共查询到19条相似文献，搜索用时 562 毫秒
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本研究根据马、驴线粒体DNA中的保守区段设计了一对引物,通过聚合酶链式反应(polymerase chain reaction,PCR)采取马、驴、牛、羊、猪、鸡、兔、鹿、火鸡等实验材料进行扩增,可以专一地扩增出马、驴源性成分的DNA片段,再经限制性内切酶Sau 3A和Alu Ⅰ的酶切鉴定可以区分马源性成分和驴源性成分,PCR扩增产物的测序结果验证了酶切鉴定结果的正确性.引物灵敏度测试结果表明该方法的检测低限均达0.3%,该对引物可以成为检测马、驴源性成分高效准确的检测工具. 相似文献
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旨在探讨模板制备方法对单细胞 PCR准确性的影响 ,采用 KOH/ DTT和液氮冻融煮沸两种不同方法裂解单个淋巴细胞 ,行 DMD基因外显子 17、19、4 4、4 5、4 8五重巢式 PCR.用 KOH/DTT法裂解的细胞扩增成功率为 98% ( 147/ 150 ) ,失败率为 2 % ( 3/ 150 ) ;用液氮冻融煮沸法裂解的细胞扩增成功率为 78% ( 117/ 150 ) ,失败率为 2 2 % ( 33/ 150 ) ,差异有显著性 (χ2 =2 8.4 ,P <0 .0 1) .结果表明单细胞 PCR模板制备与 PCR扩增失败有密切关系 ,选用合适的细胞裂解方法可提高单细胞 PCR的准确性 相似文献
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目的明确妊娠期阴道白色念珠菌的基因型是否存在差异,了解不同基因分型方法的优劣。方法对628名妊娠妇女的阴道分泌物进行念珠菌培养和鉴定,以三种方法对比研究不同孕期白色念珠菌的基因型。结果妊娠期阴道念珠菌检出率为31.52%,白色念珠菌为最常见的菌种。INT引物PCR法可将126株白色念珠菌分为A、B、C三组,A组最为多见。EcoRⅠ酶切法可将119株白色念珠菌分为A、B、C三组和9个亚组,A—1亚组最常见。RAPD法扩增后,126株白色念珠菌共获得17种基因型,相似系数SAB=0.805±0.007,不同孕期的组内、组间SAB值均大于0.80。结论妊娠期阴道白色念珠菌具有显著的基因多态性,它们是由基因型非常相似的不同白色念珠菌。INT引物PCR法简便、快速,结果稳定、可靠,适于临床推广应用;RAPD法快速、简便、经济,分辨率高,适于临床和科研应用。 相似文献
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目的:研究我国优秀冬季项目运动员肌型肌酸激酶基因(CKMM)A/G多态分布及其与优秀耐力能力的关联性。方法:应用MALDI-TOF技术测定120名我省汉族健康大学生及50名我国优秀冬季项目运动员CKMM基因A/G位点的基因型和等位基因的频率分布。结果:等位基因在对照组中的频率为A=87%,G=13%,基因型频率为A/A=76%,A/G=22%,G/G=2%;冬季项目运动员等位基因频率为A=86%,G=14%,基因型频率为A/A=72%,A/G=28%,其中名25耐力项目运动员等位基因频率为A=85%,G=15%,基因型频率为A/A=70%,A/G=30%,基因型频率和等位基因频率在对照组与耐力运动员组间差异也不显著。结论:我国耐力型冬季项目运动员的优秀耐力素质与CKMM基因N coⅠ多态性无关,该位点不能作为其耐力素质选材的遗传学标记。 相似文献
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利用随机扩增多态性DNA(RAPD)技术对14个枣Ziziphus jujuba 品种和1个野生种——泰山酸枣Z．spinosus的遗传变异进行了研究。从120个10-碱基随机引物中筛选出37个多态性引物用于正式扩增，共扩增出429条DNA带，其中多态性带214条，占49．88％。根据DNA扩增结果计算了品种及类型间遗传距离，并用UPGMA构建了聚类树状图。分析结果表明：龙爪枣 Z．jujuba var．tortuosa、葫芦枣 Z. jujuba var．lageniformis、无核枣 Z.jujuba var．anucleatus 等几个变种内的遗传距离大于变种间遗传距离，认为枣的变种划分是不自然的，宜并入其原变种；枣种下不宜设变种，对枣种下的众多品种，应根据品种间的遗传关系，直接划分品种群。 相似文献
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目的 探讨西藏地区乙型肝炎病毒及其亚型;方法 随机选择HBsAg阳性携带者34例,慢性乙型肝炎患者9例,应用FQ-PCR方法检测血清HBV-DNA含量;采用聚合酶链反应限制性酶长度多态性分析法,通过PCR扩增出目标基因片段,然后用特定的限制性内切酶进行酶切,根据酶切图谱进行基因型及亚型的测定.结果 43例HBV-DNA定量阳性血中c基因型占83.72%(36/43例);B型占6.9%(3/43);B、C混合型占4.65%(2/43);D型占4.65%(2/43).B基因型的亚型中Ba6.91%(5/43);B、C型混合型中C2亚型4.65%(2/43).结论 西藏地区HBV基因型以C型为主,B型次之,B型亚型以Ba占优势. 相似文献
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目的 用基因芯片对胎儿卵巢组织和成人卵巢组织进行HLA-DRB1分型,对于进一步了解胎儿卵巢基因型及其档案的建立进行了初步探索.方法 将6份胎儿卵巢组织分别与育龄妇女及绝经后妇女卵巢组织的基因组DNA,通过组间特异引物不对称扩增,扩增中同时用荧光素Cy3标记.扩增标记后的产物与结合在HLA-DRB1基因分型芯片上的探针进行杂交,通过荧江扫描仪对杂交产生的荧光信号值进行分析,确定样品的HLA-DRB1基因型.结果 18例卵巢组织样本中,纯合子只有1例,其余均为杂合子.其中两例胎儿卵巢组织的HLA-DRB1型别分别与一例育龄妇女卵巢组织及一例绝经后妇女卵巢组织相吻合.芯片重复率为100%.结论 基因芯片是一种理想的分型方法,具有特异性高、重复性好、操作简便、所需时间少、结果判读容易、一次可作多份样本的优点.通过这种技术对胎儿卵巢HLA-DRB1进行快速分型,可为卵巢移植手术的供受体选择节约了时间和成本,并可进一步建立胎儿卵巢组织的基因型档案,为卵巢移植工作开展奠定基础. 相似文献
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《西藏科技》2016,(7)
目的研究细胞因子诱导含SH2结构域蛋白(cytokine-inducible SH2-containing protein,CISH)基因的-292、+1320多态性位点与世居藏族人群结核病易感性的关联性。方法选取世居藏族结核病患者135例,并以143名健康体检者(藏族)作为对照,采用聚合酶链反应-限制性片断长度多态性(PCR-RFLP)方法并进行测序验证,对CISH基因-292和+1320多态性位点进行基因分型。采用SPSS 19.0统计学软件进行了数据处理,率的比较采用了χ2检验,用比值比(oddratios,OR)及其95%可信区间(Confidence interval,CI)表示相对危险度。结果对于CISH基因-292位点,A/A基因型在患者和健康人群中,比例分别占43.0%和46.1%;T/A基因型占48.1%和43.4%;T/T基因型占8.9%和10.5%;对于CISH基因+1320位点,患者和健康人群中A/A基因型占60.0%和56.4%;C/A基因型占37.8%和39.2%;C/C基因型占2.2%和4.9%;病例组和对照组中各基因型所占比例无统计学差异。结论 CISH基因-292、+1320位点的多态性与世居藏族人群的肺结核易感性可能无关。 相似文献
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《科学学与科学技术管理》2008,29(11)
项目简介该项目属农业科技领域。以技术体系、资源创新和品种选育为研究内容。国内率先创建了花椰菜自交不亲和系育种、游离小孢子培养、杂交制种等技术体系;首次研制出育种专家系统;系统地提出抗TuMV和黑腐病苗期人工接种鉴定的方法和标准;创新一批优异资源;育成系列优良品种。 相似文献
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In this paper, a novel oscillating flow polymerase chain reaction (PCR) device was designed and fabricated to amplify SPPS150 and salmonella typhi. In this new design, the samples are shuttled (oscillating flow) inside a microfluidic chip to three different temperature zones required for DNA amplification. The amplification cycle time has markedly been reduced as the reagent volume used was only about 25% of that used in conventional PCRs. Bubble formation and adsorption issues commonly associated to chip based PCR were also eliminated. Based on the performance evaluated, it is demonstrated that this oscillating flow PCR has the advantages of both the stationary chamber and continuous flow PCR devices. 相似文献
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Multiple sample DNA amplification was done by using a novel rotary-linear motion polymerase chain reaction (PCR) device. A simple compact disc was used to create the stationary sample chambers which are individually temperature controlled. The PCR was performed by shuttling the samples to different temperature zones by using a combined rotary-linear movement of the disc. The device was successfully used to amplify up to 12 samples in less than 30 min with a sample volume of 5 μl. A simple spring loaded heater mechanism was introduced to enable good thermal contact between the samples and the heaters. Each of the heater temperatures are controlled by using a simple proportional–integral–derivative pulse width modulation control system. The results show a good improvement in the amplification rate and duration of the samples. The reagent volume used was reduced to nearly 25% of that used in conventional method. 相似文献
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目的:设计获得5段hIL-18异构体,定向插入pEGFP-C1质粒形成融合基因,转染胰腺癌Bx-PC-3细胞并表达,为进一步研究改建的hIL-18蛋白功能奠定实验基础.方法:设计引物从pGEM-T-hIL-18质粒中PCR扩增得到5段不同的hIL-18片段,分别连接入pEGFP-C1载体构建不同的突变子(Mu0、Mu1、Mu2、Mu3和Mu4),转染原位胰腺癌BxPC-3细胞,以荧光显微镜观察绿色荧光蛋白的表达,RT-PCR法检测转染细胞中hIL-18表达水平.结果:(1)五种重组质粒经酶切与测序证实构建成功;(2)BxPC-3细胞转染重组质粒后可观察到绿色荧光蛋白的表达.结论:(1)成功构建了hIL-18五种异构体的绿色荧光蛋白表达载体;(2)改建的hIL-18蛋白可与绿色荧光蛋白在BxPC-3细胞融合表达. 相似文献
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A comprehensive study involving numerical analysis and experimental validation of temperature transients within a microchamber was performed for thermocycling operation in an integrated centrifugal microfluidic platform for polymerase chain reaction (PCR) amplification. Controlled heating and cooling of biological samples are essential processes in many sample preparation and detection steps for micro-total analysis systems. Specifically, the PCR process relies on highly controllable and uniform heating of nucleic acid samples for successful and efficient amplification. In these miniaturized systems, the heating process is often performed more rapidly, making the temperature control more difficult, and adding complexity to the integrated hardware system. To gain further insight into the complex temperature profiles within the PCR microchamber, numerical simulations using computational fluid dynamics and computational heat transfer were performed. The designed integrated centrifugal microfluidics platform utilizes thermoelectrics for ice-valving and thermocycling for PCR amplification. Embedded micro-thermocouples were used to record the static and dynamic thermal responses in the experiments. The data collected was subsequently used for computational validation of the numerical predictions for the system response during thermocycling, and these simulations were found to be in agreement with the experimental data to within ∼97%. When thermal contact resistance values were incorporated in the simulations, the numerical predictions were found to be in agreement with the experimental data to within ∼99.9%. This in-depth numerical modeling and experimental validation of a complex single-sided heating platform provide insights into hardware and system design for multi-layered polymer microfluidic systems. In addition, the biological capability along with the practical feasibility of the integrated system is demonstrated by successfully performing PCR amplification of a Group B Streptococcus gene. 相似文献
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Markéta ?kereňová Veronika Mikulová Otakar ?apoun Tomá? Zima 《Biochemia medica : ?asopis Hrvatskoga dru?tva medicinskih biokemi?ara / HDMB》2016,26(1):103-113
Introduction
Nowadays, on-a-chip capillary electrophoresis is a routine method for the detection of PCR fragments. The Agilent 2100 Bioanalyzer was one of the first commercial devices in this field. Our project was designed to study the characteristics of Agilent DNA 1000 kit in PCR fragment analysis as a part of circulating tumour cell (CTC) detection technique. Despite the common use of this kit a complex analysis of the results from a long-term project is still missing.Materials and methods
A commercially available Agilent DNA 1000 kit was used as a final step in the CTC detection (AdnaTest) for the determination of the presence of PCR fragments generated by Multiplex PCR. Data from 30 prostate cancer patients obtained during two years of research were analyzed to determine the trueness and precision of the PCR fragment size determination. Additional experiments were performed to demonstrate the precision (repeatability, reproducibility) and robustness of PCR fragment concentration determination.Results
The trueness and precision of the size determination was below 3% and 2% respectively. The repeatability of the concentration determination was below 15%. The difference in concentration determination increases when Multiplex-PCR/storage step is added between the two measurements of one sample.Conclusions
The characteristics established in our study are in concordance with the manufacturer’s specifications established for a ladder as a sample. However, the concentration determination may vary depending on chip preparation, sample storage and concentration. The 15% variation of concentration determination repeatability was shown to be partly proportional and can be suppressed by proper normalization.Key words: lab-on-a-chip devices, capillary electrophoresis, multiplex PCR, circulating tumour cells, Agilent DNA 1000 kit 相似文献19.
This study describes a novel microfluidic reactor capable of flow-through polymerase chain reactions (PCR). For one-heater PCR devices in previous studies, comprehensive simulations and experiments for the chip geometry and the heater arrangement were usually needed before the fabrication of the device. In order to improve the flexibility of the one-heater PCR device, two heat pipes with one fan are used to create the requisite temperature regions in our device. With the integration of one heater onto the chip, the high temperature required for the denaturation stage can be generated at the chip center. By arranging the heat pipes on the opposite sides of the chip, the low temperature needed for the annealing stage is easy to regulate. Numerical calculations and thermal measurements have shown that the temperature distribution in the five-temperature-region PCR chip would be suitable for DNA amplification. In order to ensure temperature uniformity at specific reaction regions, the Re of the sample flow is less than 1. When the microchannel width increases and then decreases gradually between the denaturation and annealing regions, the extension region located in the enlarged part of the channel can be observed numerically and experimentally. From the simulations, the residence time at the extension region with the enlarged channel is 4.25 times longer than that without an enlarged channel at a flow rate of 2 μl/min. The treated surfaces of the flow-through microchannel are characterized using the water contact angle, while the effects of the hydrophilicity of the treated polydimethylsiloxane (PDMS) microchannels on PCR efficiency are determined using gel electrophoresis. By increasing the hydrophilicity of the channel surface after immersing the PDMS substrates into Tween 20 (20%) or BSA (1 mg/ml) solutions, efficient amplifications of DNA segments were proved to occur in our chip device. To our knowledge, our group is the first to introduce heat pipes into the cooling module that has been designed for a PCR device. The unique architecture utilized in this flow-through PCR device is well applied to a low-cost PCR system. 相似文献