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为鉴别新城疫病毒(NDV)强毒株和弱毒株,本研究建立了基于新型锁核酸(LNA)探针的实时荧光RT-PCR检测方法(Duplex LNA rRT-PCR)。该方法针对NDVF基因裂解位点设计了两条新型LNA探针,通过对11株NDV株进行大将军脂duplex LNA rRT-PCR检测方法检测,验证该方法的特异性;通过对副粘病毒I型(APMV-1)和NDV中强毒株(vNDV)不同浓度病毒液进行检测,确定该方法的灵敏度,并与TaqMan实时荧光RT-PCR检测方法进行比较。结果显示本研究所建立的方法对11株NDV检测的特异性为100%(11/11),优于TaqMan实时荧光RT-PCR检测方法(10/11);所建立的duplex LNA rRT-PCR方法检测中强毒株F48E9和弱毒株LaSota的灵敏度分别为10个EID50和0.1个EID50,比美国农业部推荐的TaqMan实时荧光RT-PCR检测方法低10倍。本研究利用新型LNA探针技术,建立了鉴别NDV中强毒株与弱毒株的duplex LNA rRT-PCR检测方法,可以特异性检测NDV并有效区分中强毒株与弱毒株,适合用于鸡场和进出境动物产品中NDV的快速检测。 相似文献
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根据GenBank中鸭Ⅰ型肝炎病毒的基因序列,设计合成一对引物和一条Taq-Man探针。进行优化后,建立了能够检测鸭Ⅰ型肝炎病毒的荧光定量RT-PCR方法。该方法敏感性好,检测敏感性达到20个模板拷贝数,比常规PCR灵敏度高100倍;该方法特异性强,对番鸭细小病毒、鸭圆环病毒和鹅细小病毒等6种病原体的检测全为阴性。对广西地区鸭群收集的118份病料进行检测,结果阳性率为1.69%。结果提示:在广西地区的鸭群中,存在鸭Ⅰ型肝炎病毒的感染,建立的荧光定量RT-PCR方法可用于鸭Ⅰ型肝炎病毒的临床快速检测。 相似文献
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根据A型流感病毒基质蛋白（matrix protein,M）基因保守序列设计并合成特异性引物和TaqMan BHQ探针,建立快速检测A型流感病毒的实时荧光定量RT-PCR方法。通过RT-PCR方法克隆A型流感病毒M基因靶序列并将其连入pMD-18T载体,制备阳性标准品,优化反应条件,以10倍系列稀释的标准品绘制标准曲线,其相关系数为0.998。检测结果显示,该方法的灵敏度可达10 copies/μL或1 EID50病毒且特异性良好,除A型流感病毒外,对猪繁殖与呼吸综合征病毒、猪瘟病毒、猪圆环病毒2型、猪输血传播病毒检测结果均为阴性。该方法重复性好,批内和批间变异系数均小于3%。对40份实验感染样品的检测结果表明,该方法与病毒分离结果一致,比常规RT-PCR检测方法灵敏度更高,特异性更强。 相似文献
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Newcastle disease, induced by a type 1 Avian Paramyxovirus (APMV-1), is one of the most serious poultry diseases. APMV-1 are divided into two classes based on genetic analysis: class II strains have been recovered from wild or domestic birds and include virulent and avirulent isolates whereas class I strains have been mainly isolated from wild birds and are avirulent. Within class I, a new proposed genotype has recently been reported. The only full genome strain of this group is presently characterised from the point of view of codon usage with reference to class I and class II specificities. Class-specific residues were identified on HN and F proteins that are the two major proteins involved in cell attachment and pathogenicity. Comparison of protein patterns and codon usage for this newly identified APMV-1 strain indicates it is similar to class I viruses but contains a few characteristics close to the viruses of class II. Transmission of viruses from this recently identified divergent group from wild birds to domestic birds could have a major impact on the domestic poultry industry. 相似文献
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根据Ⅶ型新城疫病毒基质蛋白基因保守序列设计并合成特异性引物,建立快速检测Ⅶ型新城疫病毒的实时荧光定量RT-PCR方法。通过RT-PCR方法克隆Ⅶ型新城疫病毒M基因靶序列,并将其连入T-Easy载体,制备阳性标准品,优化反应条件,以10倍系列稀释的标准品绘制标准曲线,其相关系数为0.999。检测结果显示,该方法的灵敏度可达100 copies/μL,而且特异性良好,除Ⅶ型新城疫病毒外,对Ⅱ型/Ⅲ型/IV型/Ⅸ型新城疫病毒、H5亚型禽流感病毒、H9亚型禽流感病毒、鸡传染性支气管炎病毒检测结果均为阴性。本研究所建立的检测方法重复性好,批内和批间变异系数均小于5%。对40份实验感染样品的检测结果表明,其检出率为95%,而常规RT-PCR方法检出率仅为70%,表明该方法比常规RT-PCR检测方法灵敏度更高、特异性更强。 相似文献
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本研究根据GenBank发表的禽副黏病毒4型（APMV-4）基因组序列,在保守区域分别设计了特异性RT-PCR引物和荧光探针,初步建立了可用于检测APMV-4的荧光RT-PCR方法。特异性试验表明本方法特异性强,与新城疫病毒（NDV）、禽流感病毒（AIV）、传染性支气管炎病毒（IBV）、减蛋综合征病毒（EDSV）等没有交叉反应。灵敏度试验表明本方法的检测下限为1EID50,比常规RT-PCR的敏感度高100倍左右。本研究所建立的荧光RT-PCR方法具有快速、准确、灵敏等优点,可用于APMV-4样品的检测。 相似文献
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Yu-Pin LIU Fan LEE Ming-Chu CHENG Chiu-Yen CHANG Chwei-Jang CHIOU Hsiang-Jung TSAI 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2022,84(3):378
Avian paramyxoviruses (APMVs) belonging to the subfamily Avulavirinae within the family Paramyxoviridae. APMVs consist of twenty-two known species and are constantly isolated from a wide variety of avian species around the world. In this study, the APMV isolates obtained from wild birds and domestic poultry during 2009–2020 in Taiwan were genetically characterized by phylogenetic analysis of their complete fusion protein gene or full-length genome. As a result, 57 APMV isolates belonging to seven different species were obtained during this period and subsequently identified as APMV-1 (n=17), APMV-2 (n=1), APMV-4 (n=25), APMV-6 (n=8), APMV-12 (n=2), APMV-21 (n=2) and APMV-22 (n=2). Sanger sequencing was performed to provide 22 full-length genome sequences and 35 complete fusion protein gene sequences for the APMV isolates. Phylogenetic analysis showed that the recovered viruses were closely related to Eurasian strains, except five class I APMV-1 and four APMV-4 isolates were related to North America strains. Our findings provided more evidence for the intercontinental transmission of APMVs between Eurasia and North America by wild birds. In addition, according to the criteria of the classification system based on complete fusion protein gene sequences, three novel genotypes within APMV-2, APMV-12, and APMV-22 were identified. Together, this investigation provided a broader perspective on the genetic diversity, evolution, and distribution of APMVs in multiple avian host species sampled in Taiwan. 相似文献
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Pathogenesis of six pigeon-origin isolates of Newcastle disease virus for domestic chickens 总被引：6，自引：0，他引：6
The pathogenesis of six pigeon-origin isolates of Newcastle disease virus (NDV) was investigated in chickens. Four isolates were previously defined as the variant pigeon paramyxovirus 1 (PPMV-1), and two isolates were classified as avian paramyxovirus 1 (APMV-1). Birds inoculated with PPMV-1 isolates were euthanatized, and tissue samples were collected at 2, 5, and 10 days postinoculation (DPI). Birds inoculated with APMV-1 isolates died or were euthanatized, and tissue samples were collected at 2, 4, and 5 DPI. Tissues were examined by histopathology, immunohistochemistry (IHC) for the presence of NDV nucleoprotein, and in situ hybridization (ISH) for the presence of viral mRNA for the matrix gene. Spleen sections were stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and by IHC using an anti-active caspase-3 antibody (IHC-Casp) to detect apoptotic cells. Brain sections of PPMV-1-infected birds were examined by IHC to detect T and B lymphocytes and glial fibrillary acidic protein (GFAP). Histologically, birds inoculated with PPMV-1 isolates had marked lesions in the heart and brain. Presence of viral nucleoprotein and viral mRNA in the affected tissues was confirmed by IHC and ISH, respectively. Numerous reactive astrocytes were observed in brain sections stained for GFAP Among all the isolates, the IHC-Casp demonstrated that apoptosis was very prominent in the ellipsoid-associated cells of the spleen at 2 DPI. Results of the TUNEL assay indicated that apoptotic cells were prominent at 5 DPI and were more randomly distributed. The clinical signs and gross and histopathologic changes observed in the APMV-1-infected birds were characteristic of an extensive infection with highly virulent NDV evident by IHC. 相似文献
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应用ViewRNA技术特异性检测感染细胞中的不同马传染性贫血病毒株 总被引：1，自引：0，他引：1
为了对体外培养细胞感染的不同马传染性贫血病毒(EIAV)株进行特异的鉴别检测,本研究基于一种新的RNA原位杂交-ViewRNA技术,通过设计合成针对EIAVFDDV13和EIAVUK3病毒株基因组RNA的特异性探针,并结合信号放大技术和荧光标记技术,同时通过激光共聚焦显微镜进行观察,建立了可时两种不同株的EIAV基因组RNA进行细胞内原位检测的方法.研究结果表明,两组探针能够特异地与感染细胞中EIAVFDD13和EIAVUK3的病毒RNA相结合,可在两种不同EIAV株共感染的宿主细胞中实现对两种毒株的有效鉴别和定位检测.该方法为进一步研究EIAV与宿主细胞间相互作用提供了有效手段. 相似文献
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鹅新城疫病毒RT-LAMP可视化检测方法的建立 总被引：1，自引：1，他引：0
本研究拟建立一种能在基层实时、便捷诊断鹅新城疫病毒（goose Newcastle disease virus，GNDV）的检测方法。根据GenBank中公布的GNDV F基因的高度同源保守序列设计特异性引物，并在反应体系中添加钙黄绿素/氯化锰指示剂，建立可视化RT-LAMP检测方法。结果显示，建立的方法能特异地检测出GNDV及NDV Lasota疫苗株，而小鹅瘟病毒（GPV）、禽流感病毒（AIV）、鸭坦布苏病毒（DTMUV）等其他病毒均为阴性。所建立的GNDV可视化RT-LAMP检测方法对RNA的最低检测限为10 pg，反应过程不需PCR仪等复杂的仪器，50 min即可完成反应，可经肉眼观察反应体系颜色判定结果。该方法特异性强、灵敏度高，且操作安全、简便、快捷，可满足基层筛查GNDV的需求。 相似文献
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猪链球菌1、7、9型多重PCR诊断方法的建立及初步应用 总被引：3，自引：3，他引：0
为了建立猪链球菌（Streptococcus suis,SS）1、7、9型（SS1、SS7、SS9）的快速鉴别诊断和分型方法,本研究根据GenBank已登录的SS1型特异性的CPS1I基因、7型CPS7H基因、9型CPS9H基因设计引物,以标准SS1、SS7、SS9株基因组DNA为模板,建立了SS1、SS7和SS9的多重PCR检测方法,并进行了特异性、敏感性和重复性试验;利用所建立的多重PCR检测方法对11份猪链球菌的临床分离纯化菌株进行了的应用检测。结果表明,成功建立了SS1、SS7和SS9的多重PCR检测方法,该方法的检测灵敏度可达100 CFU/mL,特异性和重复性好;利用多重PCR检测方法对11份猪链球菌临床分离纯化菌株检测结果表明,11份样品中有3份样品为SS9阳性、2份样品为SS7阳性、1份样品为SS1阳性。本研究为临床上SS1、SS7、SS9的快速鉴别诊断提供了一种新方法。 相似文献
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Chicken anaemia virus
was detectable from various samples such as cell-free virus, infected
cells, unfixed liver homogenates, formalin-fixed liver homogenate or formalin-fixed paraffin-embedded (
) tissues from experimental or field infected chicks using
assay. The detection limit of the first PCR assay was 1 infected cell or 10−1·5,
50 of cell-free virus (strain A2). The nested
assay increased the sensitivity 10- or 100-fold.
was detectable in the other 14 Japanese strains isolated from 1976 to 1994 by the
assay. All the amplified products were digested with BglII, HindIII, PstI and SacI. These results suggest that the region amplified was highly conserved among the strains. The nested
assay was very sensitive. However,
was detectable in most field samples using the first
assay. Therefore, the nested
assay may not always be necessary. In contrast, the nested PCR assay was necessary to detect
in
tissues or formalin-fixed material. Use of the
assay in
detection from
tissues may be most valuable in diagnosis of diseases caused by or associated with
, because it allows detection of both microscopic lesions and
. 相似文献
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《Veterinary microbiology》1997,58(1):23-30
The polymerase chain reaction (PCR) with primers complementary to the 16S rRNA genes was used to detect avian mycoplasmas. A primer pair designed for the detection of human and rodent mycoplasmal species was examined for its ability to detect the most important avian mycoplasmas. After testing the respective reference strains, we found that Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae could be detected by PCR with this primer pair, and distinction could be made among them by restriction fragment length polymorphism (RFLP) assay with two restriction enzymes (BamHI and RsaI). For the detection of Mycoplasma gallisepticum by PCR, we needed species-specific primers. The results of the PCR- and RFLP-based identification procedures of 17 different field isolates agreed with those obtained by conventional methods. 相似文献