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1.
试验旨在利用新型荧光探针--分子信标,建立一种检测猪流感病毒的新方法。根据H3N2亚型猪流感病毒（swine influenza virus,SIV）的H3和N2基因的保守基因序列,分别设计并合成了特异性引物和分子信标探针,利用数字RT-PCR技术检测H3N2亚型SIV。结果显示,该数字RT-PCR检测方法与其他主要相关病毒均不发生交叉反应,重复性良好;对猪H3N2流感病毒而言,最低可检测到106倍稀释的病毒株。数字RT-PCR方法能够对RNA模板定量分析。在H3N2 SIV的鉴定上,数字RT-PCR方法较实时荧光定量PCR方法更灵敏、准确。 相似文献
2.
为建立一种快速、简便、准确的方法以诊断和检测H1亚型猪流感病毒(swine influenza virus,SIV),试验根据H1亚型SIV血凝素(hemagglutinin,HA)基因保守序列,分别设计并合成1对特异性引物和1条TaqMan MGB探针,建立检测H1亚型SIV的一步法实时荧光定量RT-PCR技术。结果显示,该方法的敏感性可达102拷贝/μL,除H1亚型SIV外,对H3N2亚型SIV、H9N1亚型SIV、猪瘟病毒、猪繁殖与呼吸综合征病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒的检测均为阴性,且应用该方法对疑似猪流感样品进行检测,其结果与SPF鸡胚分离病毒方法结果的符合率为94%。本试验结果表明该方法特异性强、重复性好,有望成为一种特异、敏感、快速、定量检测H1亚型SIV的方法。 相似文献
3.
WANG Bo WANG Hui-yu ZHAO Bao-hua LUO Jing WANG Cheng-min HE Hong-xuan HAN Xue-qing 《中国畜牧兽医》2017,44(10):3035-3041
To establish a rapid, accurate method to diagnose and detect H1 and H3 subtype swine influenza viruses (SIV) at the same time, the specific primers and TaqMan probes were designed according to the conserved region of the HA gene of H1 and H3 subtype SIV. A duplex Real-time RT-PCR assay was developed for detection of H1 and H3 subtype SIV. The results showed that the Real-time RT-PCR could detect 102 copies/μL of H1 and H3 subtype SIV, the sensibility was well. Coefficient of variation of Ct value between repeating groups were all below 5%, the repeatability was favorable. The results were negative for the detection of H4, H5, H7, H9 subtypes SIV, classical swine fever virus, porcine reproductive and respiratory syndrome virus, foot and disease virus and pseudorabies virus, the specificity was fine. One sample was H1 subtype SIV, and one sample was H3 subtype SIV, by the established assay, the positive rate was 1.16%. The method was highly accurate, rapid, sensitive and specific, and could provide a method for rapid detection and epidemiological investigation of H1 and H3 subtype SIV. 相似文献
4.
为建立一种快速、准确地检测H1和H3亚型猪流感病毒（swine influence virus,SIV）的方法,根据H1和H3亚型SIV血凝素（hemagglutinin,HA）基因保守序列,分别设计2对特异性引物和2条TaqMan探针,建立双重实时荧光定量RT-PCR方法。结果显示,该方法敏感性高,可检测到最低拷贝数为102拷贝/μL;重复性良好,重复孔Ct值的变异系数均在5%以下;特异性好,除H1和H3亚型SIV外,H4、H5、H7、H9亚型SIV、猪瘟病毒、猪繁殖与呼吸综合征病毒、口蹄疫病毒、伪狂犬病病毒检测均为阴性;在田间样品中成功检出1株H1和1株H3亚型SIV,检出率为1.16%。该方法准确、快速、灵敏、特异,可为H1、H3亚型SIV的快速鉴别检测及流行病学调查提供有效的技术手段。 相似文献
5.
猪流感病毒H1、H3、N1、N2亚型分型 RT-PCR方法的建立 总被引：1，自引：0，他引：1
根据GenBank中H1N1和H3N2亚型猪流感病毒(SIV)血凝素(hemagglutinin,HA)、神经氨酸酶(neuraminidase,NA)和M基因保守序列,分别设计合成了5对特异性引物,利用RT-PCR技术对SIV的型和亚型进行鉴定。结果表明,该方法的型RT-PCR可以检测出104 EID50病毒量所提取的RNA;H1、H3、N1和N2的亚型RT-PCR均可以检测出104 EID50病毒量所提取的RNA。除每对特异性引物所对应的亚型外,对其他亚型及猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)的检测均为阴性,应用该方法对临床样品进行检测,其结果与病毒分离结果符合率为100%。结果表明,该方法特异性好、敏感性高,有望成为SIV的一种特异、敏感、快速的分型检测方法,为猪流感分子流行病学的调查奠定了良好的基础。 相似文献
6.
本试验针对猪流感病毒（swine influenza virus,SIV）NP基因保守区域设计并合成6条引物,建立了SIV的环介导等温扩增（LAMP）检测方法,并进行了特异性、敏感性和重复性试验。结果显示,该方法可特异性检测H1N1、H1N2、H3N2、类禽H1N1亚型SIV及甲型H1N1流感病毒,但不能检测猪繁殖与呼吸综合征病毒、猪细小病毒、猪瘟病毒、猪伪狂犬病病毒、猪圆环病毒2型、日本乙型脑炎病毒;该方法的最低检测量为100拷贝/μL质粒DNA。结果表明建立的LAMP方法具有较高的敏感性和特异性,可用于SIV的快速检测。 相似文献
7.
8.
为建立一种同时检测H4、N2和所有亚型禽流感的方法,分别针对H4亚型禽流感病毒(AIV)HA基因、N2亚型AIV NA基因和所有亚型AIV M基因保守序列,设计筛选出3对特异性引物,优化引物之间的浓度,对三重反应体系进行特异性和敏感性验证,建立了H4、N2和所有亚型AIV三重RT-PCR检测方法,并用该法对临床样品进行检测。建立的方法能特异性扩增H4、N2和所有亚型AIV,与其他禽病病原体不发生交叉反应;对H4、N2和所有亚型AIV至少能检测到6 pg/μL。在185份临床样品的检测中,检出4份H4、10份N2和19份AIV阳性。所建立的三重RT-PCR方法特异性强、灵敏度高,为快速检测H4、N2和所有亚型AIV提供了有效的方法。 相似文献
9.
根据GenBank中H6、N1亚型禽流感病毒(AIV)的HA、NA基因序列,设计2对特异性引物和2条用不同荧光基团标记的TaqMan探针.经反应条件优化,本试验建立了检测H6N1亚型AIV的二重荧光RT-PCR方法.该法特异性强,只对H6亚型和N1亚型AIV进行特异性扩增,对其他H亚型AIV、N亚型AIV及新城疫病毒、传染性支气管炎病毒等病原体的检测均为阴性;该法敏感性好,对H6N1亚型AIV的检测限为100拷贝/μL.本试验建立的H6N1亚型AIV的二重荧光RT-PCR方法,具有快速、敏感、特异的优点,为H6N1亚型AIV的防控提供技术支撑. 相似文献
10.
LUO Si-si XIE Zhi-xun XIE Zhi-qin DENG Xian-wen LIU Jia-bo XIE Li-ji HUANG Li HUANG Jiao-ling ZENG Ting-ting ZHANG Yan-fang WANG Sheng 《中国畜牧兽医》2016,43(2):326-332
According to the sequences of HA and NA genes of H6 and N1 subtype avian influenza virus (AIV),two pairs of specific primers and two TaqMan probes with different fluorescence were designed.The duplex Real-time RT-PCR assay was developed and optimized to simultaneously detect H6 and N1 subtypes AIV in one reaction.The result showed that the specificity of this assay was high and only amplified H6 and N1 subtypes AIV,and was not cross-reactive with other H and N subtypes AIV,newcastle disease virus and infectious bronchitis virus.The detection limit of this assay was 100 copies/μL of H6N1 subtype AIV.This newly developed duplex Real-time RT-PCR assay was a rapid,specific and sensitive method for the detection of H6N1 subtype AIV,and it could provid a technical support to prevent and control H6N1 subtype AIV. 相似文献
11.
H1N1猪流感病毒环介导等温扩增快速检测方法的建立 总被引：2，自引：0，他引：2
目的:建立H1N1猪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H1N1猪流感病毒血凝素(HA)、神经氨酸酶(NA)基因序列,应用DNAStar软件MegAlign程序分析其序列,利用Primer ExplorerV4软件在序列保守区域设计LAMP引物,即外引物和内引物,同时以H1N1猪流感病毒的cDNA作为阳性模板,对试验中的几个反应条件进行优化。结果:LAMP检测方法对H1N1猪流感病毒的灵敏度达到4～6个拷贝,其引物对于H9亚型禽流感病毒、猪瘟病毒和猪圆环病毒均无非特异性扩增,表现出良好的特异性。结论:建立的H1N1猪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,为快速检测猪流感病毒提供了新方法和新思路。 相似文献
12.
逆转录环介导等温扩增技术检测H3亚型猪流感病毒 总被引：5，自引：2，他引：3
基于环介导等温扩增技术(RT-LAMP),建立了一种用于快速检测H3亚型猪流感病毒的方法。根据GenBank中收录的H3N2亚型猪流感病毒(SIV)HA基因保守区的序列,设计一组对应HA序列6个区域的4条特异性引物,建立RT-LAMP反应体系;经优化,确定了最佳的LAMP反应条件。扩增后产物形成的焦磷酸离子和溶液中的镁离子结合而形成焦磷酸镁沉淀,结果可视。同时用特异性内切酶HhaⅠ对所获得的产物进行酶切鉴定,所得的酶切产物大小与理论值完全符合。用已建立的RT-LAMP技术检测临床样品,检测结果与RT-PCR方法符合率均达到100%。结果表明,本研究建立的RT-LAMP检测猪流感病毒方法,操作简单,灵敏度高,特异性强,是一种适用于现场诊断的检测技术。 相似文献
13.
根据H9N2亚型猪流感病毒血凝素基因序列设计并合成引物,从本室分离保存的H9N2亚型猪流感病毒中扩增出血凝素基因,并将其克隆进pMD18-T载体,限制性酶切及序列测定后,进一步将其信号肽缺失并亚克隆到pGEX-KG中,使其在E.coli BL21（DE3）中与GST融合表达。SDS-PAGE显示表达的融合蛋白的相对分子质量约为90 000。Western印迹表明表达的蛋白质具有免疫学活性。表达产物位于包涵体中,包涵体经变性、复性处理,利用复性产物作为抗原,经碳二亚胺（EDAC）将表达产物和羧基化的乳胶共价偶联,建立了H9亚型猪流感病毒抗体的乳胶凝集试验的检测方法,结果表明,应用HA重组蛋白作为诊断抗原具有特异性强、敏感性高、重复性好的特点,可用于H9亚型猪流感抗体水平监测、流行病学调查和现地疫病普查。 相似文献
14.
猪流感病毒HA基因的原核表达及其ELISA检测方法的建立 总被引：1，自引：0，他引：1
根据H1N1亚型猪流感病毒血凝素基因序列设计并合成特异性引物,从本室保存的H1N1亚型猪流感病毒中扩增信号肽和跨膜区缺失的血凝素基因,并将其克隆到原核表达载体pET-28a中,在大肠埃希菌中诱导表达.对表达蛋白进行SDS-PAGE和Western blot的结果表明,HA基因在大肠埃希菌中获得表达,产物具有免疫学活性,表达蛋白分子质量约为55 ku.表达产物经过纯化作为包被抗原建立了检测H1亚型猪流感抗体的间接ELISA方法.该方法具有较高的特异性和敏感性,重复性良好.应用该方法对2006年2 584份临床猪血清进行检测,结果显示多个地区检测猪流感抗体出现阳性,平均为20.5%.在6月、7月和12月分别出现抗体水平高峰,分别高达25.4%、25.0%和35.5%. 相似文献
15.
An immunoperoxidase monolayer assay for the detection of antibodies against swine influenza virus. 总被引：3，自引：0，他引：3
Kochakorn Direksin HanSoo Joo Sagar M Goyal 《Journal of veterinary diagnostic investigation》2002,14(2):169-171
An immunoperoxidase monolayer assay (IPMA) has been developed to detect antibodies against swine influenza A virus (SIV) in pig sera. The test was evaluated by using sequential sera from pigs experimentally infected with H1N1 subtype of SIV. Two hundred field serum samples that had been examined by the hemagglutination inhibition (HI) test were also tested. Antibodies specific to SIV were detected as early as 3 days postinoculation (dpi) in the IPMA test as compared with 7 dpi by the HI test. Unlike HI, no serum treatment was required in the IPMA test. Regardless of the virus used in the test, IPMA detected antibodies to both H1N1 and H3N2 subtypes of SIV whereas HI detects antibodies against either H1N1 or H3N2, depending upon the virus used in the test. Results of this study indicate that IPMA is a useful test for screening of pig sera for SIV antibodies. 相似文献
16.
为建立简便快速检测禽流感病毒(avian influenza virus,AIV)并同时区分出H9、N2亚型的方法,本试验根据基因库中H9亚型AIV的HA基因、N2亚型AIV的NA基因及AIV的M基因序列,分别设计了3对针对这3种基因保守序列的引物,建立了AIV H9N2亚型的三重PCR检测方法。应用该方法对H9N2亚型AIV模板进行PCR扩增,可得到3条与试验设计相符的目的条带,分别为313 bp (HA基因)、451 bp (NA基因)和667 bp(M基因);对非H9亚型的N2亚型AIV模板进行扩增,出现2条特异性扩增条带,即451 bp (NA基因)和667 bp(M基因);对非H9、N2亚型AIV模板进行扩增则只出现一条目的条带,即667 bp(M基因);对其他禽呼吸道病原体进行PCR扩增,结果均为阴性。敏感性试验结果显示此三重PCR方法最低检出限为10-2 ng/μL。应用所建立的三重PCR方法对120份临床病料进行检测的结果与病毒分离鉴定结果一致。各项试验结果均表明,该方法对于禽流感病毒尤其是H9、N2亚型禽流感病毒的检测具有快捷、特异、灵敏的特点。 相似文献
17.
Real-time reverse transcription-polymerase chain reaction assays for the detection and differentiation of North American swine influenza viruses. 总被引：10，自引：0，他引：10
Jürgen A Richt Kelly M Lager Deborah F Clouser Erica Spackman David L Suarez Kyoung-Jin Yoon 《Journal of veterinary diagnostic investigation》2004,16(5):367-373
18.
Novel swine influenza virus subtype H3N1 in Italy 总被引：2，自引：0，他引：2
Ana Moreno Ilaria Barbieri Enrica Sozzi Andrea Luppi Davide Lelli Guerino Lombardi Maria Grazia Zanoni Paolo Cordioli 《Veterinary microbiology》2009,138(3-4):361-367
To date, three subtypes of swine influenza viruses, H1N1, H1N2, and H3N2 have been isolated in Italy. In 2006, a novel swine influenza virus subtype (H3N1) was isolated from coughing pigs. RT-PCR performed on lung tissues, experimental infection in pigs with the novel isolate, and cloning the virus by plaque assay confirmed this unique H and N combination. The novel isolate was also antigenically and genetically characterized. Genetic and phylogenetic analysis showed that the complete HA gene of the H3N1 strain has the highest nucleotide identity to three Italian H3N2 strains, one isolated in 2001 and two in 2004, whereas the full length NA sequence is closely related to three H1N1 subtype viruses isolated in Italy in 2004. The remaining genes are also closely related to respective genes found in H1N1 and H3N2 SIVs currently circulating in Italy. This suggests that the novel SIV could be a reassortant between the H3N2 and H1N1 SIVs circulating in Italy. 相似文献
19.
LI DAN XIE Zhi-xun SONG De-gui LI Meng XIE Zhi-qin LUO Si-si XIE Li-ji HUANG Li FAN Qing HUANG Jiao-ling 《中国畜牧兽医》2016,43(12):3101-3106
This experiment was aimed to develop a method for simultaneous detection of H9 and H6 subtype avian influenza virus (AIV).Two pairs of specific primers were designed according to the conserved regions sequences of H6 and H9 AIV HA gene,a duplex RT-PCR simultaneous detection of H9 and H6 subtype AIV was developed by optimizing the PCR system such as the concentration of different primers and annealing temperature.It showed that all samples could be amplified specific bands from H9 subtype AIV single infection samples or H6 subtype AIV single infection samples,and the samples mix infection these two subtypes AIV.No specific bands of the same sizes were amplified from genomic materials of other avian pathogens.The detection limit of the duplex RT-PCR was 5×104 copies/μL. It suggested that this duplex RT-PCR assay was a specific,sensitive,stable and repeatable method for detection of H9 and H6 subtype of AIV,and could provide technical support for the monitoring of H9 and H6 subtype AIV. 相似文献
20.
试验旨在建立可同时鉴别检测H9和H6亚型禽流感病毒（avian influenza virus,AIV）的二重RT-PCR方法。根据GenBank中H9和H6亚型AIV的HA基因保守序列,分别设计2对特异性引物,优化引物浓度与退火温度等条件,建立了可同时鉴别检测H9和H6亚型AIV的二重RT-PCR检测方法。用该法对H9和H6亚型AIV混合感染样品、H9亚型AIV单一感染样品和H6亚型AIV单一感染样品进行扩增,结果均得到对应的目的条带,而对其余亚型AIV及其他禽病病原体均未扩增出特异性条带。该法对H9和H6亚型AIV的检测下限均为5×104拷贝/μL。本研究建立的二重RT-PCR检测方法特异性强、敏感性高、稳定性和重复性良好,可同时鉴别检测H9与H6两种亚型AIV,为H9与H6亚型AIV的监测提供技术支撑。 相似文献