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PRRSV和PCV-2以及PRV多重SYBR Green-Ⅰ实时荧光PCR检测方法的建立 被引量:21
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作者 王小武 符芳 +5 位作者 柴政 孔令达 蔡雪辉 宋淑萍 许红喜 李曦 《中国兽医科学》 CAS CSCD 北大核心 2008年第6期494-499,共6页
根据GenBank中登录的猪生殖与呼吸综合征病毒(PRRSV)N蛋白基因、猪圆环病毒2型(PCV-2)rep蛋白基因和猪伪狂犬病病毒(PRV)gE基因的核苷酸序列分别设计了3对特异性引物,成功建立了同时检测PRRSV、PCV-2、PRV的多重SYBR Green-Ⅰ实时荧光PC... 根据GenBank中登录的猪生殖与呼吸综合征病毒(PRRSV)N蛋白基因、猪圆环病毒2型(PCV-2)rep蛋白基因和猪伪狂犬病病毒(PRV)gE基因的核苷酸序列分别设计了3对特异性引物,成功建立了同时检测PRRSV、PCV-2、PRV的多重SYBR Green-Ⅰ实时荧光PCR方法。敏感性试验结果显示,PRRSV、PCV-2的敏感性可达250拷贝/μL,PRV的敏感性可达500拷贝/μL。表明,该方法具有较好的特异性、重复性和敏感性,可以用于PRRSV、PCV-2和PRV的快速检测。 展开更多
关键词 猪生殖与呼吸综合征病毒 猪圆环病毒2型 猪伪狂犬病病毒 多重SYBR green-实时荧光 PCR
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猪生殖与呼吸综合征病毒的SYBR Green-Ⅰ荧光定量PCR检测 被引量:2
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作者 王小武 符芳 +3 位作者 许红喜 周艳君 童光志 李曦 《中国兽医科学》 CAS CSCD 北大核心 2008年第5期401-406,共6页
根据猪生殖与呼吸综合征病毒(PRRSV)GP5蛋白基因核酸序列设计引物,经BLAST比较发现,该引物既可以扩增较早发现的PRRSV毒株,也可以扩增近期发现的在Nsp2基因上有较大变异的PRRSV毒株,以含有该引物扩增序列的重组质粒作为标准品建立了检测... 根据猪生殖与呼吸综合征病毒(PRRSV)GP5蛋白基因核酸序列设计引物,经BLAST比较发现,该引物既可以扩增较早发现的PRRSV毒株,也可以扩增近期发现的在Nsp2基因上有较大变异的PRRSV毒株,以含有该引物扩增序列的重组质粒作为标准品建立了检测PRRSV的SYBR Green-Ⅰ荧光定量PCR方法。结果表明,该方法标准曲线的相关系数大于0.99,敏感性可以达到1×101拷贝/μL,约为普通PCR的100倍;用该方法检测人工感染PRRSV的猪全血,结果阳性检出率为100%。敏感性、特异性和稳定性试验表明,该方法具有很高的敏感性、特异性和稳定性,可以用来快速检测PRRSV。 展开更多
关键词 猪生殖与呼吸综合征病毒 SYBR green- 荧光定量PCR
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Significance of the antigenic epitopes in heterogenous nuclear ribonucleoproteins-Ⅰfor the diagnosis and prognosis of systematic sclerosis
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作者 JING XUE ZHONG QIANG YAO +2 位作者 YANG GAO MENG XUE YU LI PING ZHU 《Journal of Microbiology and Immunology》 2006年第4期300-305,共6页
To assess the presence of autoantibodies against epitopes of heterogenous nuclear ribonucleoprotein- Ⅰ (hnRNP- Ⅰ ) in systematic sclerosis (SSc) and to analyze their clinical significance, polypeptides of hnRNP-... To assess the presence of autoantibodies against epitopes of heterogenous nuclear ribonucleoprotein- Ⅰ (hnRNP- Ⅰ ) in systematic sclerosis (SSc) and to analyze their clinical significance, polypeptides of hnRNP- Ⅰ were designed by biological technical software and analyzed with both the Wonderful Biology Information System and DNA Star-Protean Analysis Software at the same time. In these ways, two polypeptides of hnRNP- Ⅰ were obtained based on their amino acid sequences, folding features, hydrophilic, curl style, dough kneading sensation and the possibility on the surface of proteins. They are named as hnRNP- Ⅰ -1 ( NVKYNNDKSRDYTRPDLPSGDSQPSLDQT, 264-292 aa) and hnRNP- Ⅰ -2 (QLP4REGQEDQGLTKDYGNSOL, 441-461 aa), simply designated as Ⅰ -1 and Ⅰ -2. The autoantibodies against hnRNPs were detected by means of ELISA using the synthetic epitopes polypeptides as antigen. It was found that the positive rate of detection for anti- Ⅰ -1 and anti- Ⅰ -2 autoantibodies were rather higher in SSc patients than that in other CTDs and the sensitivities and specificities of the testing with ELISA for anti- Ⅰ -1 and anti- Ⅰ -2 antibodies in SSc patients were 47.62%/93.43% and 38.1%/ 91.08%, without any significant difference between these two groups of testings. Also, there was no significant difference in the clinical features and laboratory findings, such as age, involvements in digestive and respiratory tracts and erythrocyte sedimentation rate etc., between the anti- Ⅰ -Ⅰ-positive and -negative groups in SSc patients. However, the hnRNP- Ⅰ -autoantibody-positive group of patients had obviously shorter duration of disease course compared with that of the autoantibody-negative group. Anti- Ⅰ -1 and anti- Ⅰ -2 autoantibodies also had no association with antinuclear antibody, anti-Sc170 and anti-centromere antibody (ACA) in SSc patients. So, it is apparent that the autoantibodies related with SSc may act through different pathways in the pathogenesis of SSc, and the hnRNP- Ⅰ autoantibody is a new type antibody occuring during the early stage of disease and appearing to have diagnostic and prognostic significances. 展开更多
关键词 Heterogenous nuclear ribonucleoprotein- (hnRNP- Systemic sclerosis Epitope Enzyme-linked immonosorbent assay (ELISA)
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实时荧光定量PCR法检测草莓镶脉病毒 被引量:5
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作者 苗立祥 荣宁宁 +4 位作者 张豫超 杨肖芳 张琴 张慧琴 蒋桂华 《浙江农业学报》 CSCD 北大核心 2016年第6期1030-1036,共7页
为了建立一种特异、灵敏、快速检测草莓镶脉病毒(Strawberry vein banding virus,SVBV)的SYBR Green-Ⅰ实时荧光定量PCR方法,根据已有的检测SVBV的引物信息,合成引物,对不同草莓品种进行PCR扩增。经过克隆、测序及序列比对得到了感染SVB... 为了建立一种特异、灵敏、快速检测草莓镶脉病毒(Strawberry vein banding virus,SVBV)的SYBR Green-Ⅰ实时荧光定量PCR方法,根据已有的检测SVBV的引物信息,合成引物,对不同草莓品种进行PCR扩增。经过克隆、测序及序列比对得到了感染SVBV的阳性植株,利用此植株PCR扩增产物构建重组质粒作为标准品,优化了PCR反应体系,标准曲线的循环阈值(Ct值)与模板浓度有良好的线性关系(R2=0.999 1);进行了灵敏度和重复性试验,敏感性可达到10~1拷贝·μL^(-1),约为普通PCR的1 000倍,重复性好;并与常规PCR方法进行了比较,成功建立了检测SVBV的实时荧光定量PCR法。用该方法检测了部分草莓品种的DNA样品,结果表明,大部分植株都带有SVBV。该方法具有很高的灵敏性和重复性,可用来快速检测草莓植株是否感染SVBV。 展开更多
关键词 草莓镶脉病毒 SYBR green- 实时荧光定量PCR
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Assessment of in vitro sensitivity of Plasmodium vivax fresh isolates 被引量:2
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作者 Poonuch Muhamad Wanna Chacharoenkul +2 位作者 Kanchana Rungsihirunrat Ronnatrai Ruengweerayut Kesara NaBangchang 《Asian Pacific Journal of Tropical Biomedicine》 SCIE CAS 2011年第1期49-53,共5页
Objective:To compare the applicability of the SYBK Grcen-Ⅰ assay with the standard schizont maturalion assay,for determination of sensitivity of Plasmodium vivax(P.vivax) to chloroquine and a new antifolale WR 99210.... Objective:To compare the applicability of the SYBK Grcen-Ⅰ assay with the standard schizont maturalion assay,for determination of sensitivity of Plasmodium vivax(P.vivax) to chloroquine and a new antifolale WR 99210.Methods:The study was conducted at Mae Tao Clinic for migrant workers,Tak Province during April 2009 to July 2010.A total of 64 blood samples(1 mL blood collected into sodium heparinized plastic tube) were collected from patients with monoinfection with P.vivax malaria prior to treatment with standard regimen of a 3-day chloroquine. In vitro sensitivity of P.vivax isolates was evaluated by schizont maturation inhibition and SYBR Green-Ⅰ assays.Results:A total of 30 out of 64 blood samples collected from patients with P.vivax malaria were successfully analyzed using both the microscopic schizont maturation inhibition and SYBR Green-I assays.The failure rates of the schizont maturation inhibition assay(50%) and the SYBR Green-I assay(54%) were similar(P=0.51).The median IC_(10)s,IC_(50)s and IC_(90)s of both chloroquine and WR99210 were not significantly different from the clinical isolates of P.vivax tested.Based on the cut-off of 100 nM,the prevalences of chloroquine resistance determined by schizont maturation inhibition and SYBR Green-I assays were 19 and 11 isolates,respectively.The strength of agreement between the two methods was very poor for both chloroquine and WR992I0.Conclusions:On the basis of this condition and its superior sensitivity,the microscopic method appears better than the SYUK Green-I Green assay for assessing in vitro sensitivity of fresh P.vivax isolates to antimalarial drugs. 展开更多
关键词 PLASMODIUM VIVAX CHLOROQUINE In vitro sensitivity Schizont maturation inhibition assay SYBR green-ⅰassay FRESH isolate Antifolate Malaria ANTIMALARIAL drug Standard regimen
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