珠海检验检疫局成功研发H7N9亚型禽流感病毒双重荧光RT-PCR检测试剂盒

不久前,珠海检验检疫局成功研发了H7N9亚型禽流感病毒双重荧光RT-PCR 检测试剂盒,并应用于活禽的H7N9禽流感病毒检测和监控,取得了良好的防控效果。该H7N9禽流感诊断试剂盒可为H7N9禽流感病毒监测和疫情排查提供快速、准确的病原学检测方法,为加强供港澳活禽检验检疫,做好供港澳家禽注册养殖场H7N9禽流感防控工作提供技术支持。     

基于荧光染料和氧化石墨烯同步荧光法同时检测多种流感病毒亚型

分别采用三种不同发射波长的荧光染料,通过共价偶联的方式分别标记三种流感病毒亚型H1N1、H5N1和H9N2的抗体,再利用氧化石墨烯猝灭所标记染料的荧光。当将荧光标记抗体和氧化石墨烯一并加入到流感病毒溶液中时,由于抗原和抗体之间的特异性相互作用,病毒会和抗体作用而使得氧化石墨烯远离荧光染料,染料的荧光得以恢复。通过恢复的荧光发射波长位置和荧光强度,可以定性和定量检测三种不同的流感病毒亚型。在最佳实验条件下,对三种流感病毒亚型H1N1、H5N1和H9N2进行同时检测,H1N1的检出限为0.48ng/mL,线性范围为1~18ng/mL;H5N1的检出限为0.46ng/mL,线性范围为1~18.5ng/mL;H9N2的检出限为0.42ng/mL,线性范围为1~16ng/mL。该方法具有较好的稳定性、重现性和灵敏度,可实现多个流感病毒亚型的分型和同时检测。     

指数线性聚合酶链式反应法制备焦磷酸测序反应的单链模板

建立了一种简单的焦磷酸测序用单链模板制备方法,以包含SNP6位点一段78bp序列为对象,采用非热启动Taq酶进行指数线性PCR扩增,通过加入甘油、BSA等PCR增强剂增加反应的效率和特异性,设计反应液A和B处理PCR产物中干扰焦测序的限制性引物、未完全反应的产物、焦磷酸和dNTPs等杂质, 处理后1～2 μL PCR产物就可直接用于焦测序检测.测定了BRCA1基因中5个乳腺癌相关的SNP位点,获得的图谱无非特异性信号,测得序列与参考序列一致,能够进行SNP分析,表明本方法可以制备高质量焦测序单链模板,且使焦测序的成本显著降低,操作更为简便,减少了操作过程中样本间的交叉污染,有利于焦测序样品预处理的自动化.     

检测禽流感H5亚型病毒的阻抗型免疫研究

研制了一种可用于H5亚型禽流感病毒快速检测的阻抗型免疫传感器。通过蛋白A将H5N1表面抗原血凝素(HA)的单克隆抗体固定于金叉指阵列微电极表面,并与待测溶液中的目标抗原H5N1进行免疫反应。在[Fe(CN)6]3"/4"溶液中进行电化学阻抗谱扫描,表征电极的表面修饰及抗原捕获过程。当H5N1病毒浓度在21～26 HA unit/50μL范围时,其浓度的对数值与叉指阵列微电极的电子传递阻抗的变化值呈线性关系,相关系数为0.9885;检出限为20 HA unit/50μL,检测时间为1 h。此传感器特异性好,灵敏度高,可以重复使用,在病原微生物快速检测领域具有良好的应用前景。     

荧光探针Ru(phen)2(dppx)2+测定H1N1禽流感病毒DNA

利用荧光探针Ru(phen)2dppx2+与ssDNA作用时不产生荧光或荧光很弱,而与dsDNA作用时荧光增强的机理,将H1N1禽流感病毒ssDNA与其完全互补ssDNA杂交形成dsDNA实现Ru(phen)2dppx2+对H1N1禽流感病毒DNA特定序列（5’-CTA CCA TGC GAA CAA TTC AAC CGA CAC TGT T-3’）的定量检测。在优化的实验条件下,测定H1N1禽流感病毒 DNA的线性范围为9.3×10-10～7.4×10-8 mol/L,线性关系：y = 3.3829x + 8.3948,R2 =0.9982,检出限为5.3×10-10 mol/L。该方法具有操作简单,检测快速,灵敏度高和选择好等优点。     

免疫磁分离技术结合阻抗测量法快速检测禽流感病毒

采用免疫磁分离和阻抗测量技术联用的方法,实现了对禽流感H5亚型病毒的特异性快速检测。将偶联生物素的H5抗体固定于链霉亲和素修饰的纳米磁珠表面,制备成免疫磁珠,用于样品中禽流感H5N1病毒的分离和浓缩。使用金叉指阵列微电极测量样品阻抗,在特征频率(100 kHz)下,阻抗模值随样品中H5N1病毒浓度增加而增大。研究了抗体浓度、免疫反应时间和磁分离时间对阻抗信号的影响,结果表明,在优化的实验条件(抗体浓度0.25 g/L、免疫反应时间60 min、磁分离时间3 min)下,纯病毒样品和拭子样品中H5N1病毒浓度分别在2!1～24HA unit/50μL和20～24HA unit/50μL范围时,病毒浓度对数值与阻抗信号值呈线性相关关系,检出限分别为0.5 HA unit/50μL和2 HA unit/50μL。     

禽流感病毒HA蛋白与人受体的分子对接

血凝素(hemagglutinin,HA)是位于禽流感病毒表面的糖蛋白。在病毒感染过程中,HA与禽类宿主细胞表面受体结合,介导病毒膜与宿主核内体膜的融合,在传染过程中发挥关键作用。自然界中的禽流感病毒处于不断演化之中,其HA的禽受体结合位点常常发生氨基酸变异。因此,当HA变异体与人受体结合能力较强时,禽流感病毒往往会发生跨种传播而感染人。为预防禽流感的跨种传播,人们迫切需要发展大规模快速检测或预测HA变异体与人受体结合亲和力的方法,以评估各种新发禽流感病毒的跨种传播能力,提前筛选出有潜在危险的病毒株。针对此问题,本研究以H7N9亚型的HA蛋白H7为研究对象,发展了一种运用分子对接的计算方法,预测HA变异体与人受体的结合亲和力。该方法的计算结果表明,H7与人受体的结合亲和力普遍弱于有较强传染人能力的H1,说明H7N9亚型病毒的跨种传播能力普遍较弱;但是,计算分析也揭示,部分新发的H7N9毒株的HA有强的人受体结合亲和力,提示在自然演化过程中,H7N9病毒有可能演化出具有较强的感染人能力的新毒株,这与2013年禽流感疫情的实际发生情况相一致。因此,本文所发展的计算方法可用于快速预测新发禽流感病毒HA与人受体的结合亲和力,为新发禽流感病毒的跨种传播风险评估提供理论依据。     

喷射式流动注射电化学发光免疫检测禽流感H9亚型

利用磁分离和生物素亲和素技术,形成亲和素化磁微球-生物素化抗体-抗原-钌标抗体的免疫夹心复合物,初步建立了体外免疫诊断试剂的制备方法,并利用喷射式流动注射电化学发光体系对制备出的禽流感病毒H9免疫复合物进行检测。实验选择最适的包被抗体,检测抗体和封闭剂,优化Ru标抗体的最佳稀释度。在生物素化的兔抗H9多抗作为亲和素化的磁微球的结合抗体,鼠抗H9单抗作为Ru(bpy)32+标记抗体,2%BSA作为封闭剂,1:50倍稀释的Ru-鼠抗H9单抗条件下,非特异性吸附最低。测定不同浓度的H9抗原,发现抗原浓度在3.125～100μg/mL范围内与电化学发光强度呈较好的线性关系。实验还测定了不同亚型的禽流感病毒、不同来源的毒株和鸡的棉拭子样品。     

面向现场的活病毒高灵敏检测

禽流感病毒因为其高传染性,易致病性以及对人和禽类带来的危害,得到越来越多的关注.目前检测禽流感病毒方法主要有核酸检测,鸡胚病原分离等~([1]).     

澳研制出快速检测禽流感的新仪器

禽流感疫情仍在不断蔓延，最让人们担心的，就是如果有一天禽流感病毒在人与人之间传播，人类该如何应对。澳大利亚科学家们最近就发明了一种检测仪，它能快速判断被检测者是否感染了禽流感病毒。     

单管高灵敏度等温扩增技术快速检测甲型H1N1流感病毒

建立了单管逆转录环介导等温扩增法(RT-LAMP)快速检测甲型H1N1流感病毒的方法.针对甲型H1N1流感病毒的M基因和HA基因的保守区,设计了两组特异性引物,分别用于筛选甲型流感病毒及鉴定甲型H1N1流感病毒.对反应体系中的关键因素进行优化,反应结果可直接通过浊度或者SYBR Green Ⅰ荧光进行判定.本方法最低可...     

Electric detection of target DNA by fabricating gold nanowire bridges on planar nanogap electrodes

For the electrical detection of target DNA (partial avian influenza virus/H1N1/HA sequence) prepared via asymmetric PCR, we fabricated DNA-templated conducting gold nanowire bridges on planar nanogap electrodes using positively charged gold nanoparticles.     

Binding interaction analysis of the active site and its inhibitors for neuraminidase (N1 subtype) of human influenza virus by the integration of molecular docking, FMO calculation and 3D-QSAR CoMFA modeling

Recently, the worldwide spread of A/H5N1 avian influenza with high virulence has highlighted the potential threat of human influenza pandemic. Tamiflu and Relenza are currently the only two anti-influenza drugs targeting the neuraminidase (NA) enzyme of human influenza virus. Reports of the emergence of drug resistance further make the development of new potent anti-influenza inhibitors a priority. The X-ray crystallographic study of A/H5N1 avian influenza NA subtypes (Russell, R. J. Nature 2006, 443, 45-49) has demonstrated that there exist two genetically distinct groups, group-1 (N1, N4, N5 and N8) and group-2 (N2, N3, N6, N7 and N9), whose conformations are substantially different. The detailed comparison of their active sites has established, heretofore, the most accurate and solid molecular basis of structure and mechanism for the development of new anti-influenza drugs. In the present study, a three-dimensional structure of N1 subtype of human influenza type A virus (N1hA) has been generated by homology modeling using the X-ray crystallographic structure of N1 subtype of avian influenza virus (N1aA) as the template. Binding interaction analysis between the active site and its inhibitors has been performed by combining ab initio fragment molecular orbital (FMO) calculations and three-dimensional quantitative structure-activity relationship with comparative molecular field analysis (3D-QSAR CoMFA) modeling. Integrated with docking-based 3D-QSAR CoMFA modeling, molecular surface property (electrostatic and steric) mapping and FMO pair interaction analysis, a set of new receptor-ligand binding models and bioaffinity predictive models for rational design and virtual screening of more potent inhibitors of N1hA are established. In addition, the flexibility of the loop-150 of N1hA and N1aA has been examined by a series of molecular dynamics simulations.     

A new method for analyzing H5N1 avian influenza virus

In this paper a novel method for phylogenetic analysis of H5N1 avian influenza virus has been proposed. At first we provide a mapping of virus protein sequence. Based on this mapping, we propose a new distance measure and make use of the corresponding similarity matrix to construct phylogenic tree without requiring multiple alignment. As an application, we construct phylogenic tree for 123 species of H5N1 avian influenza virus. The phylogeny obtained is generally consistent with evolutionary trees constructed in previous studies.     

Novel algorithm for phylogenetic analysis of proteins: application to analysis of the evolution of H5N1 influenza viruses

The highly pathogenic avian influenza virus (HPAIV) A subtype H5N1 is causing threat to human health over the years. Phylogenetic analysis is an important tool for analyzing the evolution of influenza. A novel phylogenetic algorithm based on a new protein distance measure derived from the informational spectrum method (ISM) has been presented. The new phylogenetic approach allows assessment of functional evolution of protein sequences. The new ISM-based phylogenetic approach has been found to overcome some drawbacks of other phylogenetic approaches, particularly concerning sensitivity to a single mutation, deletion and the position of the mutation. The ISM-based approach applied to hemagglutinin subunit 1 protein (HA1) of HPAIV A subtype H5N1 viruses in Egypt between 2006 and 2011, revealed clear clustering in two groups, with one growing group of H5N1 viruses after 2009 with increased number of human infections with H5N1. Four group-specific mutations are identified which are important for increased human tropism and the pandemic potential.     

A new approach to molecular phylogeny of H5N1 avian influenza viruses in Asia

In this article, we introduce a new method to analysis avian influenza virus (AIV) of subtype H5N1 and study the similarity of these sequences. We make a comparison for some nucleic acid sequences of H5N1 AIV in Asia by using the 2D and 3D graphic representation. Comparing these sequences, we structured a phylogenetic tree and discussed the evolutional relationship among these viruses. The sequences analysis shows that there are some obvious traits depending on different areas, periods, and hosts. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010