实时荧光PCR技术快速鉴别检测H5、H9、H7亚型禽流感灭活疫苗的研究

选取H5、H9、H7亚型禽流感病毒（avian influenza virus, AIV）血凝素( hemagglutinin, HA) 基因保守序列,利用Primer Express 2.0软件设计了各自亚型的特异性引物和Taqman MGB探针,利用实时荧光RT-PCR一步法建立了H5、H9、H7亚型禽流感灭活疫苗的鉴别检测方法,该方法特异性好,重复性佳,对其他疫苗无交叉反应。结果表明实时荧光PCR方法为禽流感灭活疫苗提供了一种特异、敏感、快速和简洁的鉴别检测方法。     

实时荧光PCR技术快速鉴别检测H5、H9、H7亚型禽流感灭活疫苗的研究

选取H5、H9、H7亚型禽流感病毒(avian influenza virus,AIV)血凝素(hemagglutinin,HA)基因保守序列,利用PrimerExpress2.0软件设计了各自亚型的特异性引物和Taqman MGB探针,利用实时荧光RT-PCR一步法建立了H5、H9、H7亚型禽流感灭活疫苗的鉴别检测方法,该方法特异性好,重复性佳,对其他疫苗无交叉反应。结果表明实时荧光PCR方法为禽流感灭活疫苗提供了一种特异、敏感、快速和简洁的鉴别检测方法。     

TaqMan—MGB荧光定量RT-PCR技术快速检测H5亚型禽流感病毒

建立以TaqMan-MGB荧光探针为特点的荧光定量RT-PCR方法,用于检测H5亚型禽流感病毒。针对H5亚型禽流感病毒血凝素(HA)基因保守区域设计特异性引物与TaqMan-MGB荧光探针,筛选并优化荧光定量RT-PCR反应体系与反应条件,用以提高方法的特异性、敏感性与准确性;并通过体外克隆技术建立病毒基因拷贝数进行定量分析。结果表明:引物与探针的优化浓度分别640nmol/L和480nmol/L,体系具有良好的保守性和特异性,与其他呼吸道病毒均无交叉反应。方法检测灵敏度为100拷贝/反应,标准曲线线性范围为107～102拷贝/反应,从病毒核酸提取至检测完成仅需3h左右,操作简便,重现性好。本研究建立的TaqMan-MGB荧光定量PCR方法特异、敏感、快速,适合于临床实验室进行H5亚型禽流感病毒的快速定量检测。     

Detection of influenza viruses in throat swab by using polymerase chain reaction

An assay protocol based on exploiting the polymerase chain reaction (PCR) for the direct detection of influenza virus in throat swab is described. By use of the mixture of H1 and H3 primers, it was possible to determine the subtype of the influenza A viruses simultaneously. No visible band was detected after PCR of influenza B or A (H2N2) viruses with a pair of H1 or H3 primers. The dilution experiment showed that the influenza viruses, as few as 1.3-6 plaque-forming units, were sufficient for detecting the HA gene by PCR. All throat swab samples from which influenza viruses had been isolated by conventional method were also positively detected by PCR method.     

Rapid and sensitive detection of avian influenza virus subtype H7 using NASBA

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) is an isothermal technique allowing rapid amplification and detection of specific regions of nucleic acid from a diverse range of sources. It is especially suitable for amplifying RNA. A NASBA/ECL technique has been developed allowing the detection of RNA from avian influenza virus subtype H7 derived from allantoic fluid harvested from inoculated chick embryos and from cell cultures. Degenerate amplification primers and amplicon capture probes were designed enabling the detection of low and highly pathogenic avian influenza of the H7 subtype from the Eurasian and North American lineages and the Australian sub-lineage. The NASBA/ECL technique is specific for subtype H7 and does not cross-react with other influenza subtypes or with viruses containing haemagglutinin-like genes. The assay is 10- to 100-fold more sensitive than a commercially available antigen capture immunoassay system. The NASBA/ECL assay could be used in high throughput poultry screening programmes.     

H5N1禽流感病毒环介导等温扩增快速检测方法的建立

目的：建立H5N1禽流感病毒环介导等温扩增（LAMP）快速检测方法。方法：从GenBank中获得H5N1禽流感病毒血凝素（HA）基因序列,应用DNAStar软件MegAlign程序分析其序列,利用PrimerExplorerV3软件在序列保守区域设计LAMP引物,即外引物、内引物和环引物,同时以所克隆的阳性质粒为模板,对试验中的几个重要参数进行优化。结果：LAMP检测方法对H5N1禽流感病毒的灵敏度达到4～6个拷贝,其引物对于H1、H9亚型禽流感病毒和新城疫病毒无非特异性扩增,表现出良好的H5亚型特异性。结论：建立的H5N1禽流感病毒环介导等温扩增快速检测方法灵敏度高、特异性强、重复性好,为快速检测禽流感病毒提供了新方法和新思路。     

禽流感病毒分型基因芯片的研制

[目的]禽流感病毒是一种全球重要的人和动物呼吸道病病原,快速确定其不同亚型对于全球流感监测具有重要的意义.本研究意在研制一种可同时鉴定禽流感病毒所有亚型的方法.[方法]根据GenBank上已发表的禽流感病毒不同亚型(16个HA亚型和9个NA亚型)的基因序列,设计合成了25对特异性引物和1对通用引物,然后以各亚型病毒的参考株RNA作为模板,建立扩增不同亚型的多重RT-PCR方法.参考各亚型病毒靶cDNAs区域的保守序列设计了52条亚型特异的探针,进而利用扩增的各亚型病毒的靶cDNAs对其特异性进行评价.在此基础上,将设计好的探针点制到处理好的玻片上,制备了禽流感病毒分型鉴定基因芯片,结合所建立的扩增不同亚型的多重RT-PCR方法,开发了禽流感病毒亚型鉴定基因芯片试剂.利用收集自49个地区的2653份标本对其特异性和敏感性进行了初步评价.[结果]用于评价的各亚型参考毒株均出现良好的特异性杂交信号,检测的敏感度可达2.47 PFU/mL或2.5 ng靶DNA片段,而且与禽类常见的IBV、NDV等6种病毒均无交叉反应.[结论]证明该病毒分型基因芯片具有良好的特异性、敏感性.     

新型Taq Man-MGB探针在结核分枝杆菌实时PCR检测中的应用

为建立一种比现有方法敏感、准确性高、重复性好的结核分枝杆菌DNA定性定量检测方法 ,以TaqMan探针技术为基础 ,运用TaqMan MGB探针 ,实时检测临床标本中的结核分枝杆菌DNA .用来自临床标本的DNA及克隆于载体的IS6 1 1 0序列检测所建立方法的有效性 .结果显示 ,所建立方法的最低检测限度为 1个基因拷贝 反应 ,在每反应 1 0 0 ～ 1 0 8拷贝范围内 ,Ct 值同DNA量的对数呈线性关系 .同一模板不同时间或同一时间不同管内扩增 ,所得Ct 值恒定 .用该方法检测 37例结核分枝杆菌培养阳性的痰液标本 ,敏感度为 1 0 0 % ;用该方法检测 1 6例TB系列阴性参考品 ,特异性为1 0 0 % .结果表明 ,所建立的方法是用于结核分枝杆菌定性定量检测较理想的方法     

Development of a new probe for specific and sensitive detection of 'Candidatus Phytoplasma mali' in inoculated apple trees

A new TaqMan minor groove binding (MGB) probe and new PCR conditions were designed for quick, specific and sensitive detection of ‘Candidatus Phytoplasma mali’. The new probe can distinguish a single mismatch between ‘Ca. P. mali’ and ‘Candidatus Phytoplasma prunorum’, this constituting an improvement over a previously published method. In our study, the relative position of the mismatch in the MGB probe influenced greatly the specificity of detection. Our new real‐time PCR protocol was able to detect one plasmid copy in water and 100 copies in healthy plant DNA extracts. The sensitivity of this new real‐time PCR method, three other real‐time PCR protocols and a conventional PCR with fU5/rU3 primers was compared. Periwinkles and MM106 rootstocks were grafted with infected material and surveyed over time by symptom observation, conventional PCR and real‐time PCR. Phytoplasma infection was detected by symptom observation in all periwinkles within 4 months and in 75% of the MM106 rootstocks by the end of 7 months. Conventional PCR detected phytoplasma infection in all periwinkles within 4 months and in all MM106 rootstocks within 7 months. Best results were obtained by our real‐time PCR, which detected phytoplasma infection in all grafted plants within 3 months after inoculation.     

Quantum‐dots‐based fluoroimmunoassay for the rapid and sensitive detection of avian influenza virus subtype H5N1

The continuous spread of highly pathogenic avian influenza virus (AIV) subtype H5N1 is threatening the poultry industry and human health worldwide. Rapid and sensitive diagnostic methods are required for the H5N1 surveillance. In this study, the fluorescent (FL) probe of CdTe quantum dots (QDs) was designed using covalently linked rabbit anti‐AIV H5N1 antibody. Based on these QD–antibody conjugates, a novel sandwich FL‐linked immunosorbent assay (sFLISA) was developed for H5N1 viral antigen detection. The sFLISA allowed for H5N1 viral antigen determination in a linear range of 8.0 × 10^?3 to 5.1 × 10^?1 μg mL^?1 with the limit of detection (LOD) of 1.5 × 10^?4 μg mL^?1. In comparison with virus isolation for 103 clinic samples, the sensitivity and specificity of sFLISA were found to be 93.6 and 91.1% respectively. The sFLISA supplied a novel approach to rapid and sensitive detection of AIV subtype H5N1 and showed great potential for biological applications in immunoassays. Copyright © 2009 John Wiley & Sons, Ltd.     

Real-Time PCR Detection of Vibrio vulnificus in Oysters: Comparison of Oligonucleotide Primers and Probes Targeting vvhA

We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (C_T) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 10³ V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 × 10³ CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.     

禽流感H5、H7、H9亚型多重实时荧光RT-PCR检测方法的建立

为了对致病性强、危害性大的H5、H7、H9亚型禽流感病毒进行同时集成化快速检测,通过对GenBank已报道的禽流感病毒的HA基因进行序列分析比较,设计了H5、H7、H9 3个亚型的特异性引物和分别用3个荧光基团标记的Taqman MGB核酸探针。将各个亚型引物与探针优化组合,筛选出能够同时检测禽流感病毒H5、H7、H9 3个亚型、且对Ct值和扩增效率影响不大的3组引物和探针,建立了三重实时荧光RT-PCR方法。该方法特异性好,在我们检测的样品中,没有发现假阳性和假阴性现象。同时敏感性高,检测禽流感病毒H5、H7、H9亚型的敏感性分别达到1 0001、000、500个模板拷贝数;此外抗干扰能力强,对禽流感H5、H7、H9 3个亚型的不同模板浓度进行组合,仍可有效地同时检测3个病毒亚型。所建立的方法对保存的89个禽流感病毒样品进行检测,结果与经典检测方法(病毒分离鉴定、HA、HI)的符合率达100%。用上述建立的方法与鸡胚分离法同时对新鲜采集的4 000多份临床样品进行检测,两种方法的检测结果符合率为100%。     

甲型H1N1流感病毒核酸荧光定量RT-PCR检测技术

目的:建立基于TaqMan荧光探针技术的甲型H1N1流感病毒实时定量RT-PCR方法。方法:分析H1N1流感病毒基因特性,根据新发甲型H1N1流感病毒突变基因片段设计检测引物和TaqMan荧光探针,建立荧光定量RT-PCR检测体系,体外转录制备RNA标准品,进行特异性、敏感性、重复性及盲样检测评价实验。结果:可特异性有效检测新发甲型H1N1流感病毒核酸,与H1～H16流感病毒基因无交叉反应;对RNA标准品的检测敏感性达103拷贝/μL;重复性实验中,阳性标准品Ct值变异系数(CV)10%,阴性标准品检测结果均呈阴性;12份盲样检测结果特异性好。结论:该荧光定量RT-PCR方法可作为甲型H1N1流感防控的病原快速诊断技术。