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本试验采集蒙古马新鲜粪便样品,采用酚-氯仿抽提法和2种细菌基因组DNA提取试剂盒法进行样品中细菌基因组DNA的提取。通过DNA浓度和纯度的测定,并将其作为模板扩增细菌16S rDNA V3区特异性片段对提取效果进行比较,从而找出更适合蒙古马粪便中细菌基因组DNA的提取方法。结果显示,3种提取方法得到的基因组DNA均能用于PCR扩增的模板,其中B试剂盒法提取的DNA数量较多,且纯度高,更适合用于提取蒙古马肠道微生物细菌基因组DNA及后续的蒙古马肠道微生物多样性等分子生物学试验。 相似文献
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目前,对微生物多样性的研究越来越多地从传统的培养方法转向分子生物学方法,现代分子生物学技术的蓬勃发展解决了不可培养微生物研究的难题,使肠道微生物的研究进入了一个新的发展阶段,而肠道微生物总DNA的提取是整个分子生物学方法的关键.主要总结了前人提取土壤、植物、粪便、瘤胃和肠道微生物DNA的试验方法,介绍了微生物总DNA的纯化方法,为用于分子生物学研究的肠道微生物总DNA的提取提供依据. 相似文献
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采用反复冻融法、玻璃珠法和超声波+反复珠磨的方法提取犊牛盲肠的微生物总DNA,并对以上三种方法进行比较,从中确定最佳的方法,以实现普通实验条件下成功提取符合PCR扩增要求的DNA.经紫外分光光度分析表明,超声波+反复珠磨的方法所得的DNA的A<,260>/A<,280>的比值为1.81,0.8%琼脂糖凝胶电泳结果显示,所提DNA片段分子量大于20 kb,适于酶解和PCR扩增要求.以提取的DNA样品为模板,利用细菌通用引物,对其16S rDNA进行PCR扩增,获得了1.7 kb大小特异性很好的预期条带.这是研究犊牛盲肠微生物的关键一步. 相似文献
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本试验采用反复冻融法、酶裂解法和试剂盒法分别提取猕猴肠道菌群的总DNA,根据提取的DNA浓度和纯度最终确定酶裂解法为较好的方法。将酶裂解法提取的DNA进行ERIC-PCR扩增,结果显示:同年龄组猕猴粪样的ERIC条带数量和位置变化不大;幼年组猕猴的菌群种类较丰富,但优势菌群不明显;青年组猕猴的菌群种类和优势菌群最为丰富;老年组猕猴的菌群种类最少。 相似文献
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《黑龙江畜牧兽医》2016,(16)
为优化出可获得完整瘤胃细菌总DNA的方法,以满足分子生物学研究需要,试验采用3种DNA提取方法对瘤胃细菌DNA进行提取并对提取效果进行了比较。结果表明:细菌基因组试剂盒法提取的DNA其OD_(260)/OD_(280)值在1.8以上,电泳无杂带,DNA的提取效果最佳,PCR扩增效果较好;珠磨-CTAB法提取的DNA其OD_(260)/OD_(280)值在1.8以上,电泳结果存在少量杂带;水煮法提取的DNA其OD_(260)/OD_(280)值在2.0以上,无电泳条带,DNA严重降解。推荐采用细菌基因组DNA试剂盒法和珠磨-CTAB法对瘤胃细菌进行DNA提取。 相似文献
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本研究旨在比较5种DNA提取方法对绵羊血液中布氏杆菌DNA的提取效果及对PCR检测的影响。将不同浓度的疫苗株布氏杆菌加入绵羊全血中,采用3种DNA提取试剂盒和酚/氯仿法以及碘化钠法等5种方法提取模拟的绵羊血液样品中的DNA,评价所获得DNA的浓度、纯度和完整性,并采用布氏杆菌特异性PCR进行检测。同时,对各提取方法所需时间及经济成本进行了比较。结果表明,各方法均能提取获得绵羊全血中布氏杆菌DNA,3种试剂盒和碘化钠法获取布氏杆菌DNA的效果相同,而酚/氯仿法获取布氏杆菌DNA的效率最低或存在PCR抑制剂而不适合用于绵羊血液中布氏杆菌的PCR检测。碘化钠法具有耗时较短、成本低、方便的优点,是从绵羊血液中提取布氏杆菌DNA的良好方法。本研究结果为临床绵羊血液中布氏杆菌DNA提取方法的选择提供了参考。 相似文献
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Stephen H Stauffer Adam J Birkenheuer Michael G Levy Henry Marr Jody L Gookin 《Journal of veterinary diagnostic investigation》2008,20(5):639-641
Feces are increasingly valued as practical samples for molecular diagnosis of infectious disease. However, extraction of polymerase chain reaction (PCR) quality DNA from fecal samples can be challenging because of coextraction of PCR inhibitors. Because the type and quantity of PCR inhibitors is influenced by diet, endogenous flora, and concurrent disease, it is unlikely that extraction method performance with human feces can be directly extrapolated to that of domestic cats. In the present study, 4 commercially available DNA extraction methods were examined for their influence on the sensitivity of PCR for the detection of Tritrichomonas foetus in feline stool. DNA was extracted from serially diluted feline-origin T. foetus trophozoites in the absence or presence of feline feces. The ZR Fecal DNA kit was identified as affording the greatest analytical sensitivity and reproducibility and was able to detect >/=10 T. foetus organisms per 100 mg feces in 100% of PCR reactions. Further, the identified extraction method could be completed in the shortest time of all kits tested. 相似文献
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为了探讨不同食性动物肠道菌群的多样性与相似性。本试验采用ERIC-PCR方法对成年马、梅花鹿、老虎、豹、非洲狮、黑熊、小熊猫及成年大熊猫的新鲜粪便细菌总DNA进行了指纹图谱分析。结果显示,肠道菌群的多样性指数为草食动物(1.27)〉肉食动物(1.23)〉杂食动物(1.04),而它们的优势度指数则相反,草食动物(0.32)﹤肉食动物(0.41)=杂食动物(0.41)。同一种动物的肠道菌群多样性指数差异不明显。结果表明,动物肠道菌群的多样性在一定程度上与食物有关。 相似文献
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栽培乌拉尔甘草根中黄酮类和皂甙类化合物的提取与分析 总被引:1,自引:0,他引:1
采用冷浸乙醇提取法对兰州人工引种栽培的6年生乌拉尔甘草Glycyrrhiza uralensis根中的总黄酮和总皂甙类化合物进行了提取分离,并且采用重量法和3种紫外-可见分光光度法对乌拉尔甘草根中总黄酮含量进行了测定和比较分析。结果表明,3种分光光度测定方法中,黄酮含量以直接测定法为最高,硝酸铝显色法次之,氯化铝显色法最低,直接测定法测定样品中的黄酮含量因不受显色剂干扰,测定结果相对更为准确可靠。人工栽培乌拉尔甘草相对野生甘草有效成分的含量较低,但仍可作为野生药用甘草的替代品。 相似文献
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S A Masri P T Nguyen S P Gale C J Howard S C Jung 《Canadian journal of veterinary research》1997,61(1):15-20
A rapid and specific method for the detection of pathogenic Leptospira spp. in bovine semen using the polymerase chain reaction (PCR) is described. The primers used were derived from an EcoR1/BamH1 fragment that hybridized strongly to chromosomal DNA from the hardjobovis serovar. Three different extraction methods were evaluated in this study: phenol-chloroform extraction method, proteinase K (PK) in 1% SDS, followed by phenol-chloroform, and phenol-chloroform followed by 1% cetyltrimethylammonium bromide (CTAB). A PCR product of approximately 500 base pairs (bp) in length was obtained when DNA from pure Leptospira culture was used as a template for PCR, regardless of the DNA extraction method used. The product was consistent with that predicted from the gene sequence. However, in semen seeded in vitro, as well as in semen from infected bulls, a PCR product was obtained only when the leptospiral DNA was extracted from the specimen using the CTAB method. In contrast, other methods used for DNA extraction did not generate suitable templates for the PCR procedure. This is the first PCR protocol developed to detect Leptospira in bovine semen. The PCR protocol provided a direct and unequivocal demonstration that Leptospira can be detected in semen of infected animals. The CTAB method was also used successfully in detecting Leptospira in the urine of infected animals. The PCR procedure was shown to be more sensitive than either the fluorescent antibody test (FAT) or culture for detecting the organism in urine. 相似文献
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Hanan M. Sheikh Ali A. I. Khalafalla A. H. Nimir 《Tropical animal health and production》2009,41(8):1637-1641
PCR following two methods of DNA extraction was used to confirm the growth of camel pox virus (CPV) and vaccinia virus in
cell culture and chorioallantoic membrane. Results were compared with the commonly used neutralization test. The first method
of DNA extraction was accomplished by using viral DNA in tissue culture supernatant and Chorioallantoic membrane, which was
released by initial heating for 15 min at 99°C followed by ordinary PCR. In the second method DNA was extracted by using DNA
Isolation Kit from tissue culture supernatant and used as a template. Rapid identification and differentiation of CPV and
Vaccinia virus were achieved by PCR and this assay proved to be fast and feasible, and can be an alternative to orthodox serological
methods. 相似文献
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为筛选出一种从牛凝固血中高效获得基因组DNA的方法,本研究选择64个凝固血样,分别进行匀浆破碎和手动剪碎的预处理,结合酚-氯仿抽提和试剂盒提取方法,提取基因组DNA,并选择抗凝血作为参照,对试验耗时、DNA数量和质量进行比较。结果表明:通过对牛凝血块进行匀浆破碎预处理,不仅缩短了蛋白酶K的消化时间,而且获得了高质量基因组DNA,浓度和纯度均达到抗凝血提取效果(P0.05)。酚-氯仿提取法相比试剂盒法,虽然得到更多量的DNA,但是DNA质量下降,而且试验耗时增加。本研究证实,匀浆破碎预处理后的牛凝固血样可以作为高质量基因组DNA的样本来源,并可替代抗凝血,解决生产中抗凝血不易获得和采集的问题。 相似文献
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全血中DNA的5种不同提取方法比较研究 总被引:7,自引:0,他引:7
比较几种从血液中提取DNA方法所需要的时间、样本量、提取的DNA纯度、产量以及成本等。结果表明,几种方法所提取的DNA质量都能达到分子生物学的试验要求,但在DNA的纯度、产量、试验时间和价格等方面存在差别。实验人员可根据自己的实验要求、实验室以及经济条件等选择适当的方法。 相似文献