狗獾粪便DNA提取方法的初步探讨

在京杭大运河扬州段堤坝上采集狗獾的新鲜粪便,采用预处理-酚/氯仿抽提法、NaCl改良法、异硫氰酸胍裂解法、淀粉吸附法及QIAamp试剂盒法5种方法对粪便样品中狗獾DNA进行提取,探讨狗獾粪便样品中DNA提取方法和优化条件。结果表明,在5种提取方法中,淀粉吸附法的效果明显优于其它4种方法。采用粪便裂解液快速裂解细胞后,加入淀粉去除其中的大量PCR抑制物,然后用蛋白酶K裂解、酚/氯仿/异戊醇抽提,最后使用UNIQ-10柱纯化粪便DNA,对线粒体控制区和微卫星位点的PCR扩增反应及测序结果证实该方法的可行性。以上结果表明,通过该方法获得的粪便DNA能够用于更深入的分子遗传学等学科的研究。     

土壤样品中DNA提取方法的比较

对土壤样品中提取DNA方法的有效性进行了比较研究。如果以细胞有效裂解和DNA产率为标准,用冻融进行预处理再结合SDS和溶菌酶的化学裂解方法,是效果最佳的DNA抽提方法,细胞裂解率为82%,DNA产率达20.8μg/g。为了去除PCR抑制物,将DNA样品进一步用柱纯化,回收率为80%。纯化后的DNA样品可用于16SrDNA扩增及其他分子操作。     

有效抽提乙醇中螨标本的核DNA

对如何有效地从乙醇保存的叶螨中抽提核基因组DNA作了探讨。70％乙醇保存的叶螨标本,经过干燥,TEN洗涤,蛋白酶K消化,酚—氯仿抽提,2倍体积无水乙醇沉淀等步骤,得到叶螨核基因组DNA沉淀,再用TE或无菌水溶解后,以其为模板,进行随机引物PCR扩增。结果显示.该方法抽提的DNA无Taq酶抑制物,且其纯度达到进行PCR反应对模板要求的程度。这为采用AP-PCR技术研究螨的系统分类提供了便利。     

一种从大熊猫粪便中提取DNA的改进方法

本研究描述一个改进的方法，使从大熊猫粪便中提取DNA用于PCR扩增变得更加容易。在粪便DNA的提取过程中采用一个新的预处理方法，将粪便用预冷的丙酮洗2～3次，除去粪便中含有的大量PCR抑制物，然后用蛋白酶K裂解、酚氯仿抽提，能提取到纯度很高的DNA供PCR扩增。本实验PCR扩增了大熊猫脑源性神经营养因子(BDNF)基因和线粒体细胞色素6基因片段，并进行测序分析，证实了提取的可靠性。对比本方法和未经丙酮预处理的方法提取的DNA进行PCR扩增，前者的扩增结果明显优于后者。     

分子生物学技术讲座（四）——真核基因组DNA的制备,分析及基因组DNA文库的构建

真核细胞DNA的制备通常是在有EDTA及SDS一类去污剂的存在下用蛋白酶K消化细胞后用酚抽提而实现的。用此方法得到的DNA,其大小为IOO～15Okb,恰好用于SOIJthern分析和经限制性内切酶部分消化后用人噬体载体构建基因组DNA文库。而用粘粒载体或用酵母人工染色体（YAC）载体构建基因组DNA文库则需要更大的DNA。1关于DNA的制备制备真核细胞基因组DNA的方法有许多种,但基本步骤相类似,即包括组织匀浆、细胞裂解、蛋白和RNA的去除以及用乙醇沉淀或透析等方法除去残存的其它物质。方法的选择主要根据样品的类型以及所制备的DN…     

乙醇保存的动物标本基因组DNA提取方法的比较

为提高从乙醇长期保存的动物标本中提取大分子DNA的质量,用5种不同方法对动物组织进行预处理实验,然后采用SDS／蛋白酶K裂解,酚一氯仿抽提和乙醇沉淀提取总DNA,通过0．8％琼脂糖凝胶对模板进行电泳和PCR产物作鉴定,经比较,用0．9％NaCL法、PBS法和混合液法进行预处理,消除乙醇对Taq酶的影响以及蛋白质和核酸交联问题,为提取动物基因组DNA的3种更理想方法。     

犬细小病毒的PCR诊断试剂盒的研制

目的　建立检测犬细小病毒的PCR诊断试剂盒。方法　通过在犬细小病毒 (CPV)的基因组中设计的特异性寡核苷酸引物 ,利用PCR技术研制犬细小病毒的PCR诊断试剂盒。结果　在特定的反应条件下 ,使用该试剂盒能特异地扩增含有犬细小病毒的样品 ,且能检出痕量 (10ng)的犬细小病毒DNA ,被测粪样只需进行简单煮沸就能进行PCR反应 ,该试剂盒能在常温下保存 1周以上、4℃保存 1年以上。同血凝试验 (HA〕及单克隆抗体ELISA方法比较 ,对 6 6份样品进行检测 ,显示该PCR试剂盒具有特异、灵敏等优点。结论　成功研制了犬细小病毒的PCR诊断试剂盒 ,利用该试剂盒能从DNA水平上对犬细小病毒进行监控     

一种高效可直接用于PCR分析的土壤总微生物DNA抽提方法

以CTAB-溶菌酶-蛋白酶K-冻融裂解法直接抽提土壤总微生物的基因组DNA,利用G8000沉淀和纯化DNA.结果表明,该方法是一种简便、有效可直接应用于PCR分析的土壤总微生物基因组DNA的抽提方法.采用含聚乙烯吡咯烷酮(PVP)的缓冲液预洗,添加CaCl₂和BSA,可以去除腐殖酸;用PEG8000沉淀DNA,可以提高DNA质量;采用冻融法破碎细胞,CTAB、溶菌酶和蛋白质酶K共同作用以裂解细胞,可以保证获得大片段的DNA,提高DNA产率.用该方法抽提的七子花林下土壤总微生物DNA产率为9.22 μg·g^-1,A260/A280为1.65,可适用于 PCR扩增及扩增rDNA限制酶切分析(ARDRA)技术,适宜的模板DNA浓度为0.67 ng·μl^-1.快速、有效、可直接用于PCR分析的土壤总微生物DNA提取方法的建立,为大规模的土壤微生物分子生态学研究提供了可能.     

大鼠肠内容物细菌基因组DNA提取方法的比较

目的比较两种肠内容物前处理和两种提取方法对清洁级SD大鼠肠内容物细菌基因组DNA提取效率。方法分别选用PBS多次离心漂洗、液氮破细胞两种前处理方法和酚/氯仿抽提、试剂盒过柱法两种提取方法进行组合分析,对4份肠内容物和16份含金黄色葡萄球菌肠内容物进行随机提取。结果大鼠肠内容物细菌基因组DNA含量和纯度测定结果显示,与PBS反复离心相比,液氮研磨前处理能显著提高大鼠肠内容物基因组DNA。荧光定量PCR表明,液氮研磨前处理较PBS反复离心能更好地收集细菌基因组DNA,其Ct值最低。结论研究结果表明,采用液氮研磨试剂盒法在大鼠肠内容物DNA提取中是较为优良的方法,该方法为建立实验动物中微生物的定量PCR检测方法打下了基础。     

植物叶片基因组DNA快速提取方法

为了满足分子生物学研究中大量植株基因需要PCR检测的需求,建立了一种无需研磨、无需有机试剂抽提的快速提取植物叶片基因组DNA的方法。通过比较基因组DNA的浓度及PCR检测结果,获得了最佳提取条件,即约50 mg叶片加入150μL含1%β-巯基乙醇的TE提取液,快速破碎1 min。破碎后的样品4℃13 500×g离心1 min,上清于-20℃冷冻后室温融解,4℃、13 500×g离心1 min,离心后收集的上清溶液即可用于PCR检测。整个提取过程仅需10-12 min,具有样品用量小、操作简单、廉价、高效等优点。使用此方法提取的烟草、水稻、大豆、玉米、油菜和花生的基因组DNA均可用于PCR扩增,并可成功扩增长度为3 244 bp的基因片段。此方法提取的基因组DNA也可用于对未知基因的扩增,获得4条新的花生actin基因序列。     

大熊猫幼兽腹泻粪便分离出的轮状病毒鉴定

11 只5 ～ 11 月龄的断奶大熊猫幼兽先后患一种急性传染性、顽固性腹泻病,1 只大熊猫幼兽在发病4 d 后死亡,相同饲养条件下由母兽哺育的同龄大熊猫幼兽未见发病。取发病大熊猫幼兽粪便、血液以及呕吐物进行细菌学和寄生虫学检查,未检出病原;用轮状病毒粪便抗原ELISA 快速诊断,受检的8 只大熊猫幼兽腹泻便都多次检出阳性结果。采集发病大熊猫的病料进行病毒病原分离,从腹泻大熊猫幼兽的粪便中分离出病毒颗粒,通过电镜形态、理化特性和中和试验鉴定,确认分离病毒为轮状病毒。      

An ARMS-based technique for sex determination of red panda (Ailurus fulgens)

Molecular sexing is a key component in the investigation of wild populations. In this study, we developed a fast, accurate and reliable amplification refractory mutation system (ARMS) technique for sex determination of red panda based on the exon 4 of the ZFX/ZFY gene. The amplicons were distinguished simply by agarose gel electrophoresis, exhibiting one fragment in females (X: 300 bp) and two in males (X: 300 bp, Y: 166 bp). Robustness of this ARMS system was confirmed by testing both 43 captive red pandas using DNA samples with known-sex and 10 wild red pandas using faecal DNA samples with unknown sex.     

对大熊猫数量调查方法中咬节区分机制的准确性评价

在目前所广泛应用的大熊猫的数量调查方法中,咬节区分是一种重要的区分机制。本文收集了王朗国家级自然保护区内的同一个熊猫个体的粪便咬节数据,并结合已经发表的同个体粪便咬节数据,对应用咬节区分机制时正确判断的比率进行了评估。通过对17组124团粪便的522个同组内粪便咬节平均值差值的分析,发现在王朗应用2mm的判断阈值时,正确判断的比率为92.9%;卧龙野生种群的正确判断比率为71.2%(1组12团66个差值);圈养种群的正确判断比率为77.6%(7组139团1555个差值),显示咬节区分机制中的2mm阈值具有相当高的准确性。同一团粪便中咬节取样量不足会增加错判的比率,以100咬节测量为对照,30咬节取样会增加3.7%的错判比率,34咬节的测量会增加2.6%的错判比率。     

基于野外痕迹点和GIS技术定量评估步道对大熊猫活动的影响

道路建设不仅直接导致野生动物死亡,还能对栖息地形成阻碍效应,导致小种群出现或隔离,增加物种灭绝的风险。生态学家在道路对野生动物影响研究中的一个重要进展是道路影响域(road-effectzone)的提出,但影响域既不能反映道路影响的变化性,也难以满足栖息地评估对数据的要求。为此,我们以大熊猫(Ailuropoda melanoleuca)为例来探讨道路影响的定量评估方法。在佛坪和长青保护区内选择了3条步道,获取了步道周边1,042个大熊猫的痕迹点数据,通过GIS计算各痕迹点到步道的距离,统计距离步道每20m内的痕迹点数量,以此作为其活动频率。在距步道每100m处设置检测点,通过非参数检验比较检测点前后活动频率分布的变化,寻找道路对大熊猫活动影响的突变点,确定影响变化的阈值距离和评价标准。研究发现在距离步道1,000m内,随距离的增加,大熊猫活动频率逐渐增大,大熊猫有明显的回避效应;在距步道500m、1,000m处发现活动频率发生了显著变化,为影响的阈值距离。本研究基于痕迹点和阈值距离的评估方法可以反映道路影响连续、渐变的特点,使定量、准确评估其影响成为可能。     

Major histocompatibility complex class II variation in the giant panda (Ailuropoda melanoleuca)

Habitat destruction and human activity have greatly impacted the natural history of the giant panda (Ailuropoda melanoleuca). Although the genetic diversity of neutral markers has been examined in this endangered species, no previous work has examined adaptive molecular polymorphisms in the giant panda. Here, the major histocompatibility complex (MHC) class II DRB locus was investigated in the giant panda, using single-strand conformation polymorphism (SSCP) and sequence analysis. Comparisons of DNA samples extracted from faecal and blood samples from the same individual revealed that the two materials yielded similar quantities and qualities of DNA, as well as identical SSCP patterns and allelic sequences, demonstrating the reliability of DNA isolation from panda faeces. Analysis of faecal samples from 60 giant pandas revealed relatively low number of alleles: seven alleles. However, the alleles were quite divergent, varying from each other by a range of 7-47 nucleotide substitutions (4-25 amino acid substitutions). Construction of a neighbour-joining tree and comparisons among DRB alleles from other species revealed that both similar and highly divergent alleles survived in the bottlenecked panda populations. Despite species-specific primers used and excellent faecal DNA isolated, a lower level of heterozygosity than expected was still observed due to inbreeding. There were three types of evidence supporting the presence of balancing selection in the giant panda: (i) an obvious excess of nonsynonymous substitutions over synonymous at the antigen-binding positions; (ii) trans-species evolution of two alleles between the giant panda and other felids; and (iii) a more even distribution of alleles than expected from neutrality.     

Population genetics reveals high connectivity of giant panda populations across human disturbance features in key nature reserve

The giant panda is an example of a species that has faced extensive historical habitat fragmentation, and anthropogenic disturbance and is assumed to be isolated in numerous subpopulations with limited gene flow between them. To investigate the population size, health, and connectivity of pandas in a key habitat area, we noninvasively collected a total of 539 fresh wild giant panda fecal samples for DNA extraction within Wolong Nature Reserve, Sichuan, China. Seven validated tetra‐microsatellite markers were used to analyze each sample, and a total of 142 unique genotypes were identified. Nonspatial and spatial capture–recapture models estimated the population size of the reserve at 164 and 137 individuals (95% confidence intervals 153–175 and 115–163), respectively. Relatively high levels of genetic variation and low levels of inbreeding were estimated, indicating adequate genetic diversity. Surprisingly, no significant genetic boundaries were found within the population despite the national road G350 that bisects the reserve, which is also bordered with patches of development and agricultural land. We attribute this to high rates of migration, with four giant panda road‐crossing events confirmed within a year based on repeated captures of individuals. This likely means that giant panda populations within mountain ranges are better connected than previously thought. Increased development and tourism traffic in the area and throughout the current panda distribution pose a threat of increasing population isolation, however. Maintaining and restoring adequate habitat corridors for dispersal is thus a vital step for preserving the levels of gene flow seen in our analysis and the continued conservation of the giant panda meta‐population in both Wolong and throughout their current range.     

ERIC-PCR分子杂交技术分析大熊猫肠道菌群结构

目的了解大熊猫肠道微生物区系结构的相似性和稳定性,并找出大熊猫肠道微生物群落结构的变化与健康状况的关系。方法对上海动物园及上海野生动物园所饲养的3只大熊猫2次采集的粪便样品进行微生物群落总DNA的抽提,并以此为模板获得反映肠道微生物群落结构特征的ERIC—PCR和Southern杂交指纹图谱,比较各DNA样品指纹图谱的相似性指数。结果除国庆(大熊猫)的第1次采集的样品(当时处于腹泻状态),其他各DNA样品的ERIC-PCR及Southern杂交指纹图谱的相似性都达到85％～100％;佳斯及川川(大熊猫)2个个体2次采集的样品之间ERIC指纹图谱的相似性分别为93％和87％,而国庆腹泻时的样品与健康时的样品之间则为71％。结论大熊猫不同个体之间肠道微生物群落结构比较相似,而且同一个体在不同时期表现出比较高的稳定性,但当个体的健康出现问题时肠道优势菌菌群结构有一定波动。所采用的DNA提取方法、ERIC—PCR和Southern杂交指纹图谱的高度重复性证明了之一分子生态学技术在大熊猫肠道微生物区系动态监测中的可行性。     

唐家河自然保护区大熊猫种群数量及栖息地的移动

本文根据大熊猫季节性活动规律特点,应用海拔高度路线调查法,全面收集唐家河自然保护区大熊猫的比较新鲜粪便,计获１２４个粪样材料,提取ＤＮＡ作ＤＮＡ指纹图,经微机检测,保护区有大熊猫３７只,个体数量与粪样采集量之比为１∶３３５１４,有６个家系,雌兽２１只,雄兽１６只。并搞清了保护区生态环境质量,箭竹遭受病虫害的程度,洪水对栖息地的影响,特别对引起大熊猫数量减少及栖息地移动等原因,作了较为详细的分析。     

A novel method for collection and preservation of faeces for genetic studies

Quantitative and qualitative measurements of DNA were used to compare faecal sample storage in ethanol and silica with a novel method (two‐step) in which samples are soaked in ethanol and then desiccated with silica. Silica‐preserved samples had the lowest DNA concentrations. The two‐step method yielded significantly more DNA in high quality samples (average DNA concentrations > 100 pg/µL with all storage methods). However, for lower quality samples, the ethanol and two‐step methods performed similarly. The amounts and rates of sample degradation were not strongly affected by storage method and neither was the percentage of target DNA (< 1%) obtained from the samples.