共查询到20条相似文献，搜索用时 906 毫秒
1.
依据牛１．７０９卫星ＤＮＡ序列设计了１对引物，建立了ＰＣＲ鉴定生、熟牛肉的方法。应用本方法特异地扩增出预期的牛２１８ｂｐＤＮＡ片段，其检测敏感度对生牛肉达３３．６ｆｇＤＮＡ，对熟牛肉和高压牛肉为０．３２ｐｇ。运用该引物均可扩增出水牛、牦牛、奶牛、黄牛肉单一的相同大小的ＤＮＡ条带，而对马、山羊、绵羊、骆驼、鹿、猪等１５种动物肉的ＤＮＡ扩增则呈阴性。扩增片段经ＨａｅⅢ酶切分析确认，所得１２９、７９、１０ｂｐ片段与微机分析结果一致。利用本法对１０３份生、熟牛肉及其制品进行鉴定，检出率为１００％。对各种样品检测，均可在６ｈ内完成。 相似文献
2.
玉米品种真实性和纯度鉴定的SSR标记多重PCR体系优化 总被引：7，自引：2，他引：5
为了建立稳定可靠的玉米真实性和纯度鉴定ＳＳＲ 标记,对ＤＮＡ 提取方法、ＳＳＲ 引物和多重ＰＣＲ 反应程序进行了优化。结果表明,用预热到７５℃以上的研钵和９５℃的１．５×ＣＴＡＢ提取缓冲液进行材料研磨,可得到纯度高、完整性好的ＤＮＡ,并且提取成本较低。利用软件ＰｒｉｍｅｒＰｒｅｍｉｅｒ５．０和Ｏｌｉｇｏ６．７２对玉米指纹鉴定的ＳＳＲ 核心引物进行重新分析与设计,建立了２１对ＳＳＲ 通用引物构成的８组多重ＰＣＲ 复合扩增体系和３步法扩增程序,均能在统一的ＰＣＲ 扩增条件下进行,扩增片段之间不存在交叉现象,扩增条带清晰,扩增结果稳定,这一扩增体系检测效率比单对ＳＳＲ 引物提高２．６倍以上。 相似文献
3.
噬菌体肽库的构建及抗狂犬病病毒肽的筛选 总被引：1，自引：0，他引：1
体外合成编码６肽的随机ＤＮＡ片段以及用于随机。ＤＮＡ片段扩增的１对ＰＣＲ引物，经ＰＣＲ扩增ＢｇｌⅠ酶切扩增产物，得到编码６肽的随机ＤＮＡ克隆片段，将随机ＤＮＡ克隆片段与噬菌体表达载体（ＦＵＳＥ５）相连接，连接产物电转化ＭＣ１０６１宿主菌，经抗性和插入复活筛选，获得１．６×１０＾８个独立克隆。文库经ＰＣＲ扩增、斑点杂交、序列测定等综合鉴定，结果表明已成功构建了噬菌体６肽表面表达文库。用生物素化的抗狂 相似文献
4.
用聚合酶锭反应检测鸭瘟病毒的研究 总被引：7，自引：0，他引：7
根据已发表的鸭瘟病毒基因序列，设计并合成了一对引物，其扩增跨幅为６０２ｂｐ。用这对引物对６株鸭瘟病毒ＤＮＡ进行扩增，均获得了与设计大小相符的明亮条带，为阳性率人而对其它６株禽病的原体基因扩增不出任何条带，为阴性。敏感试验表明可检测到１００ｆｇ的鸭瘟病毒ＮＤＡ模板。 相似文献
5.
聚合酶链反应检测猪细小病毒的研究 总被引：18，自引：1，他引：17
本研究成功地建立了检测猪细小病毒（ＰＰＶ）的聚合酶链反应（ＰＣＲ）技术。以１６个核苷酸引物对首次完成了ＰＰＶＤＮＡ结构蛋白ＶＰ１编码区２２８ｂｐ片段的扩增。同样数目的另一引物对，也成功扩增了Ｖｐ２编码区１５８ｂｐＤＮＡ片段。ＰＰＶＢＭ－１株与其它国内分离株（广西株、天津株和哈尔滨株）均出现相同扩增结果。琼脂糖凝胶电泳显示了特异条带（ＶＰ１２２８ｂｐ．Ｖｐ２１５８ｂｐ），而伪狂犬病病毒、犬细小病毒均未扩增，表明了本方法的特异性。同时，限制性内切酶分析（Ｖｐ１２２８ｂｐ及Ｖｐ２１５８ｂｐ分别含单一ＰｖｕⅡ、ＥｃｏＲⅠ位点），也证实了特异性。本方法敏感性高，可检出１０ｆｇ模板ＤＮＡ。既可作样品的快速检测，又能检查细胞系的污染。具有特异、简便、快速的优点。 相似文献
6.
二花脸猪和杜洛克猪的RAPD分析 总被引：8，自引：0，他引：8
利用６０个随机引物对二花脸猪（太湖猪的一个类群）和杜洛克猪的基因组ＤＮＡ进行ＲＡＰＤ分析,其中４８个引物可扩增出清晰的ＲＡＰＤ带型,８个引物可扩增出多型式片段。一个引物（ＯＰＢ１３）可在两品种间扩增出一条稳定的重复性好的多态片段,其大小约为１８９０ｂｐ。结果表明,二花脸猪、杜洛克猪群内遗传距离指数分别为０．０３７１、０．０３４５,遗传多样性指数分别为０．４８６、０．４３１,两品种间存在着ＤＮＡ分子 相似文献
7.
应用聚合酶链反应检测鉴别火鸡支原体方法的建立 总被引：8，自引：0，他引：8
根据基因库中火鸡支原体（ＭＭ）１６Ｓ ｒＲＮＡ基因序列（Ｇｅｎｅｂａｎｋ Ｕ０４６４９）,设计了一对跨幅为８５０ｂｐ的引物,用这对引物对４禽侏火鸡支原体标准菌株和６种其它禽病病原体进行ＰＣＲ扩增,结果４禽株鸡支原体菌株均得到片段大小与预期设计相一致的８５０ｂｐ的ＰＣＲ扩增产物,而其它６种禽病病原体（包括ＭＧ、ＭＳ和ＭＩ）的扩增结果则均为阴性,该ＰＣＲ能检出１００ｆｇ的火鸡支原体的ＤＮＡ模板。 相似文献
8.
两对寡核苷酸引物通过聚合酶链反应扩增大肠杆菌志贺样毒素Ｉ（ＳＬＴ Ｉ）和志贺样毒素Ⅱ（ＳＬＴⅡ）基因,在单一反应中,两对引物分别扩增出１３０ｂｐ（ＳＬＴⅠ）和３４６ｂｐ（ＳＬＴ Ⅱ）的ＤＮＡ 片段,扩增片段经地高辛标记寡核苷酸探针Ｓｏｕｔｈｅｒｎ 印迹杂交分析,证实为ＳＬＴ Ⅰ和ＳＬＴ Ⅱ基因产物。同一扩增反应中应用ＳＬＴⅠ和ＳＬＴⅡ复合引物进行扩增,不同基因型志贺样毒素大肠杆菌从样品中鉴定出。８６９份牛、猪腹泻粪样ＤＮＡ 样品通过ＰＣＲ进行了测定,其中,３％ 检出志贺样毒素大肠杆菌,与牛出血性腹泻、猪水肿病有关。 相似文献
9.
旋毛虫各隔离种快速诊断方法的研究 总被引：1，自引：0，他引：1
提取４个旋毛虫隔离种的ＤＮＡ，应用Ｊｅａｎ设计的引物序列进行了ＰＣＲ扩增。结果黑龙江猪旋毛虫和Ｔ．ｓｐｉｒａｌｉｓ均显示出家畜型旋毛虫特异的６０２ｂｐ目的ＤＮＡ基因片段，而犬旋毛虫和Ｔ．ｎａｔｉｖａ未见此片段。扩增结果显示，黑龙江猪旋毛虫相当于Ｔ．ｓｐｉｒａｌｉｓ，犬旋毛虫相当于Ｔ．ｎａｔｉｖａ。 相似文献
10.
11.
It is essential to isolate high-quality DNA from muscle tissue for PCR-based applications in traceability of animal origin. We wished to examine the impact of cooking meat to a range of core temperatures on the quality and quantity of subsequently isolated genomic (specifically, nuclear) DNA. Triplicate steak samples were cooked in a water bath (100 degrees C) until their final internal temperature was 75, 80, 85, 90, 95, or 100 degrees C, and DNA was extracted. Deoxyribonucleic acid quantity was significantly reduced in cooked meat samples compared with raw (6.5 vs. 56.6 ng/microL; P < 0.001), but there was no relationship with cooking temperature. Quality (A(260)/A(280), i.e., absorbance at 260 and 280 nm) was also affected by cooking (P < 0.001). For all 3 genes, large PCR amplicons (product size >800 bp) were observed only when using DNA from raw meat and steak cooked to lower core temperatures. Small amplicons (<200 bp) were present for all core temperatures. Cooking meat to high temperatures thus resulted in a reduced overall yield and probable fragmentation of DNA to sizes less than 800 bp. Although nuclear DNA is preferable to mitochondrial DNA for food authentication, it is less abundant, and results suggest that analyses should be designed to use small amplicon sizes for meat cooked to high core temperatures. 相似文献
12.
Rajapaksha WR Thilakaratne ID Chandrasiri AD Niroshan TD 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2002,49(7):322-324
A polymerase chain reaction (PCR) assay was developed to differentiate meat of Ceylon spotted deer (Axis axis ceylonensis), Ceylon hog deer (A. porcius oryzus), Ceylon sambhur (Cervus unicolor unicolor) and barking deer (Muntiacus muntijak malabaricus) from meat of cattle, goat, buffalo, pig, dog and sheep. A set of primers was designed according to the sequence of the mitochondrial cytochrome b gene of C. elaphus canadensis and by PCR amplification about 450 bp band was observed for all four animal species and these primers were not cross reacted with DNA of other animal species tested in the study under the tested reaction conditions. A band of 649 bp size was observed for all animal species when DNA was amplified with the universal primers and that indicated the presence of mitochondrial DNA in the samples. Further, the results indicated that this technique was sensitive enough to differentiate rotten meat, at least 5 days after the killing of an animal. Under these PCR conditions, the DNA of bacteria, which is involved in decomposition of meat, was not amplified with both universal and specific primers. However, the method was not sensitive enough in differentiating cooked meat of these species. Slaughtering of these four wild animal species is banned, but the animals are being killed illegally. Lack of meat identification methods has been identified as one of the major constraints to implement legal procedures and conserve biodiversity in the country. 相似文献
13.
在提取黄牛肉、牦牛肉和水牛肉总DNA的基础上,设计通用引物进行PCR扩增,电泳回收PCR产物后双向测序,再通过构建系统进化树鉴别牛肉的物种来源。PCR扩增获得的牦牛、水牛12S rDNA基因片段大小都为440 bp,黄牛12S rDNA基因片段大小都为439 bp。参照引用的不同牛种12S rDNA基因序列,构建的系统进化树能够清晰地鉴别测序样品的牛种来源。因此,结合运用PCR扩增和DNA测序技术是一种精确可靠的方法,能够有效地运用于牛肉的种源鉴别。 相似文献
14.
We developed and evaluated a PCR procedure to detect pork in heated and unheated meat, sausages, canned food, cured products, and patés using a faster, more specific, and more sensitive method than others previously described. Isolation of a new DNA-specific porcine repetitive element was performed by nonspecific PCR amplification. After analyzing this repetitive sequence, a pair of primers were synthesized. To confirm the effectiveness and specificity of this fragment, 55 pig blood DNA samples (from differents breeds) were tested and positive results were obtained. With 200 samples tested from other species, the specific pork amplification was not detected. Using this method, we can partially quantify degree of contamination, depending on the PCR amplification cycles, detecting up to 0.005% pork in beef and 1% pork in duck paté using 30 and 20 PCR amplification cycles, respectively. The amount of porcine DNA detected in cattle DNA was 1.25 and 250 pg when using 30 and 20 amplification cycles, respectively. Pork has been identified in both heated and unheated meat products, sausages, canned food, hamburgers, and patés. In conclusion, specific PCR amplification of a repetitive DNA element seems to be a powerful technique for the identification of pork in processed and unprocessed food, because of its simplicity, specificity, and sensitivity (with 30 amplification cycles we can detect 0.005% pork). Furthermore, it is a very fast method, because 1% pork contamination can be detected with 20 PCR cycles. The procedure is also much cheaper than other methods based on RFLP-PCR, immunodiffusion, or other techniques that need expensive equipment. 相似文献
15.
Bystroń J Molenda J Bania J Kosek-Paszkowska K Czerw M 《Polish journal of veterinary sciences》2005,8(1):37-40
The aim of this work was to determine the contamination of raw poultry meat with enterotoxigenic strains of S. aureus, using the PCR method. PCR is a rapid and sensitive method, which can show the presence in food of enterotoxigenic strains of S. aureus on the basis of specific gene sequences and detect the potential source of contamination before enterotoxins are produced. No coagulase-positive staphylococci strains were found in 65 samples of chicken parts, but these bacteria were present in 11 of 23 examined samples of minced turkey meat (48%). Using the primers for enterotoxin genes A to C, 4 of the 11 isolated S. aureus strains showed a positive result in the PCR. Three of the isolates represented the SEB gene and remaining one the SEC gene. The results obtained showed that PCR is sensitive and rapid method which may be used for detection and identification of enterotoxigenic S. aureus. 相似文献
16.
17.
本研究基于非洲猪瘟病毒（African swine fever virus, ASFV）vp72基因设计引物,建立了能够快速检测非洲猪瘟病毒的环介导恒温扩增技术（loop-mediated isothermal amplification, LAMP）。将LAMP与OIE参考的PCR检测方法进行比较,并且应用LAMP对非洲猪瘟参考实验室提供的非洲猪瘟病毒17个毒株的基因组以及国内收集的50份猪的基因组、30份蜱的基因组进行检测。结果显示,本研究设计的引物具有良好的特异性和敏感性,所建立的LAMP能够成功扩增非洲猪瘟病毒17个毒株的基因组,而野外收集的猪和蜱的基因组检测均为阴性。因此,本研究所建立的方法能够用于非洲猪瘟的快速诊断以及防控。 相似文献
18.
猪瘟病毒野毒株RT-LAMP可视化检测方法的建立 总被引：2，自引：0，他引：2
本研究旨在建立一种可视化检测猪瘟病毒(CSFV)野毒株的反转录.环介导等温扩增方法(RT-LAMP).根据CSFV的NS5B基因序列设计一套RT-LAMP引物,以样品的cDNA为模板,利用Bsf DNA聚合酶,在62℃恒温条件下进行扩增,扩增产物中加入sYBR Green I染料直接或在紫外光下观察判定扩增结果.该方法可检测出不同基因型的CSFV厂野毒株,其检出极限为2.5 TCID_(50)的CSFV,与实时荧光定量RT.PCR方法的敏感性相当;特异性试验表明,该方法对猪瘟免化弱毒疫苗株(HCLV)、牛病毒性腹泻病毒以及其它常见猪源病毒均无扩增反应;通过对126份不同样品进行检测比较,该方法与实时荧光定量RT-LAMP检测方法的符合率达100%.与引物.探针能量转移PCR方法的符合率为98.4%.该方法无需特殊仪器,是一种适用于基层的快速、简便的CSFV野毒鉴别检测方法. 相似文献
19.
Simone Mancini Sara Minieri Arianna Buccioni Margherita Marzoni Fecia di Cossato Claudia Russo Gisella Paci 《Animal Science Journal》2019,90(5):680-689
Tannins were recently evaluated as feed additives in order to increase antioxidant compounds in animal diet, mainly to enhance resistance to lipid oxidation in meat. Rabbit meat is one of the most susceptible animal products, thus the main aim of this study was to evaluate the capacity of tannins to elongate shelf life of rabbit meat. Ninety hybrid rabbits were fed with three different diets: basal diet (control, C) and basal diet supplemented with 0.3% or 0.6% of tannins mix. Meat samples were refrigerated as raw at 4°C up to 11 days and analysed both as raw and cooked for physical‐chemical characteristics, fatty acids profile, lipid oxidation and antioxidant capacity. Results showed that dietary tannins affected meat colour of raw samples (mostly yellowness). Lipid peroxidation (TBARS) of raw samples was lower in tannins group than C group; a further inhibition of peroxidation was showed also in cooked samples only by the highest dose of tannins mix. Moreover, antioxidant capacity (ABTS) of raw samples increased with the percentage of tannins. In conclusion, supplementation with 0.6% of tannins mix seems to positively affect the lipid peroxidation and antioxidant capacity of meat without modifying the intrinsic characteristics of rabbit meat. 相似文献
20.
非洲猪瘟病毒环介导等温扩增快速检测方法的建立 总被引：7，自引：1，他引：6
本试验利用环介导等温扩增技术（LAMP）建立了一种快速高效检测非洲猪瘟病毒的方法。整个反应在水浴锅中恒温进行,不需要其他昂贵仪器,30 min即可得到阳性结果,灵敏度是OIE标准PCR方法的100倍,并且与其他常见猪DNA病毒无交叉反应。该方法非常适合在野外现场或缺乏试验条件的边远地区检测非洲猪瘟病毒。 相似文献