新蚕药蝇僵灵的防治效果试验

蝇僵灵是一种能防治多化性蚕蝇蛆和家蚕真菌性病害的新型多功能蚕药。对多化性蚕蝇蛆卵、蛆的最低有效杀灭浓度为 5 0 0倍 ;对家蚕真菌性病害病原孢子最低有效防治浓度为 6 0 0倍。生产实用浓度 375倍 ,对多化性蚕蛆蝇及真菌性病害的防治效果均达 95 %以上。对家蚕的半中毒剂量 (KD50 ) 4龄为 0 746 4μL/头或 0 92 75 μL/g,5龄 3 9390 μL/头或 1 886 0 μL/g ;全部中毒最低剂量 (MED) 4龄为 1 6 0 0 0 μL/头或 2 0 2 0 0 μL/g ,5龄为 12 80 0 0 μL/头或 6 0 95 2 μL/g ,最大安全剂量 (MPD) 4龄为 0 2 0 0 0 μL/头或 0 2 5 41μL/g,5龄为 0 80 0 0 μL/头或 0 333 3μL/g。蝇僵灵原药可自然贮放 2 5a,稳定性良好     

蚕微卫星DNA体外自我扩增条件初步研究

采用一种类似PCR的基因组自我引发PCR(GSP PRC)法 ,初步研究了家蚕、野桑蚕、天蚕、蓖麻蚕和柞蚕的微卫星DNA的体外基因组自我引发PCR扩增条件。质量浓度为 10 0mg/L的基因组DNA经 10 0℃变性 15min后 ,与等量相同浓度未变性的DNA混合作为模板 ,在 80 μL扩增体系 [含 0 2mmol/LdNTPs、5 0mmol/LKCl、10mmol/LTris HCl(pH 9 0 )、4mmol/LMgCl2 、1 2 5UTaqpolymerase]中加入 1μL此混合模板 ,在92℃、1min→ 5 5℃、2min→72℃、2min ,30～ 90次累积循环的扩增条件下 ,成功地得到了PCR产物。反应体系中 ,基因组DNA的适宜质量浓度为 1 2 5mg/L。GSP PCR产物经琼脂糖凝胶电泳后的部分片段回收后 ,在同样条件下 ,30～ 6 0个循环可得到同样大小范围的产物。GSP PCR产物经 6 %的变性测序胶电泳 ,得到典型的梯状带 ,表明为微卫星DNA。     

竞争性RT-PCR检测铜对Ctr1 mRNA表达的影响

为了应用竞争 RT- PCR法检测铜对猪传代肾细胞 (PK15 )中特异性铜转运蛋白 Ctr1基因 m RNA表达水平的影响 ,经 2次克隆 ,将筛选出一段 4 0 0 bp的核苷酸序列 ,插入 RT- PCR扩增出的 5 30 bp Ctr1目的片段中 ,构建了插入突变型 Ctr1- c DNA竞争模板。再将竞争模板与各试验组 c DNA同时进行 PCR扩增。结果 ,猪肾 PK15细胞 Ctr1基因m RNA表达量在 7.8～ 31.2 μmol/ L Cu范围内随铜添加浓度的升高减少 ,当铜添加浓度达到 6 2 .5 μmol/ L 时 ,其表达量又有所回升 ,呈双相反应。     

枫泾猪发情周期子宫性腺激素受体含量的变化

以长白猪为对照 ,测定了枫泾猪发情周期血浆中雌二醇和孕酮及其子宫受体含量。发情期血浆雌二醇的峰值 ,枫泾猪为 ( 57.0 4± 3 .60 ) ng/ L,长白猪为 ( 55.85± 3 .0 4 ) ng/ L,两者无显著差异 ( P >0 .0 5)。孕酮最高水平和间情期平均水平 ,均为枫泾猪高 ( P <0 .0 1 )。孕酮最高水平 ,枫泾猪为 ( 2 4 .1 1± 1 .2 4 ) μg/ L,长白猪为 ( 1 6.67± 0 .2 1 ) μg/ L;孕酮间情期平均水平 ,枫泾猪为 ( 1 8.1 1± 1 .2 4 ) μg/ L,长白猪为 ( 1 1 .0 7± 0 .61 )μg/ L。枫泾猪发情期和间情期的细胞质雌二醇受体 ( CER)分别为每毫克 DNA( 3 87± 2 1 .2 )、( 3 52± 1 8.2 )fmol,其离解常数 ( kd)值分别为 ( 4.8± 0 .4 )、( 3 .8± 1 .0 ) nmol;细胞核雌二醇受体 ( NER)分别为每毫克DNA( 1 2 69.58± 1 56.4 2 )、( 578± 2 1 .4 ) fmol,其 kd值分别为 ( 5.8± 1 .4 )、( 3 .4 8± 1 .0 ) nmol;细胞质孕酮受体 ( CPR)分别为每毫克 DNA( 3 4 3 .0± 51 .4 )、( 1 48± 4 1 .0 ) fmol;细胞核孕酮受体 ( NPR)分别为 ( 3 2 4± 6.9)、( 1 1 5± 2 .4 ) fmol。发情期和间情期子宫 NER含量较长白猪高 ( P <0 .0 5) ,说明此时枫泾猪子宫对雌二醇具有较高的敏感性     

垂穗披碱草ISSR反应体系的正交优化

以垂穗披碱草(Elymus nutans)基因组DNA为模板,对ISSR-PCR反应体系中5个因素(TaqDNA聚合酶、Mg2+、模板DNA、dNTPs、引物)进行优化试验.建立垂穗披碱草稳定的ISSR-PCR反应体系及最佳扩增程序.在25 μL反应体系中,最佳反应体系为2.5 μL 10×buffer(不含Mg2+)...     

带有绿色荧光蛋白外源DNA转染绵羊成纤维细胞的研究

为了优化建立转染绵羊成纤维细胞系的外源基因转染体系,本研究利用带有绿色荧光蛋白（GFP）的外源基因,采用阳离子脂质体法,探讨了DNA用量、脂质体用量和转染时间等因素对转染绵羊成纤维细胞的影响。结果表明,在500 μL转染培养基中成纤维细胞达到85%汇合时,添加由50 μL DMEM中含有0.2 μg DNA和50 μL DMEM中含有2.0 μL LipofectamineTM 2000的两种溶液混合构成的转染液并转染12 h,可获得最高的转染效率。当转染12 h后,用基础培养液培养至第24小时,细胞核周区出现强绿色荧光,而当转染后48～64 h观察时,细胞核周区的荧光强度明显弱,而远离核周区的胞质中的荧光强度增加。     

燕麦AFLP反应体系的建立与优化

在利用扩增片断长度多态性(AFLP)技术研究燕麦Avena sativa遗传多样性过程中,从引物筛选、酶切连接、扩增条件、检测方法等几个方面进行优化,建立燕麦AFLP最适反应体系为:800ng/μL DNA 用10 U EcoRⅠ和4 U MseⅠ在37 ℃ 7 h,65 ℃ 4 h温浴双酶切; 16 ℃下连接过夜;预扩增取连接产物1 μL,10 μmol/L预扩引物各0.6 μL, 10×buffer 2μL, dNTPS 2μL,ddH2O 补平至20 μL;取5μL稀释 20 倍后的预扩增产物,50 ng/μL EcoRⅠ、MseⅠ选扩引物各0.8μL,总体系20 μL进行选择性扩增.在此优化体系下,可以得到重复性好、稳定且多态性高的燕麦AFLP条带.     

大肠杆菌β-内酰胺酶耐药基因blaTEM,blaSHV,blaCTX-M三重PCR检测方法建立

本研究以我国大肠杆菌中常见的3种β-内酰胺酶耐药基因(bla,TEM blasHV和blaCTX-M)作为目的基因,设计3对特异性引物,建立了blaTEM,bla SHV和blaCTX-M耐药基因三重PCR检测方法.三重PCR反应体系为:10×PCR缓冲液2.5 μL,MgCl2(25 mmol/L) 2.5 μL,dNTP (2.5 mmol/L)4 μL,blaTEM,blasHV和blaCTX-M上下游引物(25 μmol/L)分别为0.25 μL、0.5 μL、1.0μL,Taq DNA聚合酶(2.5 U/μL)0.4μL,模板2.5μL,反应总体积为25 μL.反应参数为:94℃5 min,94℃50 s,55℃55 s,72℃1 min,32个循环,72℃延伸5 min.本方法的建立为大肠杆菌中β-内酰胺酶耐药基因的快速检测及分子流行病学调查提供了新的手段.     

多个顺式作用元件调控t-PAcDNA乳腺定位表达

由牛 β-酪蛋白 ( β-Casein)启动子与大鼠乳清酸蛋白 ( WAP)启动子融合及串联构建成 2个组织纤溶酶原激活剂 ( t-PA) c DNA乳腺定位表达载体 p SVL-βΔWAP-PA和 p SVL-WAPβ-PA。将这 2种表达质粒分别注入怀孕家兔乳腺导管中 ,溶圈试验结果显示 ,2种质粒均能在家兔乳腺中表达 ,且以分娩后第 5天的乳汁中表达水平最高 ,分别为 70 0 μg/ L和 50 0 μg/ L。本试验为进一步研究 t-PA基因乳腺定位表达调控和建立 t-PA乳腺生物反应器奠定了基础     

脂质体法转染水牛肾脏成纤维细胞条件的优化

试验旨在研究脂质体介导基因转染水牛肾脏成纤维细胞时如何获得较高的转染效率。本研究将以绿色荧光蛋白基因为标记基因,利用Lipofectamine LTX介导外源DNA转染水牛肾脏成纤维细胞,探讨脂质体用量、质粒用量、质粒大小、转染时间、细胞初始接种密度对转染效率的影响。结果表明,4 μg pmaxGFP质粒DNA与4 μL脂质体转染6 h,其细胞活性达98%,可获得较满意的转染效率。     

Isolation and detection of Fasciola hepatica DNA in Lymnaea viatrix from formalin-fixed and paraffin-embedded tissues through multiplex-PCR

Detection of Fasciola hepatica infection in Lymnaea viatrix through analysis of histological cuts is based upon morphological characters of the parasite during the intra-mollusk phase of parasitism. At this stage, trematode forms are very similar and, thus, very difficult to differentiate. Specific detection may also be impaired by the presence of other helminthes in the mollusk. Histological samples are usually fixed in formalin, embedded in paraffin, sectioned and HE stained. In the current study, a method for the extraction of DNA from formalin-fixed, paraffin-embedded tissues was standardized by means of deparaffinizing with xylol and digesting with proteinase K. Extracted DNA was amplified in a multiplex-PCR, by using simultaneous primers in a single reaction under high stringency conditions. Results showed specific amplification of DNA from the trematode and the snails. The technique was sensitive enough to detect F. hepatica infections in L. viatrix, in histological sections in which the presence of larval stages could not be observed through brightfield microscopy. The profiles generated were: stair bands referring to F. hepatica DNAmt amplification; a band of 1200 bp referring to L. viatrix ITS and another of 1300 bp referring to F. hepatica ITS and other trematodes. Multiplex-PCR has shown to be a fast, safe, highly sensitive and specific method, which is able to amplify DNA from fixed tissues, despite a low DNA quantity and its degradation caused by fixation processes. Such methodology may be useful in studies on fascioliasis epidemiology, enabling the use of material from histological collections.     

A novel haemoplasma species identified in archived primate blood smears

In order to confirm a microscopic diagnosis of 'eperythrozoonosis' made over 40 years ago in a captive owl monkey (Aotus trivirgatus), DNA was extracted from archived fixed and stained blood smears and subjected to generic haemotropic mycoplasma (haemoplasma) quantitative real-time PCR (qPCR) and a human glyceraldehyde-3-phosphate dehydrogenase qPCR as an amplification control. The qPCRs confirmed the extraction of host DNA from the samples and the presence of a haemoplasma species. Partial 16S rRNA and ribonuclease P ribosomal gene fragments were amplified by PCR, cloned and sequenced. Sequence data and phylogeny showed the owl monkey haemoplasma to lie in the haemominutum clade of haemoplasmas, most closely related to 'Candidatus Mycoplasma kahaneii'. This study confirms the use of generic haemoplasma qPCRs to successfully amplify haemoplasma DNA from fixed, stained and archived blood smears from the early 1970s and provides molecular confirmation of the existence of a novel haemoplasma species in an owl monkey, for which the name 'Candidatus Mycoplasma aoti' sp. nov. is proposed.     

Detection of Leptospira interrogans DNA and antigen in fixed equine eyes affected with end-stage equine recurrent uveitis.

Equine recurrent uveitis (ERU) is the most frequent cause of blindness in horses worldwide. Leptospira has been implicated as an etiologic agent in some cases of ERU and has been detected in fresh ocular tissues of affected horses. The objective of this study was to determine the presence of Leptospira antigen and DNA in fixed equine ocular tissues affected with end-stage ERU. Sections of eyes from 30 horses were obtained. Controls included 1) 10 normal equine eyes and 2) 10 equine eyes with a nonrecurrent form of uveitis. The experimental group consisted of 10 eyes diagnosed with ERU based on clinical signs and histologic lesions. Sections were subjected to immunohistochemical staining with an array of rabbit anti-Leptospira polyclonal antibodies. DNA extractions were performed by using a commercial kit designed for fixed tissue. Real-time PCR analysis was completed on extracted DNA. The target sequence for PCR was designed from alignments of available Leptospira 16S rDNA partial sequences obtained from GenBank. Two of 10 test samples were positive for Leptospira antigen by immunohistochemical assay. Zero of 20 controls were positive for Leptospira antigen. All test samples and controls were negative for Leptospira DNA by real-time PCR analysis. Leptospira was detected at a lower frequency than that previously reported for fresh ERU-affected aqueous humor and vitreous samples. Leptospira is not frequently detectable in fixed ocular tissues of horses affected with ERU when using traditional immunohistochemical and real-time PCR techniques.     

Southern blot analysis of strain variation in Campylobacter mucosalis.

A panel of three DNA probes were derived at random from a genomic DNA library of Campylobacter mucosalis strain E8384-4. Each probe hybridized specifically to C. mucosalis DNA from bacteria fixed to nylon membranes. The probes did not hybridize to DNA from other Campylobacter species or to other bacteria even at 100-fold higher amounts. Each probe hybridized to all of 24 isolates of C. mucosalis which had been collected over time from different geographic locations. Southern blot analysis of selected C. mucosalis isolates was carried out to determine if the probes would be useful for differentiating among various isolates. It indicated that restriction fragment length polymorphisms (RFLPs) exist at the loci identified by our probes. These differences were used to characterize seven C. mucosalis isolates recovered from pigs in Minnesota. The results suggest that RFLP analysis may be a useful tool for epidemiological studies of C. mucosalis.     

家蚕后部丝腺离体线粒体的制备及其电镜制样技术的改良

为研究家蚕(Bomb yxmori)线粒体DNA(mt DNA),我们用一种简便的提取分离技术获得了纯净完好的离体线粒体制品。在离体线粒体电镜制样过程中,发现琼脂予包埋、乙醇脱水处理将破坏线粒体膜结构。改用线粒体沉淀直接固定,丙酮脱水处理电镜样品,线粒体膜结构得到保存,效果良好。     

Quantitation of sheep-associated malignant catarrhal fever viral DNA by competitive polymerase chain reaction.

A single-step, competitive polymerase chain reaction technique was developed to quantitate sheep-associated malignant catarrhal fever (SA-MCF) viral DNA. The assay employed coamplification of a fixed quantity of target DNA with graded amounts of a competitor, generated by truncation of the target sequence lying between the 2 primer binding sites. The assay yielded a linear response (r = 0.98) for DNA measurement within the range of 30-300,000 copies. Amplification efficiency analysis by coamplification of target and competitor in equal copy numbers for various numbers of cycles showed that the relative abundance of the coamplified products remained constant with increasing cycle numbers up to 40. Reproducibility was assessed by repetitively assaying a set of blind-coded samples from a variety of animals and tissues. Results indicated that the assay is reliable and reproducible for quantitation of SA-MCF viral DNA in samples from asymptomatically infected sheep and from animals with clinical SA-MCF.     

An improved method for discriminating between Japanese Nagoya chickens and others

Recently, we reported a method for identifying the Nagoya, a Japanese chicken breed. Here we describe an improved method suitable for use in conventional laboratories. Five microsatellite markers fixed in the Nagoya breed were amplified by multiplex polymerase chain reaction (PCR). The PCR products were digested with CEL I nuclease and electrophoresed in a ready‐made gel. The fragment‐length polymorphisms of each marker were detected clearly in the gel, and the resultant exclusion probabilities were almost equal to that in our previous data. This method can be used in laboratories without a DNA sequencer for routinely discriminating between the Nagoya breed and other chickens.     

大熊猫粪便DNA纯化及其PCR检测

建立了二氧化硅法纯化大熊猫粪便中DNA的技术 ,所得DNA可用于PCR扩增 ,而传统的有机溶剂提取的DNA不能去除粪便中抑制PCR反应的成分 ,使扩增结果呈假阴性。     

Detection of Toxoplasma gondii in Crassostrea spp. oysters cultured in an estuarine region in eastern Amazon

The aim of the present study was to detect DNA of Toxoplasma gondii in Crassostrea spp. oysters cultured in the state of Pará, Brazil. A total of 400 oysters were directly collected from a fixed rack system. Gills, gastrointestinal tract (GIT) and intervalvular liquid were separated and grouped into pool samples of 10 animals, resulting in 40 samples each of gills, GIT and intervalvular liquid. DNA extraction was performed using a commercial kit, and T. gondii DNA was detected by nested PCR using the primers Toxo3 and Toxo4, which produced an amplification product of 155 bp of the T. gondii gene B1. Nucleotide sequencing was performed for positive samples, and the obtained sequences were identified by comparison with sequences in GenBank. The DNA of T. gondii was detected in 5.8% (7/120) of the pool samples, of which 7.5% (3/40) was in the GIT, 5% (2/40) in the gills, and 5% (2/40) was in the intervalvular liquid. The obtained sequences presented 100% identity and overlap with T. gondii DNA sequences. This is the report of detection of T. gondii DNA in oysters from genus Crassostrea spp. originating from the state of Pará, eastern Amazon.     

Determination of sheep prion gene polymorphisms from paraffin-embedded tissue.

Amino acid polymorphisms of the prion protein (PrP) greatly influence the susceptibility of sheep to scrapie. Selective breeding to increase the prevalence of PrP gene alleles associated with scrapie resistance is a flock management practice that is important for scrapie control programs. Determination of sheep PrP alleles typically has required extraction of DNA from host tissues that are freshly derived or stored frozen. We describe application of a DNA extraction procedure for formalin-fixed, paraffin-embedded tissues (PET) for the purpose of PCR amplification and nucleotide sequencing of relevant codons (136-171) of the sheep PrP gene. Tissues derived from 96 sheep were studied. The DNA sequence identity was confirmed in 87 of 94 matched samples of PET and frozen tissue specimens. DNA from brainstem PET of 2 sheep, from which fresh tissue was not available, was amplified and sequenced after formalin fixation for 7-70 days. This method will allow retrospective analysis of PrP genetics of sheep subsequent to postmortem diagnosis of scrapie when nonfixed tissue is unavailable for DNA extraction; however, it is not recommended that submission of fixed tissue supplant collection of fresh tissues for the purpose of determining PrP gene polymorphisms.