Epidemiology of typical and atypical rotavirus infections in New Zealand pigs

Polyacrylamide gel electrophoresis (PAGE) and enzyme-linked immunosorbent assay (ELISA) were employed to investigate the epidemiology of typical and atypical rotavirus infections in five piggeries. Of 152 faecal samples examined, 46 (30 per cent) were positive by ELISA for group A rotavirus. Rotaviruses with electrophoretic patterns resembling groups A, B and C were detected. At least two and up to five different rotavirus electrophoretypes (typical and/or atypical) were detected in each of the five piggeries. Out of 152 faecal samples examined, 28 (18 per cent) contained rotaviruses with group A electrophor etypes, 9 (6 per cent) with group C but only 1 with Group B. Six samples contained both group A and group C rotaviruses. No common electrophoretypes of group A or C rotaviruses were detected in these five piggeries. The PAGE technique was also used to analyze group A rotavirus isolated sequentially from another piggery over a three year period. A singIe electrophoretype was found during the first two years, but in the third year a different electrophoretype was detected.     

一种不经热浴从双孢蘑菇子实体中提取DNA的新方法(英文)

介绍一种不经热浴快速从双孢蘑菇子实体中提取DNA的新方法,并与传统的CTAB和SDS法相比较,结果显示:该方法得到的DNA完整性好,时间短,步骤少,有毒试剂用量少,在提取DNA的质量、得率、时间及完整性等方面都优于传统的CTAB和SDS法,为食药用菌子实体DNA的提取参考。     

紫云英根瘤菌7653R基因文库的5个互补结瘤菌株的重组质粒分析

对紫云英根瘤菌７６５３Ｒ基因文库的５个重组质粒，即ｐＮＲ１０２，ｐＮＲ１０３，ｐＮＲ１０８、ｐＮＲ２０３，ｐＮＲ２１３进行各种内切酶的酶切分析。结果证实它们均含有１．７ｋｂ的ＥｃｏＲⅠ－ＢｇｌⅡ双酶切片段和１．９ｋｂ的ＥｃｏＲⅠ－ＳａｃⅠ双酶切片段，而ｐＮＲ２１３的外源片段最大，包含有其余质粒所具有的各种片段。以ｐＮＲ１０８的外源片段作探针，进行ＤＩＧ杂交，发现探针与其余４个重组质粒的外源片段有强     

玉米多胞质雄性不育系mtDNA的RAPD分析

 用210个10碱基随机引物对玉米多胞质雄性不育系Mo17-CMS-19A（T组）、Mo17-CMS-Shang26（S组）、Mo17-CMS-C（C组）和它们的保持系Mo17以及对新选育的尚未知其不育类型的糯玉米不育系Tai-A和它的保持系C103等6个品系的线粒体基因组进行了系统的RAPD分析。试验结果如下：（1）对其中40个引物的扩增产物进行了聚类分析，绘制出6个品系的树式遗传关系图，并计算出了它们之间的遗传距离。其中，Tai-A和5个品系间关系较远，遗传距离为24.9；Mo17-CMS-19A和Mo17-CMS-C间关系最近，其遗传距离为6。（2）用OPAC-02、OPAN-05、OPG-19、OPT-09和OPT-12等5种引物的扩增产物制作了6个品系的RAPD指纹，其中OPAC-02#-（680）、OPAC-02#-（1053）、OPAN-05#-（680）、OPAN-05#-（370）、OPT-12#-（1230）、OPT-09#-（800）和OPG-19#-（290）结合起来可以分别作为鉴别Mo17-CMS-Shang26、Mo17-CMS-C、Mo17-CMS-19A、Tai-A、Mo17、C103的分子标记，为快速、方便检测6个品系 提供了可靠的分子生物学手段。用限制性内切酶PstI酶解6种材料的mt基因组，从酶切图谱上发现Tai-A和其它5个品系间差异显著，而其它5个品系间差异相对较小。综合酶切和PAPD分析结果，推测Tai-A是与Mo17-CMS-19A、Mo17-CMS-Shang26、Mo17-CMS-C等细胞质雄性不育组型不同的新细胞质雄性不育类型。     

鳙小卫星pBC174的序列结构特性分析

用双脱氧DNA测序法对鳙 (Aristichthysnobilis)小卫星 pBC174进行序列测定。其长度为 1873bp。在这段序列中 ,A T含量高达 6 4 % ,从 15 11碱基处开始 ,含有多聚腺嘌呤 多聚胞嘧啶 ,即 (AC) 13 的短重复序列的隐蔽微卫星。序列中含有 2 0种限制性内切酶的 5 7个酶切位点 ,97个不同的正向重复序列 ,其中 71个 8bp、10个 9bp、10个 10bp、2个 11bp和 4个 17bp。根据序列分析 ,认为小卫星 pBC174是由富含A的重复序列TAAAGAAA和富含T的重复序列TTTTTACA等 2个不同的核心序列组成。     

不同地区牛瑟氏泰勒虫主要抗原32k蛋白编码基因酶谱分型

提取日本和澳大利亚等不同地区牛红细胞感染瑟氏泰勒虫的ＤＮＡ，利用其主要表面抗原３２ｋ蛋白编码基因的５′和３′端附近序列合成一对引物，经ＰＣＲ方法扩增出的９００ｂｐ大小的基因片段，连接入ｐＢＲ３２２质粒中，转化入大肠杆菌ＪＭ１０９中，经四环素及氨苄青霉素培养筛选出２０～３０个克隆，再用ＰＣＲ方法扩增出每个克隆的靶基因，经ＨｉｎｄⅢ，ＢｇＩＩ和ＫｐｎⅡ限制性内切酶消化后电泳，可分出１型，２型和３型不同     

适于家蚕AFLP标记的酶切反应系统

ＡＦＬＰ作为一种新的分子标记技术，其成功的关键依赖于ＤＮＡ的酶解效果，本文使用ＰｓｔＩ、ＴａｑＩ两种限制性内切酶，选用不同的酶切方式与不同的反应离子的组合对家蚕ＢＣ１代基因组ＤＮＡ进行酶切、通过人工接头连接、二次扩增及ＰＡＧＥ电泳、银染检测，结果显示是两酶在２５ｍＭＴｒｉｓ－Ａｃｅｔａｔｅ（ＰＨ７．８）、１００ｍＭＫＡｃ、１０ｍＭＭＧＡｃ２、１ｍＭＤＴＴ反应系统中能得到多态性丰富、重复性好的ＤＮＡ指纹图谱。同时，表明寻求适宜的限制酶缓冲组分是十分重要的。     

A detection assay forCampylobacter fetus in bovine semen by restriction analysis of PCR amplified DNA

A rapid screening assay forCampylobacter fetus in bull semen was developed using the polymerase chain reaction (PCR) and restriction endonuclease analysis (REA) to complement isolation by culture. An oligonucleotide primer pair (C₁/C₂) from the hypervariable region of 16S rRNA ofC. fetus was used to amplify a 362 base pair fragment by PCR. The PCR/REA assay, which is completed in 10 hours, detected as few as threeC. fetus subsp.venerealis cells in experimentally infected raw bull semen and in semen diluted with milk or egg yolk Tris (EYT). All the strains tested, of both subspecies ofC. fetus, were amplified, as were some otherCampylobacter species. Restricting the amplified products byAluI differentiatedC. fetus from the other organisms. There was no visible product generated by PCR fromC. sputorum subsp.bubulus, a saprophytic organism found in the prepuce of bulls, or from seven other species of bacteria found in semen. A modification of the PCR assay, using another primer pair (C₃/C₂) and two temperature PCR cycling conditions, increased the probability of detectingC. fetus subsp.venerealis. PCR amplification followed by REA could be used to screen bovine semen rapidly forC. fetus. In most cases, sequencing of C₁/C₂ PCR generated products would be preferable for distinguishing between the two subspecies ofC. fetus.Abbreviations AI artificial insemination - bp base pair - Cff Campylobacter fetus subsp.fetus - Cfv Campylobacter fetus subsp.venerealis - EYT egg yolk Tris - MH Mueller-Hinton - PCR polymerase chain reaction - REA restriction endonuclease analysis - TE Tris-EDTA     

恶性疟原虫3D7株AP核酸内切酶的原核表达及多克隆抗体的制备

为研究恶性疟原虫(Plasmodium falciparum)3D7株AP核酸内切酶(AP endonuclease)的功能,通过PCR的方法扩增PF3D7_0305600(292~1 137 bp),构建克隆载体。测序正确后,将该片段插入到pET-28a和p GEX-4T-1,构建重组表达载体p ET-28a-Ae、p GEX-4T-1-Ae。将表达载体转入BL21-Codon Plus表达重组蛋白r-Ae-His和r-Ae-GST,经SDS-PAGE和Western blot鉴定,r-Ae-His免疫家兔制备多克隆抗体,并用ELISA检测抗血清效价,Western blot检测抗体特异性。结果表明:表达的r-Ae-His分子量约为39 ku,r-Ae-GST分子量约为60 ku。制备的多克隆抗体能够特异性地识别天然蛋白,可用于恶性疟原虫3D7株AP核酸内切酶的后续研究。     

茶毛虫核型多角体病毒基因组酶切分析及质粒文库的构建

应用限制性内切酶图谱分析法,估算茶毛虫核型多角体病毒基因组大小为１３１．０９ｋｂ;通过随机克隆,将ＥｐＮＰＶ基因组８条ＢａｍＨ Ｉ酶切片段中的６条,１３条Ｐｓｔ Ｉ酶切片段中的７条,２６条ＥｃｏＲ Ｉ酶切片段中的７条,２１条ＨｉｎｄⅢ酶切片段中的７条,１４条Ｘｂａ Ｉ酶切片段中的９条分别克隆至载体ＰＴＺ１９Ｒ,构建了ＥｐＮＰＶ基因组的部分质粒文库。