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11.
西尼罗热病毒RT-PCR检测方法的建立 总被引:3,自引:0,他引:3
参考Genebank发表的西尼罗热病毒(West Nile virus,WNV)E糖蛋白基因序列,自行设计合成一对引物,对WNV进行RT—PCR扩增,产物经琼脂糖电泳分析,呈现一条约400bp的条带,将其克隆入pMD18-T—Vector载体中,并进行序列测定,与已发表的WNV基因比较发现,核苷酸的同源性为99.7%,证实为WNV的E基因,通过对样品多次检测,都能扩增出一条约400bp的条带,表明该方法比较稳定。 相似文献
12.
Kai WANG Hongze SHAO Zhihua PEI Guixue HU 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2016,78(1):125-128
The aim of this experiment was to develop a loop-mediated isothermal amplification (LAMP)
assay and to research the recent epidemiology of contagious ecthyma in Jilin Province,
China, using the assay. A LAMP assay targeting a highly conserved region of the F1L gene
was developed to detect contagious ecthyma virus (CEV). Three hundred and sixty-five cases
from 64 flocks in 9 different areas of Jilin Province, China, from 2011 to 2014 were
tested using the LAMP assay. The results showed that the sensitivity of the LAMP assay was
100 copies of the standard plasmid, which is 100-fold higher than the sensitivity of PCR.
No cross-reactivity was observed with capripoxvirus, fowlpox virus, foot-and-mouth disease
virus serotype O, foot-and-mouth disease virus serotype Asia I and bluetongue virus. The
average positive rate was 19.73% (72/365), and the positive rate was highest in lambs aged
1–6 months. Our results demonstrated that CEV infection was very widespread in the flocks
of Jilin Province and that the LAMP assay allows for easy, rapid, accurate and sensitive
detection of CEV infection. 相似文献
13.
不同的样本特性和提取方法对获得微生物总DNA的质量有重要影响。文章基于高含固率木质纤维素厌氧发酵物腐殖酸、酚类物质含量高、质地均一性差、微生物浓度低的特点,研究了4种方法提取不同高含固率粪秸厌氧发酵物中微生物总DNA的效果。结果表明,常规的十二烷基磺酸钠法(sodium dodecyl sulfate,SDS)、十二烷基磺酸钠和溴化十六烷基三甲铵结合法(sodium dodecyl sulfate and cetyltrimethyl ammonium bromide,SDS-CTAB)和商业的粪便试剂盒法提取的DNA质量均较差,SDS法和试剂盒法未能获得聚合酶链式反应(polymerase chain reaction,PCR)扩增目的条带,SDS-CTAB法得到的条带较模糊;改进SDS-CTAB法获得的DNA杂质少、纯度高,具有较好的稳定性,A260/A280和A260/A230值分别为1.74~1.86和1.65~1.86,每克样品的DNA浓度在50 ng·μL^-1以上,电泳条带单一齐整、清晰明亮,PCR扩增的目的条带清晰度高,适宜后续分子生物学技术的分析。林格氏液洗脱、聚乙烯吡咯烷酮-40(Polyvinyl Pyrrolidone-40,PVP-40)洗涤液除杂以及裂解液和多种酶联合破壁是改进SDS-CTAB法获得该类专一性样本高质量微生物总DNA的关键步骤。 相似文献
14.
RJ Barnewall IB Marsh PMV Cusack F Galea N Sales JC Quinn 《Australian veterinary journal》2023,101(6):254-257
Bovine respiratory disease (BRD) exerts a major impact on the beef cattle industry nationally and worldwide, with a range of aetiological factors impacting its pathogenesis. Previous research has focussed on an increasing number of bacteria and viruses that have been shown to play a role in eliciting disease. Recently, additional agents have been emerging as potential contributors to BRD, including the opportunistic pathogen Ureaplasma diversum. To determine if U. diversum was present in Australian feedlot cattle and if that presence was linked to BRD, nasal swabs were collected from a cohort of 34 hospital pen animals and compared to 216 apparently healthy animals sampled contemporaneously at feedlot induction and again after 14 days on feed at an Australian feedlot. All samples were subjected to a de novo polymerase chain reaction (PCR) assay targeting U. diversum in combination with other BRD agents. U. diversum was detected at a low prevalence in cattle at induction (Day 0: 6.9%, Day 14: 9.7%), but in a significantly greater proportion of cattle sampled from the hospital pen (58.8%). When considering the presence of other BRD-associated agents, co-detection of U. diversum and Mycoplasma bovis was most common in hospital pen animals receiving treatment for BRD. These findings suggest that U. diversum may be an opportunistic pathogen involved in the aetiology of BRD in Australian feedlot cattle, in combination with other agents, with further studies are warranted to identify if a causal relationship exists. 相似文献
15.
16.
为研究PRRSV N蛋白的结构、功能以及N蛋白在病毒致病中的作用,以临床分离PRRSV毒株E11105为研究对象,采用Primer Premier 5.0设计一对特异性引物,经RT-PCR扩增出N基因片段,利用相关分子生物学软件对N基因序列进行分析;将N基因克隆连接到pColdⅠ原核表达载体上,经PCR、双酶切鉴定及序列测定后,得到重组质粒pColdⅠ-N,将pColdⅠ-N转入大肠埃希菌BL21(DE3)感受态细胞中,IPTG诱导表达后用SDS-PAGE蛋白电泳及Western blot验证分析。结果显示,分离株E11105N基因与北美洲型代表株VR-2332、欧洲型代表株Lelystad virus(LV)、中国2006年暴发的高致病性PRRSV代表株JXA1、中国代表株CH-1a的核苷酸序列同源性分别为93.3%、34.7%、99.2%、95.4%,氨基酸序列同源性分别为94.4%、15.3%、94.4%、99.2%;系统进化树显示,E11105株N基因与美洲型代表株VR-2332、中国高致病性JXA1毒株的亲缘关系较近;分离株E11105N基因所编码蛋白不存在跨膜区;二级结构主要以α-螺旋和无规则卷曲为主,分别占20.33%和63.41%;预测该蛋白可能存在5个较为明显的B细胞优势抗原表位。SDS-PAGE蛋白电泳结果表明,重组N蛋白主要存在于菌体沉淀中,分子质量约为16.7ku;Western blot结果显示,带His标签的重组表达蛋白能被His单克隆抗体识别,显色后条带约为16.7ku,与SDS-PAGE蛋白电泳的条带大小一致。 相似文献
17.
18.
By using RACE (rapid amplification ofcDNA ends) based homologous cloning strategy, we have successfully isolated the genomic and full-length cDNA sequences of a gene encoding typical DFR (dihydroflavonol-4-reductase) from black-seeded Brassica campestris L. var. oleifera DC.. The gene, designated BcDFR here, is 1 722bp in length and harbors 5 introns with typical splice sites of plant DFR genes. BcDFR cDNA is 1311bp in length with a 1 158bp ORF as well as a 25bp 5‘ UTR and a 128bp 3‘ UTR. The encoded BcDFR protein is 385 aa with a calculated Mw of 42.85kD and a pI value of 5.55. The nucleotide and amino acid sequences of this gene share extensive homologies to plant DFR genes of wide origins especially high similarities to Cruciferous DFR genes. Sequence analyses such as phylogenetic analysis, conserved domain search and substrate specificity region detection all indicated that BcDFR gene is a quite potentially biofunctional gene. Its cloning enables us to further dissect the possible relatedness between DFR gene and Brassica seed coat color traits and to create transgenic novel yellow-seeded rapeseed germplasm through antisense- or RNAi-suppression of DFR gene expression in black-seeded elite cultivars. 相似文献
19.
以冻融法快速纯化高活力基因工程Taq DNA聚合酶 总被引:1,自引:0,他引:1
用含有TaqDNA聚合酶基因的pTaq表达质粒转化E.coli DH5α菌株,IPTG诱导表达Taq DNA聚合酶,利用该酶的热稳定性,反复2次-70℃,75℃交替冻融以裂解细胞释放胞浆;以高速离心除去冻融变性的细胞碎片及核酸蛋白的复合物以达到快速纯化Taq DNA聚合酶的目的。PCR扩增反应和SDS-PAGE分析表明所制备的Tap DNA聚合酶的纯度,活力,敏感性,特异性均达到试验要求。该方法具有快速简便的优点。 相似文献
20.
Wang De-fu Zhang Xi-mei Guo Shang Shen Shao-fei Long Dan-dan Li Ling-yu Niu Yan-bing 《东北农业大学学报(英文版)》2021,28(3):58-65
Using double-stranded RNA(dsRNA) technology and sequence-independent amplification(SIA), the molecular identification on infected Rehmannia glutinosa in the field with mosaic symptoms was performed and the whole-genome of the Rehmannia mosaic virus(ReMV) Shanxi isolate(ReMV-SX) was sequenced. Sequencing analysis showed that the virus that infected Rehmannia glutinosa was Rehmannia mosaic virus(ReMV). The full-length of the obtained ReMV-SX sequence(GenBank accession no. JX575184) was 6 395 nt, containing four open reading frames(ORFs). The sequence homology analysis of the complete nucleotide sequence showed that ReMV-SX was 93.8%-97.0% homologous to ReMV in Tobamovirus subgroup I, while only 49.8%-58.9% homologous to the isolates in subgroups II and III of the same genus. Phylogenetic analysis showed that ReMV-SX and ReMV-Henan formed a separate branch and had the closest genetic relationship. The results laid the foundation for ongoing researches in the taxonomic status and evolution of ReMV and for further investigating the pathogenic mechanism of ReMV infecting Rehmannia glutinosa. 相似文献