毒的高灵敏生物传感器,并与智能包装相结合用于实时监测,这对禽流感的防控具有重要意义。基于杂交链式反应（HCR）信号放大策略,以AgNCs作为荧光信号基团,构建了一种无标记“turn on”型荧光生物传感器用于检测代表H5N1病毒的H5N1基因序列。该传感器以H5N1 DNA作为触发剂引发HCR过程,使AgNCs产生强的荧光信号变化。研究表明,当H5N1 DNA浓度在0.2~800.0 nmol/L内,该传感器具有良好的响应信号,且在0.2~200.0 nmol/L之间的荧光强度与H5N1 DNA浓度呈线性相关,线性方程为y=10.982C+567.435（R2=0.992 73）,检测限为176 pmol/L。核酸传感体系具有通用性,通过简单调整目标序列,可实现对不同目标物的特异性灵敏检测。该研究有望为高灵敏分析禽流感病毒标志物的通用传感平台设计提供思路。     

弓形虫H11基因克隆表达及其临床应用研究

提取弓形虫速殖子总RNA,RT-PCR扩增H11基因片段并构建重组表达载体,诱导GST-H11融合蛋白大量表达,并经亲和层析柱纯化,通过免疫印迹和酶联免疫吸附两种方法检测弓形虫特异性兔抗血清和人阳性血清,结果显示GST-H11融合蛋白检测两者血清敏感性和特异性均较高,提示H11融合蛋白可望用作诊断性抗原应用于弓形虫患者临床检测。     

“新城疫、禽流感病毒快速检测技术研究”取得重大突破

１２月１９日，被国家科教领导小组批准列为“十五＂国家重大科技专项的“新城疫、禽流感病毒快速检测技术研究”，通过了国家质检总局组织的专家鉴定。这个项目是由国家科技部立项，国家质检总局负责组织管理，北京出入境检验检疫局和深圳匹基生物工程公司的科研人员共同研究完成的。新城疫（Newcastledisease，ND）是由新城疫病毒（NDV）引起的一种急性、败血性、高度接触性传染病，传播迅速、发病率和死亡率高，有时高达１００％，曾给养鸡业造成了不可估量的经济损失。禽流感是由A型流感病毒引起的一种禽类的感染和／或疾病综合症。…     

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。     

Upconversion Luminescence Resonance Energy Transfer (LRET)‐Based Biosensor for Rapid and Ultrasensitive Detection of Avian Influenza Virus H7 Subtype

Avian influenza viruses (AIV) with good adaptation and various mutations have threatened both human and animals’ health. The H7 subtypes have the potential to cause pandemic threats to human health due to the highly pathogenic characteristics. Therefore, it is quite urgent to develop a novel biosensor for rapid and sensitive detection of H7 subtypes. In this work, a biosensor based on luminescence resonance energy transfer (LRET) from BaGdF₅:Yb/Er upconversion nanoparticles (UCNPs) to gold nanoparticles (AuNPs) has been developed for rapid and sensitive H7 subtypes detection. The amino modified capture oligonucleotide probes are covalently linked to poly(ethylenimine) (PEI) modified BaGdF₅:Yb/Er UCNPs. The thiol modified oligonucleotides with H7 hemagglutinin gene sequence are conjugated to surfaces of AuNPs. The hybridization process between complementary strands of H7 Hemagglutinin gene and its probe brings the energy donor and acceptor into close proximity, leading to the quenching of fluorescence of UCNPs. A linear response is obtained ranging from 10 pm to 10 nm and the limit of detection (LOD) is around 7 pm with detection time around 2 hours. This biosensor is expected to be a valuable diagnostic tool for rapid and sensitive detection of AIV.     

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.     

Composites by rapid prototyping technology

The use of rapid prototyping (RP) technology for rapid tooling and rapid manufacturing has given rise to the development of application-oriented composites. The present paper furnishes succinct notes of the composites formed using main rapid prototyping processes such as Selective Laser Sintering/Melting, Laser Engineered Net Shaping, Laminated Object Manufacturing, Stereolithography, Fused Deposition Modeling, Three Dimensional Printing and Ultrasonic Consolidation. The emphasis of the present work is on the methodology of composite formation and the reporting of various materials used.     

Detection of mortality clusters associated with highly pathogenic avian influenza in poultry: a theoretical analysis

Rapid detection of infectious disease outbreaks is often crucial for their effective control. One example is highly pathogenic avian influenza (HPAI) such as H5N1 in commercial poultry flocks. There are no quantitative data, however, on how quickly the effects of HPAI infection in poultry flocks can be detected. Here, we study, using an individual-based mathematical model, time to detection in chicken flocks. Detection is triggered when mortality, food or water intake or egg production in layers pass recommended thresholds suggested from the experience of past HPAI outbreaks. We suggest a new threshold for caged flocks—the cage mortality detection threshold—as a more sensitive threshold than current ones. Time to detection is shown to depend nonlinearly on R₀ and is particularly sensitive for R₀<10. It also depends logarithmically on flock size and number of birds per cage. We also examine how many false alarms occur in uninfected flocks when we vary detection thresholds owing to background mortality. The false alarm rate is shown to be sensitive to detection thresholds, dependent on flock size and background mortality and independent of the length of the production cycle. We suggest that current detection thresholds appear sufficient to rapidly detect the effects of a high R₀ HPAI strain such as H7N7 over a wide range of flock sizes. Time to detection of the effects of a low R₀ HPAI strain such as H5N1 can be significantly improved, particularly for large flocks, by lowering detection thresholds, and this can be accomplished without causing excessive false alarms in uninfected flocks. The results are discussed in terms of optimizing the design of disease surveillance programmes in general.     

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.     

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.     

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.