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1.
白斑综合征病毒(WSSV)3种PCR检测方法的灵敏度比较   总被引:1,自引:0,他引:1  
为了探讨不同PCR检测方法的灵敏度,分别利用TaqMan实时定量PCR、世界动物卫生组织(OIE)公布的巢式PCR引物(简称OIE)、黄海水产研究所种质资源与工程育种研究室(GB)设计的引物(简称GB)及2种巢式PCR对应的一步法PCR,对具有不同白斑综合征病毒(White Spot Syndrome Virus,WSSV)含量的中国明对虾(Fenneropenaeus chinensis)样品进行检测.结果显示,当使用已知病毒含量的标准品进行检测时,TaqMan实时定量PCR方法可以检测到l0个WSSV拷贝;OIE巢式PCR与GB巢式PCR方法分别可检测到104和103个WSSV拷贝;单独使用OIE巢式PCR的外引物和内引物扩增时,分别可检测到5×104和2.5×104个WSSV拷贝;单独使用GB巢式PCR的外引物和内引物进行一步法PCR扩增时,分别可检测到104和5×103个WSSV拷贝.使用上述PCR方法分别对44份未知WSSV含量的样品进行验证,定量PCR方法检测阳性率为84.09%,OIE巢式PCR与GB巢式PCR方法检测的阳性率分别为18.18%和27.27%;单独使用OIE巢式PCR的外引物和内引物扩增检测的阳性率均为15.91%;单独使用GB巢式PCR的外引物和内引物扩增检测的阳性率分别为18.18%和20.45%.根据以上结果,PCR方法检测WSSV的灵敏度由高到低依次为:定量PCR、巢式PCR、一步法PCR.  相似文献   

2.
为了建立适用于Os HV-1不同变异株的检测方法,在牡蛎疱疹病毒(Os HV-1)3个变异株全基因组序列比对的基础上,筛选到牡蛎疱疹病毒基因组中高度保守的DNA聚合酶(DNA polymerase)基因,据此设计巢式PCR引物,优化PCR反应体系和条件,建立了基于Os HV-1 DNA聚合酶基因的巢氏PCR检测方法(P-n PCR检测方法),利用P-n PCR与Cn PCR检测方法对不同年份和宿主来源的Os HV-1疑似感染样本进行检测。结果显示,Pn PCR检测方法能稳定地检出100拷贝/μL的病毒DNA;P-n PCR较C-n PCR检测方法具有更强的特异性和更高的检出率。研究表明,本研究建立的P-n PCR检测方法适用于Os HV-1不同变异株的检测,可为该病毒的检测和流行病学调查提供可靠的技术支持。  相似文献   

3.
采用PCR方法克隆了传染性胰腺坏死病毒(IPNV) VP3四段相互重叠的基因片段L1、L2、L3和L4,将PCR产物分别连接到原核表达载体pGEX-6P1和pET32a上,经酶切、PCR、测序鉴定,获得重组质粒pGEX-6P1 -VP3(L1)、pET32a-VP3( L2)、pGEX-6P1-VP3( L3)和pGE...  相似文献   

4.
猪圆环病毒2型(Porcine circovirus type 2,PCV2)可以引起猪生殖和呼吸道疾病、断奶仔猪综合征、皮炎和肾炎等,给全球养猪业造成了严重的经济损失。快速、精准的检测方法对该病毒的防控尤为重要。对近年来国内外已发表的PCV2快速检测方法的原理、特点及检测应用进行综述,主要介绍了基于PCR的检测方法(常规PCR、巢式PCR、荧光定量PCR、多重PCR、纳米PCR、数字PCR及其他基于PCR的方法)、核酸等温扩增技术(环介导等温扩增、重组酶介导等温核酸扩增、重组酶聚合酶扩增)、基因芯片、原位杂交、液相芯片系统以及免疫学检测方法(免疫层析技术、酶联免疫吸附试验、琼脂扩散试验、斑点测点法、表面等离子体共振技术)。此外,也对相关方法的未来发展方向进行了展望,以期为我国有效防控PCV2传播和感染提供科学而全面的参考。  相似文献   

5.
<正>鲫造血器官坏死病毒(CYH-2)、鳜鱼虹彩病毒(ISKNV)和白斑综合征病毒(WSSV)是危害高邮市水产养殖生产的三种重要病毒,严重制约我市水产养殖健康发展,目前常用的检测方法为聚合酶链式反应(PCR),此检测方法精确性较高,但用时较长。为了更好地服务于养殖生产,更快速、方便地检测这三种病毒,试验采用恒温荧光PCR仪快速检测方法进行检测,并用聚合酶链式反应(PCR)进  相似文献   

6.
多聚酶链反应(PCR)研究与应用的飞速发展使其成为分子生物学实验室重要的工具,实时定量PCR(Real-time quantitative PCR)技术是20世纪90年代中期发展起来的一种新型核酸定量技术,该技术以其特异性强、灵敏度高、速度快、污染少等优点,促进了PCR技术的发展.实时定量PCR技术在水生动物研究中有着广泛的应用,目前主要集中在病原体检测、定量分析以及基因表达差异等方面.本文对实时定量PCR技术的原理、特点及5种主要的荧光化学作一介绍,对其目前在水生动物研究中的应用进行了概述,并探讨了该技术存在的问题和应用前景.  相似文献   

7.
鳜(Siniperca chuatsi)传染性脾肾坏死病毒(infectious spleen and kidney necrosis virus,ISKNV)是鳜暴发性传染病的主要病原,给鳜养殖业造成了严重的经济损失,因此快速、灵敏的检测方法对鳜养殖业的发展具有非常重要的意义.根据鳜ISKNV主要衣壳蛋白(MCP)基因序列设计了2对引物,利用巢式PCR方法对病鱼脾肾组织进行扩增,建立了ISKNV快速特异的巢式PCR检测方法.应用该方法对含MCP基因的质粒进行倍比稀释后检测扩增的灵敏度可达到5 fg;巢式PCR检测的灵敏度是一步法PCR的104倍以上.  相似文献   

8.
采用人工合成的 6个串联重复寡聚核苷酸单引物 ,以及 2个加锚的二核苷酸引物 ,利用SPAR和ASSR技术 ,对中华绒螯蟹 (Eriocheirsinensis)基因组进行PCR扩增 ,结果发现 ,对于GC含量丰富的三、四核苷酸引物如(CGA) 5、(GACA) 4 ,在不同退火温度的PCR反应中 ,都能扩增出清晰的条带 ,可以作为PCR扩增引物 ;反之 ,GC含量 <5 0 %的 (CATA) 4 和 (GATA) 4 很难检测到扩增产物 ;在基本PCR条件下 ,2个加锚的二核苷酸引物能扩增出可分辨的条带 ,但随着退火温度的升高 ,其中部分条带消失 ;对于不加锚的二核苷酸引物如 (AC) 8、(GA) 8,无论怎样改变退火温度 ,终难形成清晰条带。将不同引物扩增产物分别克隆到T Vector上 ,选取其中 9个阳性克隆进行序列测定 ,发现在 (GACA) 4 引物扩增的 2个片段中 ,序列结构相似 ,除引物序列外 ,都出现了 2~ 3个内部重复序列和高含量的AT ;其余 7个片段在引物序列间都未见内部重复序列  相似文献   

9.
鲤浮肿病(carp edema virus disease,CEVD)是在锦鲤中发现的一种传染性病毒病,近年来在全球传播迅速,对鲤养殖业造成严重损害。本研究设计CEV环介导等温扩增(loop-mediated isothermal amplification,LAMP)引物,进行特异性实验;选取套式PCR、荧光PCR和LAMP三种检测方法,组织10家实验室进行室间比对测试:制备含CEV病毒核酸的测试样品,评价其均匀性和稳定性,通过测试结果分析方法检出限。结果显示,套式PCR的核酸样品检测限可达6.8×10~(-10) g。  相似文献   

10.
对虾IHHNV荧光定量PCR检测方法的建立   总被引:1,自引:0,他引:1       下载免费PDF全文
根据GenBank中对虾传染性皮下和造血器官坏死病毒(IHHNV)保守基因序列(AF218226),设计合成了1对引物和1条TaqMan探针,建立了检测IHHNV的荧光定量PCR技术.将建立的荧光定量PCR检测方法与常规PCR对比.结果显示,所建立的荧光定量PCR方法灵敏度可达2个拷贝,比常规PCR灵敏度高1 000倍.对保存的15份经常规PCR检测为IHHNV阳性的DNA样品进行荧光定量PCR检测,结果都为阳性,检测的病毒含量为2.15×107~4.21×104拷贝/μL.用该方法对不同浓度的样品进行了重复检测,表明该方法具有良好的重复性,可满足IHHNV的临床诊断需要.  相似文献   

11.
Although single nucleotide polymorphisms (SNPs) are important resources for population genetics, pedigree analysis and genomic mapping, such loci have not been reported in Pacific abalone so far. In this study, a bioinformatics strategy was adopted to discover SNPs within the expressed sequences (ESTs) of Pacific abalone, Haliotis discus hannai , and furthermore, polymerase chain reaction direct sequencing (PCR-DS) and allele-specific PCR (AS-PCR) were used for SNPs detection and genotype scoring respectively. A total of 5893 ESTs were assembled and 302 putative SNPs were identified. The average density of SNPs in ESTs was 1%. Fifty-two sets of sequencing primers were designed from SNPs flanking ESTs to amplify the genomic DNA, and 13 could generate products of expected size. Polymerase chain reaction direct sequencing of the amplification products from pooled DNA samples revealed 40 polymorphic SNP loci. Using a modified tetra-primer AS-PCR, seven mitochondrial and six nuclear SNPs were typed and characterized among 37 wild abalones. In conclusion, it is feasible to discover SNPs from number limited ESTs and the AS-PCR as a simple, robust and reliable assay could be a primary method for small- and medium-scale SNPs detection in abalones as well as other non-model organisms.  相似文献   

12.
为开发三疣梭子蟹(Portunus trituberculatus)增殖放流分子标志技术,本研究在前期获得三疣梭子蟹线粒体基因组24个SNP位点的基础上,采用高分辨率熔解曲线(HRM)检测技术对4个用于增殖放流的家系(A、B、C和D)(代表约80~120万个放流个体)进行了鉴别工作。结果显示,含有SNP位点的22条PCR扩增序列中,有9条SNP位点扩增产物在亲代(母本)及其子代的28个个体之间具有基本一致的熔解峰,且子代个体间Tm的均一性较好,无明显差异;进一步以序列已知的野生型及其突变体作为同一SNP引物扩增片段,在各家系间分析HRM标准曲线,这9个SNP可以成功用于三疣梭子蟹4个放流家系的鉴别。在这9个SNP位点中,可鉴定C家系的有5个,可鉴定B家系的有2个,可鉴定A、D家系的各1个。该研究结果为三疣梭子蟹种质资源的鉴定及标志放流工作的开展提供了技术支持。  相似文献   

13.
High‐resolution melting (HRM) has been considered as a fast and simple single‐nucleotide polymorphism (SNP) scanning and genotyping method for identifying differences in the shapes of melting curves between different genotypes. Fifty‐six SNPs were developed in the Pacific oyster, Crassostrea gigas by mining expressed sequence tags database, using the HRM method. The frequency of the SNPs was estimated at 1 per 113 bp of contig sequences. Analysis of segregation in a full‐sib family showed that 28 SNPs were polymorphic, with 15 in accordance to expected Mendelian ratios. Linkage grouping of the 28 markers resulted in six linkage groups. The combined power of exclusion of the 42 SNPs, which conformed to Hardy–Weinberg equilibrium, was greater than 99.98%, while the average polymorphic information content was 0.2223. The simulation results showed that the success rate of parentage analysis could be 97% with the 42 SNPs. These SNPs will be useful for pedigree analysis, association studies, and marker‐associated selection of this species .  相似文献   

14.
利用2b-RAD技术对119尾黄条鰤(Seriola lalandi)个体进行测序,共获得黄条鰤SNP分子标记26665个,对黄条鰤个体的体质量和全长这2个重要生长性状进行全基因组关联分析,筛选与体质量和全长性状相关的SNP位点和候选基因。结果显示,黄条鰤体质量性状中共筛选到17个体质量显著关联的SNP位点,找到17个可能的候选基因,全长性状共筛选到12个潜在显著关联位点,找到12个可能的候选基因。利用KEGG数据库对可能的候选基因进行Pathway分析,得知候选基因主要参与了细胞或组织生长发育相关的代谢通路调控过程,可能是影响黄条鰤生长性状密切相关的重要候选SNP位点和功能基因,结果可为今后黄条鰤种质资源可持续利用和育种提供遗传信息资料积累。  相似文献   

15.
We describe herein the discovery of 3905 putative single-nucleotide polymorphisms (SNPs) from the alignment of 4968 sequences in a bay scallop Argopecten irradians irradians expressed sequenced tag (EST) database. The observed frequency of SNPs was estimated at one every 118.2 bp of contig sequences. Thirty of the SNPs were chosen for validation by tetra-primer amplification refractory mutation system-polymerase chain reaction procedure, and 17 of them were polymorphic with minor allele frequency ranging from 0.016 to 0.484. BLASTX gave significant hits for all but one of the 17 genotyped SNP-containing contigs, 11 of which were located in coding regions and all resulted in a synonymous substitution. Parentage simulation demonstrated that SNP markers were more sensitive to the number of parents compared with microsatellites, thus more SNPs were needed to counteract low polymorphism. A table of optimal codons was deduced from the analysis of the A. i. irradians EST dataset to explain the frequency difference for a specific SNP. It seems that the selection of codon usage may be partly responsible for the frequency difference between the two alleles of the coding SNPs. These are the first SNPs developed for the bay scallop and will provide a useful complement to currently available genetic markers.  相似文献   

16.
基于EST序列的鲤鱼生长相关SNP发掘   总被引:1,自引:0,他引:1  
以本研究室454测序获得的EST序列和公共数据库中下载的共255,768条鲤鱼EST序列为源序列,鉴定出与生长相关的EST序列5,375条;采用QualitySNP软件,在31个EST重叠群中检测到69个可信度高的与生长相关的SNP,SNP发生频率为0.29/100个碱基。其中包括转换型35个,颠换型30个和缺失型4个。非同义SNP为46个,同义SNP为23个,二者比值为2.0。对所鉴定的SNP进行注释表明,这些包含SNP的生长相关基因归为20类,数量最多的为血纤维蛋白溶酶原基因4个、其次是载脂蛋白基因和磷酸甘油醛脱氢酶基因分别为3个。本研究所开发的SNP将促进鲤鱼生长性状的分子基础研究,为鲤鱼分子育种服务。  相似文献   

17.
Polymorphisms in the growth hormone (GH) gene that is associated with the growth rate of farmed fish have been the target of many breeding programmes. The present study aimed to identify single nucleotide polymorphisms (SNPs) in GH gene regions to evaluate the association of SNP variations with the growth rate of two Nile tilapia: Oreochromis niloticus (Linnaeus, 1758) strains. The targeted regions were amplified, sequenced, aligned and screened for the presence of SNPs; thereafter, performance tests were used to check for the association between SNPs and weight. Allele and genotype frequencies were estimated for each SNP and genotype. Genotype blocks or sets of SNP genotypes and frequencies were also estimated. Association between SNPs and growth rate was statistically evaluated using a univariate linear mixed model that included both fixed and random effects. A total of 10 SNPs were identified, nine in the proximal promoter and one located in the 5′ UTR, forming 10 genotype blocks. In all weight recordings, five genotype blocks were significantly associated with the highest weights. Single nucleotide polymorphisms 6‐10 were also found to be significantly associated with growth (p‐value < .05). Genotypes with higher additive genetic values for weight were identified in the Chitralada strain, suggesting a possible impact of these additive effects of the GH SNP genotype on the growth rate of Nile tilapia. These findings may be used as part of marker‐assisted selection in tilapia breeding programmes.  相似文献   

18.
磁珠富集法随机筛选大菱鲆基因组SNP标记   总被引:1,自引:0,他引:1  
利用磁珠富集法随机筛选了大菱鲆(Scophthalmus maximus)基因组7820bp的序列,获得了35个SNP标记,SNP标记的含量约为0.448%。超过68%的SNP标记由碱基转换造成,不足29%的SNP标记由碱基的颠换造成。使用磁珠富集法对目的基因KC70的检测发现,725bp的片段上发现7个SNP标记。此结果证实,该方法不仅能够随机筛选基因组SNP标记,还能筛选目的基因的SNP标记。  相似文献   

19.
利用2b-RAD技术对119尾黄条[鱼师](Seriola lalandi)个体进行测序,共获得黄条[鱼师]SNP分子标记26665个,对黄条[鱼师]个体的体质量和全长这2个重要生长性状进行全基因组关联分析,筛选与体质量和全长性状相关的SNP位点和候选基因。结果显示,黄条[鱼师]体质量性状中共筛选到17个体质量显著关联的SNP位点,找到17个可能的候选基因,全长性状共筛选到12个潜在显著关联位点,找到12个可能的候选基因。利用KEGG数据库对可能的候选基因进行Pathway分析,得知候选基因主要参与了细胞或组织生长发育相关的代谢通路调控过程,可能是影响黄条[鱼师]生长性状密切相关的重要候选SNP位点和功能基因,结果可为今后黄条[鱼师]种质资源可持续利用和育种提供遗传信息资料积累。  相似文献   

20.
为筛选刀鲚(Coilia ectenes)生长性状遗传改良的分子遗传标记,基于现有刀鲚转录组数据,利用基因克隆技术获得养殖刀鲚肌肉生长抑制素(myostatin,MSTN)基因的全长序列,采用PCR产物直接测序和SNaPshot技术分别进行基因多态性的筛选和分型,并将不同基因型与刀鲚的生长性状(体长、体高和体质量)进行关联分析。结果显示,养殖刀鲚MSTN基因的DNA序列主要由2个内含子和3个外显子组成,经SNP位点筛选,仅在第1内含子上筛选到2个颠换突变位点(g.564G>T和g.755G>T)和1个转换突变位点(g.786C>T);将3个SNP位点与生长性状进行关联分析发现,SNP位点g.786C>T与刀鲚的体长、体高和体质量呈显著性负相关,具有TT基因型个体的体长、体高和体质量显著低于CC型和CT型(P<0.05),而SNP位点g.564G>T和g.755G>T的不同基因型仅与刀鲚的体高具有一定程度的相关性;3个SNP位点对养殖刀鲚的群体遗传分析显示,刀鲚养殖群体的观测杂合度(H O)、期望杂合度(H E)和多态信息含量(PIC)分别为0.280~0.480、0.332~0.455和0.277~0.351,3个SNP位点均属于中度多态(0.25T有望作为剔除劣质刀鲚繁育亲本的候选分子标记。  相似文献   

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