共查询到19条相似文献，搜索用时 328 毫秒
1.
2.
金钱鱼(Scatophagus argus)是我国东南沿海名优养殖鱼类, 具有 XY 性别决定系统, Dmrt1 是其性别决定候选基因。金钱鱼生长具有性别二态性, 雌鱼生长快于雄鱼。目前缺乏快速鉴定金钱鱼遗传性别的分子标记, 阻碍了其性别控制育种技术的建立。本研究以公布的金钱鱼基因组数据, 在 Dmrt1 附近设计多对标记引物, 并通过 PCR 扩增验证标记的性别特异性。其中, 标记引物 Dmrt1-Marker-4-F/R 在雌鱼中仅扩增出一条 593 bp X 染色体条带, 而在雄鱼中能扩增出 593 bp 和 693 bp 两条条带, 分别来自 X 和 Y 染色体, 表明该标记为共显性标记。利用该标记检测我国南海沿岸 3 个不同地理群体 213 尾金钱鱼的遗传性别与表型性别完全一致。此外, 快速 DNA 提取试剂盒提取的片段较短 DNA 样品也可用于该对标记引物准确鉴定遗传性别。本研究建立了一种快速、准确、经济可靠的金钱鱼遗传性别鉴定方法, 将旨为促进金钱鱼性别控制育种技术的建立, 并为金钱鱼性别决定与分化机制研究提供依据。 相似文献
3.
《淡水渔业》2015,(6)
以患鲤疱疹病毒2型(Cyprinid herpesvirus 2,Cy HV-2)疾病的异育银鲫(Carassius auratus gibelio)肾脏为实验材料,采用-20℃冷冻、100%乙醇、Dafano's液、10%福尔马林、Zenker's液、Müller's液、2.5%戊二醛、Helly's液、Mossman's液、Bouin's液和Carnoy's液不同方法进行保存,通过微量分光光度计检测和琼脂糖凝胶电泳探讨不同保存方法对病毒DNA提取效果、常规PCR扩增效果和巢式PCR扩增效果的影响。结果表明:从病鱼肾脏中提取DNA时,除100%乙醇保存方法与-20℃冷冻保存组一样可以提取高质量的DNA外,其它保存方法对DNA提取有不同程度的影响,100%乙醇保存方法和保存的材料可以用作于鲤疱疹病毒2型DNA的提取;常规PCR扩增时,100%乙醇、Zenker's液、2.5%戊二醛、Helly's液、Bouin's液和Carnoy's液保存方法与-20℃冷冻保存组具有相同的效果,在362 bp处出现明亮一致的清晰单一目的条带,这6种保存方法及其保存的材料可以用作鲤疱疹病毒2型的常规PCR扩增;巢式PCR扩增时,100%乙醇、Dafano's液、10%福尔马林、Zenker's液、Müller's液、2.5%戊二醛、Helly's液、Mossman's液、Bouin's液和Carnoy's液所有保存方法与-20℃冷冻保存组具有相同的效果,在339 bp处出现明亮一致的清晰单一目的条带,这些保存方法及其保存的材料均可以用作于鲤疱疹病毒2型的巢式PCR扩增,在采用巢式PCR进行检测和诊断患鲤疱疹病毒2型疾病上具有应用价值。 相似文献
4.
比较了酚-氯仿法、煮沸法、试剂盒法3种基因组DNA提取方法对WSSV DNA提取效率、纯度及病毒检测结果的影响.结果表明,3种方法的DNA提取效率平均值分别为101.5、372.6、21.5 ng/μl;OD260/OD280范围分别是1.979～2.175(平均值为2.070)、1.699～1.932(平均值为1.796)、1.784～2.075(平均值为1.951);OIE巢式PCR阳性率分别为60%、50%、70%;TaqMan定量PCR检测的病毒阳性率均为100%,病毒拷贝含量分别为916.0～2.23×106、63.3～1.78×106、479.7～2.70×106Copies/μl DNA. 相似文献
5.
6.
根据CCVORF6基因序列,设计合适的引物,扩增ORF6基因,分别将其克隆到自杀性DNA疫苗载体pS-FV与常规DNA疫苗载体pcDNA3.1(+)中,转化感受态细胞DH5α后提取质粒,构建斑点叉尾鮰(Ictalurus punc-tatus)自杀性DNA疫苗ps-ORF6与常规DNA疫苗pcd-ORF6,利用转化后的大肠杆菌菌液为模板进行PCR扩增、提取质粒酶切鉴定以及序列测定等方法证实重组质粒构建正确。将重组质粒转染人胚肾细胞(293T),间接免疫荧光试验表明ORF6均获得表达,但自杀性DNA疫苗的表达效果不如常规DNA疫苗,该研究为这2种疫苗进一步的鱼体试验奠定了基础。 相似文献
7.
根据CCVORF6基因序列,设计合适的引物,扩增ORF6基因,分别将其克隆到"自杀性"DNA疫苗载体pS-FV与常规DNA疫苗载体pcDNA3.1(+)中,转化感受态细胞DH5α后提取质粒,构建斑点叉尾鮰(Ictalurus punc-tatus)"自杀性"DNA疫苗ps-ORF6与常规DNA疫苗pcd-ORF6,利用转化后的大肠杆菌菌液为模板进行PCR扩增、提取质粒酶切鉴定以及序列测定等方法证实重组质粒构建正确。将重组质粒转染人胚肾细胞(293T),间接免疫荧光试验表明ORF6均获得表达,但"自杀性"DNA疫苗的表达效果不如常规DNA疫苗,该研究为这2种疫苗进一步的鱼体试验奠定了基础。 相似文献
8.
鳙鱼不同组织基因组DNA提取方法的探讨 总被引：2，自引：0，他引：2
分别采用试剂盒和常规苯酚/氯仿抽提法提取新鲜的鳙鱼肌肉、肝脏、尾鳍以及用乙醇保存的3种组织基因组DNA,并对其进行综合分析。结果表明,对3种组织的新鲜样品2种方法均能提取高质量的DNA,而用乙醇保存的3种组织,DNA样品质量最好的是尾鳍,其次是肌肉,肝脏最差;总的来看,试剂盒法提取的DNA纯度较好,但是浓度低,而苯酚法得到的DNA浓度高,质量好,且相对成本低。综合比较,苯酚/氯仿抽提法提取乙醇保存的尾鳍组织基因组DNA是一种行之有效的方法,可以在鱼类分子研究中推广应用。 相似文献
9.
蛋种鸡不同产蛋阶段种蛋对孵化效果影响的研究 总被引：1，自引：0，他引：1
本试验选择了４个不同产蛋阶段的鸡群,分别收集种蛋进行孵化。结果显示：不同产蛋阶段的种蛋与其受精率、受精蛋孵化率和健雏率有着密切的关系。以产蛋高峰期的种蛋孵化效果最优,高峰后期次之,再次为高峰前期,产蛋后期为最差。 相似文献
10.
半滑舌鳎雌性特异扩增片段长度多态性标记的筛选与应用 总被引：3，自引：2，他引：1
半滑舌鳎(Cynoglossus semilaevisGünther)为东北亚特有的名贵冷温性比目鱼类,为我国养殖业的新宠。半滑舌鳎雌鱼生长速度是雄性的2~3倍,若能实现单雌化养殖将大大提高养殖业的经济效益。本研究利用扩增片段长度多态性(AFLP)技术,应用64个引物组合,检测了半滑舌鳎(Cynoglossus semilaevisGünther)雌雄基因组DNA的多态性,筛选与半滑舌鳎性别相关的AFLP分子标记。实验经过3轮筛选和验证,4个引物组合扩增出7个雌性个体出现频率为100%的DNA片段,我们认为这7个标记是半滑舌鳎雌性特异的AFLP标记,分别命名为CseF382、CseF575、CseF783、CseF464、CseF136、CseF618和CseF305。同时,将标记CseF382成功转化为SCAR标记,测定了该标记的DNA序列,建立了半滑舌鳎遗传性别鉴定的PCR技术,为半滑舌鳎性别决定机制的研究和性别控制奠定了重要基础。 相似文献
11.
12.
Kirsten Meyer Sven Michael Bergmann Marian van der Marel Dieter Steinhagen 《Aquaculture Research》2012,43(6):835-842
Since virus isolation is seldom successful, KHV infection is commonly detected by PCR examination. A number of different PCR assays have been described in recent years. However, at present no commonly accepted PCR method is used amongst different laboratories. The aim of this study was to check if the examination of infected fish by different PCR methods yielded comparable results. We used tissue samples of three KHV‐infected koi, one KHV‐infected common carp, one KHV‐infected goldfish and one non‐infected common carp. DNA was extracted with DNAzol Reagent, High Pure PCR Template DNA Preparation Kit and QIAamp DNA Mini Kit. The DNA was tested by PCR with different combinations of published primer sets –KHV‐F and ‐R, KHV‐Gray‐2F and ‐2R and KHV‐TKf and ‐TKr – plus different DNA polymerases – a standard Taq DNA polymerase, a Platinum (hotstart) Taq DNA polymerase and a Platinum (hotstart) Pfx DNA polymerase with proofreading activity. The different extraction methods produced DNA solutions with different yields of DNA and different degrees of homogeneity. Also, the sensitivity of the PCR depended on the choice of the primer set and polymerase. Not all infected fish could be identified with all methods; there were large differences in the sensitivity between methods. 相似文献
13.
14.
对传统对虾白斑综合征病毒(WSSV)的PCR检测方法中的病毒模板DNA的提取方法加以改进,建立了一种快速检测克氏原螯虾(Procambarus clarkii)WSSV的PCR方法。本方法将克氏原螯虾肝胰腺组织匀浆液冻融3次进行差速离心后,在病毒沉淀中加入50μL蛋白酶K溶液,室温消化5 min,沸水中煮10 min后,10000 r/min高速离心5 min即可取上清液用于PCR扩增,结果显示:与传统的病毒模板DNA提取方法相比,本方法的PCR结果特异性强,扩增效率高,准确率高,可应用于快速大规模检测克氏原鳌虾中的WSSV。 相似文献
15.
16.
R Sudhakaran T Mekata T Kono K Supamattaya N T H Linh Y Suzuki M Sakai T Itami 《Journal of fish diseases》2009,32(7):611-617
White spot syndrome virus (WSSV) is an important shrimp pathogen responsible for large economic losses for the shrimp culture industry worldwide. The nucleic acids of the virus must be adequately preserved and transported from the field to the laboratory before molecular diagnostic analysis is performed. Here, we developed a new method to isolate WSSV-DNA using Flinders Technology Associates filter paper (FTA matrix card; Whatman) without centrifugation or hazardous steps involved. FTA technology is a new method allowing the simple collection, shipment and archiving of nucleic acids from haemolymph samples providing DNA protection against nucleases, oxidation, UV damage, microbial and fungal attack. DNA samples prepared from 10-fold dilutions of moribund shrimp haemolymph using FTA matrix cards were analysed using semi-quantitative and quantitative polymerase chain reaction (PCR) and were compared with two commercially available DNA isolation methods, the blood GenomicPrep Mini Spin Kit (GE Healthcare) and the DNAzol (Invitrogen). Sequence analysis was performed for the DNA samples prepared using the various isolation procedures and no differences in the sequence among these methods were identified. Results based on the initial copy number of DNA prepared from the GenomicPrep Mini Spin Kit are a little more sensitive than the DNA prepared from FTA matrix cards, whereas the DNAzol method is not suitable for blood samples. Our data shows the efficiency of retention capacity of WSSV-DNA samples from impregnated FTA matrix cards. Matrix cards were easy to store and ship for long periods of time. They provide ease of handling and are a reliable alternative for sample collection and for molecular detection and characterization of WSSV isolates. 相似文献
17.
18.
A dihydropteroate synthase gene from the chromosomal DNA of the fish-pathogenic bacteria Lactococcus garvieae (formerly Enterococcus seriolicida ) was cloned. This gene was then chosen as the target for polymerase chain reaction (PCR). The designated PCR primer set only amplified a 709-bp DNA fragment from L. garvieae strains, and did not amplify the same molecular size fragment from related species of L. lactis, Enterococcus faecalis, E. faecium or β-haemolytic Streptococcus sp. The kidney tissue of yellowtail, Seriola quinqueradiata (Temminck and Schlegel), a species which is naturally infected with L. garvieae , and also kidney tissue samples of healthy yellowtail were stored in TNES-Urea. The DNA was extracted from tissue samples by a modification of the standard method and by a boiled-extraction method. In particular, template DNA was utilized within 30 min following extraction and purification by the boiled-extraction method. These species-specific PCR primers could amplify a L. garvieae target sequence from yellowtail which were naturally infected with L. garvieae . The total procedure for the diagnosis of L. garvieae infections in fish, from the point of DNA extraction to observation in an agarose gel following electrophoresis, can be performed in less than 4 h. 相似文献
19.
利用GyrB基因的特异性引物建立沉积环境中气单胞菌属细菌的实时荧光定量PCR检测方法。将已知浓度的气单胞菌菌液加入灭菌的沉积物样品中,作为模拟沉积物样品。通过选择和优化沉积物DNA提取方法、特异性引物、标准曲线模板,建立沉积环境中气单胞菌属细菌准确检验的实时荧光定量PCR方法,同时验证该方法的特异性、灵敏性、重复性。结果表明,采用改进的溶菌酶-SDS温和裂解法提取沉积物DNA,以扩增GyrB基因片段的IAF和IAR为特异性引物,并直接以模拟沉积物样品DNA为标准品构建标准曲线,可以建立适用于定量检测沉积环境中气单胞菌属细菌的实时荧光定量PCR方法。该方法可灵敏、特异、准确地定量检测刺参养殖池塘底泥中气单胞菌属中不同种的细菌,检出效率可达103CFU/g。统计分析显示,变异系数为0.21%-0.80%,均小于5%,表明重复性良好。 相似文献