共查询到18条相似文献，搜索用时 797 毫秒
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RNA提取是诺如病毒检测的关键步骤,而目前贝类中诺如病毒RNA提取、检测方法的比较与评价常囿于缺乏量值明确、无生物安全隐患的标准样品作为参考依据。本研究将前期制备的 GⅡ型诺如病毒装甲RNA (3.0×1010拷贝)作为标准样品,人工污染牡蛎(Ostrea gigas tnunb)消化腺匀浆物,用4种常见RNA提取方法：TRIzol试剂、Viral RNA Kit、High Pure Viral Nucleic Acid Kit、柱式病毒RNAOUT试剂盒,分别提取RNA,经实时荧光RT-PCR检测后,利用标准曲线进行定量分析,分别计算4种方法对装甲RNA的回收率。结果显示,对于匀浆样本,TRIzol法对装甲RNA的回收率最高(6.80±0.89)%,显著高于Viral RNA Kit (4.51±2.28)%,二者的回收率又显著高于High Pure Viral Nucleic Acid Kit (0.24±0.05)%与柱式病毒RNAOUT试剂盒(0.11±0.02)% (P?0.05);对于冻干样本,Viral RNA Kit对装甲RNA的回收率最高(8.71±0.17)%,显著高于TRIzol试剂(7.12±0.64)%,二者的回收率又显著高于High Pure Viral Nucleic Acid Kit (0.33±0.12)%与柱式病毒RNAOUT试剂盒(0.06±0.01)% (P?0.05)。研究表明,TRIzol试剂与Viral RNA Kit对牡蛎消化腺样本中人工添加的装甲RNA均有良好的回收效果,同时也提示装甲RNA可作为一种良好的标准样品用于不同RNA提取试剂盒方法的评价与比较研究。 相似文献
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为提取高质量的坛紫菜叶状体总RNA,对常用的几种植物RNA提取方法(CTAB法、SDS法、异硫氰酸胍法)按照去除多糖、多酚的方法进行了改良,并对分离的总RNA根据吸光值、电泳图谱及RT-PCR检测等结果与两种试剂盒(离心柱试剂盒法,RNAiso法)的提取结果进行了比较。结果表明,SDS法、异硫氰酸胍法和RNAiso法3种方法提取的RNA纯度低,质量差,部分RNA已经发生了降解。而改良CTAB法和离心柱试剂盒法提取的总RNA质量可靠,完整性好,纯度高,并且成功去除了可能影响逆转录酶活性的物质,可以进一步应用于cDNA文库构建,基因表达分析等后续实验,但这两种方法各有优缺点,应根据实际情况选用合适方法进行坛紫菜叶状体总RNA的分离。 相似文献
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获得高质量的RNA是逆转录聚合酶链式反应(RT-PCR)、Northern blot以及转录组测序(RNA-Seq)等分子生物学研究的基础,由于草鱼脂肪细胞中油脂含量较高,从中提取高质量RNA存在困难。本研究以草鱼(Ctenopharyngodon idellus)成熟脂肪细胞为材料,通过增加离心和抽提次数等方式对传统总RNA提取方法进行优化,采用琼脂糖凝胶电泳、核酸定量分析及基因扩增等方法验证所得RNA的完整性、纯度及质量。结果表明,经此方法提取的草鱼成熟脂肪细胞总RNA的A260 nm/A280 nm比值在1.95到2.00之间,28S和18S条带完整,草鱼LPL和β-actin基因扩增条带清晰。认为采用本方法获得的草鱼脂肪细胞RNA样品质量较高,能够用于后续分子生物学研究。 相似文献
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草鱼体组成的数学描述 总被引：1，自引：1，他引：0
为了对草鱼体组成进行定量描述,本研究从中外文数据库收集并采纳了51个草鱼营养生理相关研究的数据,数据点约3700个,草鱼体质量为1.52~694.80 g。通过数据整理、相关性分析和线性回归分析,结果显示,草鱼蛋白质含量和内脏重(y,g)与体质量(x,g)间的线性关系分别为y=0.1604x–0.3645,R2=0.994;y=0.1059x–0.3097,R2=0.9875。随着草鱼体质量增加,草鱼脂肪和灰分含量(尤其是脂肪含量)受饲料组成的影响逐渐增加。草鱼全鱼每沉积1 g蛋白质伴随着4.57 g水分保留,而每沉积1 g脂肪会导致水分含量减少0.95 g。草鱼肝脏每沉积1 g脂肪会导致其水分含量减少0.66 g,说明草鱼不同组织沉积脂肪导致的水分损失率不尽相同。本研究亦表明,肠系膜是草鱼脂肪沉积的重要部位,肠系膜、肝脏和肌肉脂肪的积累是全鱼脂肪含量上升的重要原因,全鱼脂肪累积伴随着内脏重的增加。本研究的执行有利于定量描述草鱼体组成规律,为草鱼的生产和销售提供指导作用。 相似文献
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应用Trizol一步法对尼罗罗非鱼(Oreochromis niloticus)外周血白细胞总RNA提取方法进行优化,通过β-ac-tin基因反转录对所分离到的总RNA进行质量验证,并对mRNA的冻干浓缩进行了探讨,确定了一套快速、高效的尼罗罗非鱼外周血白细胞总RNA分离和mRNA浓缩体系。结果显示:获得的总RNA纯度较高,OD260/OD280均在1.9~2.0之间;甲醛变性电泳显示,总RNA完整性良好,28S与18S比值为2∶1;总RNA的分离量与起始白细胞量成正比,每毫升Trizol最大裂解量为1×108个白细胞,可得到总RNA量约为90μg;总RNA的分离量在前四个小时随着沉淀时间的增长而增加,并成功进行了β-actin基因的反转录。mRNA的分离率为1.08%,通过冻干浓缩成功使其浓度提高9倍。结果表明:分离到的总RNA质量可充分满足下游分子生物学实验要求,所建立的mRNA浓缩方法简单易行,可完全解决mRNA起始浓度低的难题。 相似文献
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为了精确且批量测定草鱼肠系膜脂肪（肠脂）沉积量，避免传统人工刮取称量方法耗时费力、量化粗糙等问题，本研究利用脂溶性染料油红O可特异性着色脂肪的特性，探索开发出一种便捷定量草鱼肠脂含量的新方法。测试发现，麻醉解剖200尾平均体质量约80 g的草鱼，取出整个内脏团简单处理并塞入PIT个体识别标记后，样品集中进行多聚甲醛固定、无水乙醇脱水、油红O染液定染，可在维持样品组织完整的基础上，实现肠脂组织的特异性、均一化批量染色处理。各染色样品再分别通过无水乙醇溶剂完全萃取，萃取液吸光度测定，并依据绘制的标准曲线（y = 0.0276x + 0.0403, R2 = 0.9997），即可精确获得个体肠脂沉积的相对含量，是以萃取出的油红O质量来表示。对比发现，样品肠脂组织染色-萃取量化结果与传统刮取称量数据保持了较高的相关性（n = 20，r = 0.80）。进一步的统计分析显示，对于同塘养殖体质量变异系数8.93%的草鱼群体（n = 200），萃取测定的肠脂沉积量变异系数达到24.49%，预示该性状具有丰富变异特征及遗传改良潜力。相关与聚类分析显示，肠脂沉积量与内脏质量相关性最高（r = 0.60），并且聚为一类，符合二者同属脏器关联指标的预期。多元线性回归分析显示，利用简单易测的形态指标只能解释肠脂沉积量的少量变异（R2 = 0.20），表明基于表型性状的拟合回归方程进行间接预测的效果不佳，直接测定是该性状精准量化的有效途径。本研究为草鱼体脂性状改良提供了一种性状精确测定方法。 相似文献
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草鱼出血病病毒854株基因组SDS—PAGE分析及其核酸类型鉴定 总被引：3，自引：0，他引：3
十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结果表明:草鱼出血病病毒854株基因组由11个分段的核酸片段组成,总分子量约为14.46×10~6道尔顿。其中最大片段的分子量约为2.45×10~6道尔顿;最小片段的分子量约为0.38×10~6道尔顿。其基因组核酸片段电泳图谱具有病毒株的特异性.核酸酶敏感性试验表明该病毒的核酸为RNA。 相似文献
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Effects of dietary non‐protein energy source levels on growth performance,body composition and lipid metabolism in herbivorous grass carp (Ctenopharyngodon idella Val.) 
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下载免费PDF全文 Xiaoze Guo Xu‐Fang Liang Liu Fang Xiaochen Yuan Yi Zhou Jin Zhang Bin Li 《Aquaculture Research》2015,46(5):1197-1208
A study was conducted to determine the effects of dietary non‐protein energy sources on growth, tissue lipid accumulation and lipid metabolism‐related genes expression of grass carp. Triplicate groups of fish were fed for 9 weeks on four isonitrogenous (300 g kg?1) experimental diets with four levels of non‐protein energy (6.52 kJ g?1 control diet, 5.32 kJ g?1 high‐CEL diet, 8.46 kJ g?1 high‐CHO diet and 8.53 kJ g?1 high‐LIP diet respectively). Increasing dietary non‐protein energy source levels did not improve the growth, and the high‐CEL diet reduced the growth of grass carp. The high‐CHO diet tended to induce high hepatosomatic index, with high fat and glycogen content of liver. However, the high‐LIP diet caused the high mesenteric fat index, but did not increase liver fat. The mRNA abundance and activities of hepatic lipogenic enzymes were significantly increased in the high‐CHO diet group, whereas the opposite tendencies were observed in the high‐LIP diet group. Peroxisome proliferator‐actived receptor‐α (PPARα) in liver and PPARγ in mesenteric adipose tissue were up‐regulated in the high‐CEL diet group. Lipoprotein lipase (LPL) gene expression was significantly increased both in liver and mesenteric adipose tissue of fish fed the high‐LIP diet, while the LPL gene expression was up‐regulated in liver but down‐regulated in mesenteric adipose tissue of fish fed the high‐CEL diet. These findings suggest that an increase in dietary non‐protein energy sources alters the genes expression of lipid metabolism and increased lipid deposition. 相似文献
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Taylor W. Guest Reginald B. Blaylock Andrew N. Evans 《Fish physiology and biochemistry》2016,42(1):1-6
The corticosteroid hormone cortisol is the central mediator of the teleost stress response. Therefore, the accurate quantification of cortisol in teleost fishes is a vital tool for addressing fundamental questions about an animal’s physiological response to environmental stressors. Conventional steroid extraction methods using plasma or whole-body homogenates, however, are inefficient within an intermediate size range of fish that are too small for phlebotomy and too large for whole-body steroid extractions. To assess the potential effects of hatchery-induced stress on survival of fingerling hatchery-reared Spotted Seatrout (Cynoscion nebulosus), we developed a novel extraction procedure for measuring cortisol in intermediately sized fish (50–100 mm in length) that are not amenable to standard cortisol extraction methods. By excising a standardized portion of the caudal peduncle, this tissue extraction procedure allows for a small portion of a larger fish to be sampled for cortisol, while minimizing the potential interference from lipids that may be extracted using whole-body homogenization procedures. Assay precision was comparable to published plasma and whole-body extraction procedures, and cortisol quantification over a wide range of sample dilutions displayed parallelism versus assay standards. Intra-assay %CV was 8.54 %, and average recovery of spiked samples was 102 %. Also, tissue cortisol levels quantified using this method increase 30 min after handling stress and are significantly correlated with blood values. We conclude that this modified cortisol extraction procedure provides an excellent alternative to plasma and whole-body extraction procedures for intermediately sized fish, and will facilitate the efficient assessment of cortisol in a variety of situations ranging from basic laboratory research to industrial and field-based environmental health applications. 相似文献
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Abstract. An improved technique for isolating infectious pancreatic necrosis virus (IPNV) is described. It involves trypsinization of the suspect tissue, separation of the trypsinized cells and their co-cultivation with indicator RTG-2 cells. This co-cultivation method is compared with homogenization, followed by membrane filtration, a procedure routinely employed by most disease diagnostic laboratories. From the two IPN-infected farms studied, IPNV could be isolated by homogenization from only 50 and 66%, respectively, of the fish from which virus was detected by co-cultivation. This difference in sensitivity can be explained in two ways, viz: (a) some of the virus is being retained by the membrane filter during filtration to remove contaminants following homogenization; (b) the presence of neutralizing factors in the tissue homogenate are neutralizing the virus and thus no cytopathic effect ensues. No correlation between serum neutralizing antibody levels and the differences in sensitivity of the two methods was found. The co-cultivation method is as practical as, and certainly no more time-consuming than, the homogenization technique. 相似文献
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The object of this study was to improve the isolation procedure of hyaluronan and to compare characteristics of hyaluronan
from the eyeball of bigeye tuna Thunnus obesus with other sources. General sources of hyaluronan are from Streptococcus zooepidemicus and rooster comb. Hyaluronan can be also obtained from the vitreous of fish eyes. Pure hyaluronan of higher molecular weight
was obtained by the following improved extraction procedure: the frozen vitreous of a tuna eyeball was used to avoid contamination
with blood, muscle tissue, and other factors; extracting was carried out over a long time period under cold conditions; cetylpyridinium
chloride was used in order to separate mucopolysaccharides containing hyaluronan in the initial procedure without the process
of removing fat and protein by reagents. The hyaluronan obtained was characterized by gel permeation chromatography, dynamic
light scattering measurements, and viscometry. The characteristics of hyaluronan from tuna eyeballs were similar to those
from other sources. However, the viscosity was lower. The possible reason could be ascribed to the wide distribution of molecular
size in the vitreous humor of fish eye. 相似文献
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三角帆蚌外套膜细胞体外培养优化及体内植入培养对细胞生长的影响 总被引：2，自引：1，他引：1
以DMEM为基础培养基, 通过优化改良培养基及缓冲液的配方, 对三角帆蚌(Hyriopsis cumingii Lea)外套膜进行细胞培养, 并且以显微观测、RNA/DNA的比值作为该细胞增殖的评价指标, 分别对体外培养细胞的迁出时间、速度、数量以及增殖活力进行测定。分别取体外培养第24、108、120 小时的细胞进行Hoechst DNA荧光标记, 然后将3组标记细胞植入三角帆蚌外套膜中在蚌体内培养, 在植入后第24、72、120、168、216 小时运用RNA/DNA指标检测活体培养的细胞增殖活力。结果表明, 经过优化后的缓冲液和培养基更有利于细胞从外套膜组织中迁出, 迁出速度和细胞数量显著增加(P<0.05), 且细胞的活力随着培养时间的延长而逐渐增大, 培养至108 h时, 细胞活力达到最大, RNA/DNA比值为 24.53, 在108 h后显著下降(P<0.05), 显微观测的细胞生长状况与RNA/ DNA指标测定相吻合。对体外培养的细胞植入蚌体外套膜中再进行培养时发现, 对体外培养活力较好的细胞, 活体内环境可增大细胞的活力, RNA/DNA比值最高达到25.45, 但在活体培养168 h 后活力显著下降(P<0.05); 而对体外培养活力较差的细胞, 活体内环境对细胞活力影响不显著(P>0.05)。本研究旨在为三角帆蚌外套膜细胞增殖的深入研究及建株提供基础性资料。
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根据鲤热休克蛋白70(Heat shock protein,HSP70)序列(AY120894)设计并合成一对引物,以草鱼(Cteno-pharyngodon idella)肝胰脏组织总RNA为模板,RT-PCR扩增获得草鱼HSP70基因cDNA部分序列,并进行了组织表达差异性研究。结果显示:所获为序列为480 bp,获得GeneBank登陆号为FJ483832。序列测序结果显示,HSP70扩增序列与鲤、斑马鱼、鲋的同源性为:93%、91%、93%。另外,所获序列HSP70在草鱼脂肪、肌肉、肠、脑、粘液、性腺、鳔、肝胰脏、心脏、脾脏、鳃、鳍12个组织的表达存在差异,HSP70在草鱼这12个组织中均检测到表达,其中在鳍中表达最高,极显著高于其他组织(P<0.01);在鳔中表达次之,且与脑、心脏、性腺中表达差异不显著;在粘液中表达最低。 相似文献