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1.
鸡β-actin基因实时荧光定量PCR方法的建立   总被引:12,自引:1,他引:11       下载免费PDF全文
根据GenBank上鸡β-actin基因的序列,在保守区域设计并合成一对引物,采用SYBR Green I染料建立了荧光定量PCR法(real-time PCR)。以常规PCR产物为标准品,建立了标准曲线,并进行了熔解曲线分析。结果表明,标准曲线Ct值检测范围为12~31,扩增效率为95.1%;熔解曲线分析结果显示产物特异的单个峰,其Tm为88±0℃,检测周期从RNA提取到荧光定量PCR结束只需4 h。本试验建立的鸡β-actin基因实时荧光定量PCR法扩增效率高、检测范围广、检测周期短,为β-actin基因作为内参基因进行鸡功能基因与病原基因表达的定量分析奠定了基础。  相似文献
2.
Background — The harp seal (Phoca groenlandica) is one of the most important predators in the Northeastern Atlantic ecosystem. Establishing biochemical reference intervals is important for evaluating the health status of harp seals kept in captivity and for evaluating the effects of environmental changes on the health of populations in the wild. Objective — The purpose of this study was to determine reference values for serum biochemical parameters in wild adult harp seals using readily available current methods. Methods — Blood samples were obtained from 14 adult female harp seals and 9 suckling pups on the pack ice of the Greenland Sea in early March 1998. Seven seals were humanely killed on the ice by permission of the Norwegian Directory for Fisheries and in conjunction with several other research projects. The seals were sampled within 15 minutes postmortem. Remaining seals were captured alive and sampled via the extradural intravertebral vein. Serum biochemical parameters were measured using a Technicon Axon analyzer and included electrolytes (sodium, chloride, potassium, magnesium, and calcium), substrates (free fatty acids, triglycerides, fructosamine, and glucose), end products (urea and uric acid), and proteins (total protein, globulins, and albumin). Serum protein electrophoresis also was done. Data were tested for normality and reference limits were calculated as mean ±1.96 × SD. Results between groups were compared using 2‐tailed t‐tests. Results — Serum levels of glucose and triglycerides were lower, but serum levels of urea were higher in dead animals than in animals that were captured alive. Serum levels for 7 of 17 parameters were significantly different in pups compared with adults. Separate reference intervals were calculated for adult seals and seal pups. Conclusion — Both sampling method and age should be considered when evaluating the results of analysis of serum parameters in wild and captive harp seals.  相似文献
3.
家兔GAPDH基因实时荧光定量RT-PCR方法的建立   总被引:4,自引:0,他引:4       下载免费PDF全文
根据GenBank登录的家兔GAPDH基因保守区域CDS序列设计1对引物,通过反应体系及反应条件优化,成功建立了检测家兔GAPDH基因的SYBR Green Ⅰ荧光定量RT-PCR方法。结果表明,该方法检测GAPDH的最低拷贝数为32拷贝/μL,在较广的范围内(3.2×101~3.2×107 拷贝/μL)有良好的线性关系(r=0.999);熔解曲线分析显示扩增产物的特异性单峰,其Tm为(87±0.2) ℃;5个不同浓度标准品组内试验变异系数为1.67%~4.73%,组间试验变异系数为2.66%~8.74%。该方法具有快速、灵敏、高通量及可重复性强等优点,为GAPDH基因作为内参基因进行家兔功能基因与病原基因表达的定量分析提供了方法学基础。  相似文献
4.
应用心电图分析肉鸡腹水症   总被引:3,自引:2,他引:1  
对两批肉鸡(一批120只,另一批60只)进行心电图监测,每周测1次,每次每批15只,最后对确定为腹水症的肉鸡心电图进行统计分析。结果表明,腹水症病鸡心电图Ⅱ、Ⅲ导联的平均组合向量(MRV)、心功能指数(CI)和Ⅱ导联S波振幅(Ⅱ-S)极显著地高于心扩张病鸡和对照组肉鸡(P<0.01);心扩张病鸡亦极显著地高于对照组肉鸡(P<0.01)。因此,肉鸡心电图Ⅱ、Ⅲ导联的MRV、CI和Ⅱ-S可作为诊断肉鸡  相似文献
5.
Method comparison in the clinical laboratory   总被引:2,自引:0,他引:2  
Studies comparing a new method with an established method, to assess whether the new measurements are comparable with existing ones, are frequently conducted in clinical pathology laboratories. Assessment usually involves statistical analysis of paired results from the 2 methods to objectively investigate sources of analytical error (total, random, and systematic). In this review article, the types of errors that can be assessed in performing this task are described, and a general protocol for comparison of quantitative methods is recommended. The typical protocol has 9 steps: 1) state the purpose of the experiment, 2) establish a theoretical basis for the method comparison experiment, 3) become familiar with the new method, 4) obtain estimates of random error for both methods, 5) estimate the number of samples to be included in the method comparison experiment, 6) define acceptable difference between the 2 methods, 7) measure the patient samples, 8) analyze the data, and 9) judge acceptability. The protocol includes the essential investigations and decisions needed to objectively assess the overall analytical performance of a new method compared to a reference or established method. The choice of statistical methods and recommendations of decision criteria within the stages are discussed. Use of the protocol for decision-making is exemplified by the comparison of 2 methods for measuring alanine aminotransferase activity in serum from dogs. Finally, a protocol for comparing simpler semiquantitative methods with established methods that measure on a continuous scale is suggested.  相似文献
6.
Background: The ADVIA 120 is a laser-based hematology analyzer with software applications for animal species. Accurate reference values would be useful for the assessment of new hematologic parameters and for interlaboratory comparisons.

Objective:


Objective: The goal of this study was to establish reference intervals for CBC results and new parameters for RBC morphology, reticulocytes, and platelets in healthy dogs and cats using the ADVIA 120 hematology system.

Methods:


Methods: The ADVIA 120, with multispecies software (version 1.107-MS), was used to analyze whole blood samples from clinically healthy dogs (n=46) and cats (n=61). Data distribution was determined and reference intervals were calculated as 2.5 to 97.5 percentiles and 25 to 75 percentiles.

Results:


Results: Most data showed Gaussian or log-normal distribution. The numbers of RBCs falling outside the normocyticnormochromic range were slightly higher in cats than in dogs. Both dogs and cats had reticulocytes with low, medium, and high absorbance. Mean numbers of large platelets and platelet clumps were higher in cats compared with dogs.

Conclusions:


Conclusions: Reference intervals obtained on the ADVIA 120 provide valuable baseline information for assessing new hematologic parameters and for interlaboratory comparisons. Differences compared with previously published reference values can be attributed largely to differences in methodology.  相似文献
7.
用RT-PCR法,以口蹄疫病毒China/99感染的牛舌水泡皮为材料,扩增目的cDNA,与pGEM-T Easy载体连接并转化JM109菌株,再经重组质粒电泳、PCR和EcoR1酶切鉴定。序列测定和分析结果表明,猪源毒在3A基因内缺失10个密码子,与牛源毒的核苷酸和氨基酸序列差异较大。A-G和T-C的转换率较高,而且A-G转换导致氨基酸变异的几率大于T-C转换,它们是影响氨基酸稳定的因素之一。China/99P3区编码产物在第8、120、121、127、132、193、493、501和538位具有特征性氨基酸,可能与该毒株的表型如毒力等有关。3A基因突变率较高,3B、3C和3D较低,3D最为保守,这对维持3C和3D蛋白酶和3B的引物功能至关重要。  相似文献
8.
家蚕谷胱甘肽-S-转移酶基因(BmGSTd1)的诱导表达定量分析   总被引:2,自引:1,他引:1  
谷胱甘肽-S-转移酶(GST)在机体的解毒代谢和抗氧化中起重要作用。为探究家蚕谷胱甘肽-S-转移酶基因(BmGSTd1)在家蚕体内的解毒和抗性功能,采用实时荧光定量PCR方法,对BmGSTd1基因在正常饲养及添食NaF家蚕5龄幼虫不同组织中的转录水平进行检测,并采用家蚕Actin3、GAPDH、28S rRNA 3种内参照基因对检测结果进行归一化处理。BmGSTd1基因在家蚕5龄幼虫各组织的转录水平存在差异,且采用不同内参照基因结果不一致:分别以家蚕Actin3、GAPDH、28S rRNA基因为内参照,该基因转录水平最高的组织分别为中肠、丝腺、脂肪体。5龄第2天幼虫添食NaF对体内各组织中BmGSTd1基因的转录水平具有诱导作用,且在不同组织中诱导表达的情况不同,采用以上3种内参照基因进行归一化处理的结果数据表明,中肠和脂肪体组织中的诱导转录水平最高,可能与这2种组织是家蚕主要的解毒器官有关。研究认为,检测基因在不同组织中的转录水平,采用合适的内参照基因对保证检测结果的可靠性非常重要。  相似文献
9.
氰戊菊酯对照品的质量检测及赋值   总被引:1,自引:1,他引:0  
为研制兽用氰戊菊酯国家对照品。取氰戊菊酯原油精制得氰戊菊酯原料,经质量检查,最大紫外吸收波长为277 nm、熔点为53-54℃、水分为0.02%,用正相和反相两种高效液相色谱法测得纯度为99.8%,按质量平衡法进行含量赋值为99.8%,不确定度为±0.1%。  相似文献
10.
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