一种快速检测植物病毒RNA蓄积量的竞争PCR方法

用Oligo6.6软件在pUC18上设计1对比目的基因多100～200 bp的内参引物,摸索了目的基因和内参共同扩增的竞争PCR反应条件,测定了嘧肽霉素处理对烟草组织中烟草花叶病毒RNA相对含量的影响,同时用间接ELISA方法验证了该方法.试验结果表明:竞争PCR的方法能够快速、相对准确的测定植物组织中病毒RNA蓄积量的变化情况.     

Physical location of terminal markers belonging to five linkage groups in maize RFLP map using in situ hybridization

Ten terminal or subterminal RFLP markers belonging to linkage groups 1, 3, 5, 6, and 10 in maize RFLP map were physically locted onto maize mitotic chromosomes with in situ hybridization. All biotinylated probes from 600 to 2 250 bp were detected by DAB staining. The markers belonging to linkage groups 1, 3, 5, 6, and 10 correspondingly located at the chromosomes 1, 3, 5, 6, and 10. All of the tested markers except bnl6.25 and umc44 were duplicated sequences. Each of them was also labeled on another chromosome besides on the chromosome corresponding to its linkage group. The marker bnl3. 04 was triplicated sequences and the signals were detected on three nonhomologous chromosomes. In the tested ten markers, there were only four located at the ends of corresponding chromosomes. Others were located at sites midway along the chromosome arms or near the centromeres. The region covered by two terminal or subterminal markers in each of linkage groups 1, 3, 5, and 6 occupied 80.02%, 38.25%, 82.30% and 51.16% of the region of both short and long arms in chromosomes 1, 3, 5, and 6 respectively. Only two terminal markers of linkage group 10 covered the whole chromosome 10. In some linkage groups, two terminal or subterminal markers covered a short genetic distance but were physically distant, while two covering a longer genetic distance were physically closer. Supported by The National Natural Science Foundation of China and the Doctorate Vesting Point Foundation of the Education Committec of the People's Republic of China Mao Ninghui: born in 1986, used to be an MS student of Wuhan University in 1992–1995 and now is working in Fudan University, Shanghai 200433     

稻瘟菌毒素胁迫下水稻根部生理变化及相关基因表达研究

以丽江新团黑谷和谷梅二号为研究对象,利用稻瘟菌粗毒素分别对两者的幼苗根部处理0、6、12、24、48h,并进行地上部和地下部生理指标和相关基因表达分析研究.结果表明,毒素处理后,除SOD外,POD和CAT的活性均升高,MDA含量也升高.与水杨酸和茉莉酸途径相关的8个基因与本底水平相比都表现出上调或下调的趋势,可见这两个途径都得到了表达.特别是48h时,PAL、JIOsPR10、PR1b和PR4在稻瘟病抗性品种谷梅二号的根或叶中的表达量远高于敏感品种丽江新团黑谷,说明这4个基因的表达在调节谷梅二号抗稻瘟菌毒素中具有重要作用.     

多氯联苯胁迫下红树蚬荧光定量PCR内参基因的筛选

【目的】筛选出多氯联苯(PCBs)胁迫下红树蚬(Polymesoda erosa)的实时荧光定量PCR(qRT-PCR)最适内参基因,为进一步开展分子毒理学研究奠定基础。【方法】以β?actin、18S rRNA、GAPDH和#?tubulin为候选内参基因,qRT-PCR测定PCBs胁迫下4个候选内参基因在红树蚬外套膜、鳃、闭壳肌和肝胰腺组织中的表达水平,应用geNorm、NormFinder和BestKeeper软件分析其表达稳定性。【结果】geNorm软件分析表明β?actin表达最稳定;NormFinder软件分析发现,外套膜、鳃和肝胰腺组织中β?actin的稳定性最好,闭壳肌组织中β?actin(0.454)表达稳定性略微低于#?tubulin(0.425),排第2位;BestKeeper软件分析表明,外套膜和鳃组织中β?actin最稳定,肝胰腺和闭壳肌组织中GAPDH最稳定,β?actin排位第2位。【结论】筛选β?actin为红树蚬在PCBs胁迫下的qRT-PCR最适内参基因。     

一种利用微波炉加热快速提取细菌DNA用于PCR扩增的方法

利用微波加热原理,探索一种快速、简单、高效提取细菌DNA用于常规PCR快速检测的方法。在微波炉额定功率800 W条件下分别对大肠杆菌、金黄色葡萄球菌、单增李斯特氏菌、副溶血弧菌、沙门氏菌以及志贺氏菌6种常见食源性病原菌进行微波处理,离心取上清,获取细菌DNA,将样本DNA进行PCR扩增及琼脂糖凝胶电泳进行验证。并进一步采用微波加热方法针对6种病原菌最低检出浓度和混合提取6种病原菌DNA的PCR检测进行分析。6种病原菌在微波炉中加热40-130 s的上清DNA样本均能用于常规PCR扩增。针对检出浓度的分析表明,副溶血弧菌的最低检出浓度为103 cfu/ml,金黄色葡萄球菌为104 cfu/ml,肠出血性大肠杆菌为105 cfu/ml,肠炎沙门氏菌和福氏志贺氏菌的最低检出浓度为106 cfu/ml,针对单核细胞增生李斯特氏杆菌的的最低检出浓度为107 cfu/ml。进行6种混合菌微波共提取时,其中5种病原菌的特异性基因都能扩出,并且条带很亮。该微波提取方法具有快速,简单,高效的特点,能满足大部分食源性病原菌的常规PCR检测需求,大大节约了时间和成本,具有广泛的适用性,为细菌快速分子检测提供了简便手段。     

牛肉及其制品中肉类掺假的荧光PCR鉴别体系优化

根据可能用于牛肉及其制品掺假的肉类原料(猪肉、鸡肉和鸭肉),设计了4对引物和探针.经过测试其具有良好的特异性后,对影响各自聚合酶链式反应(polymerase chain reaction,PCR)扩增的5个影响因素包括Mg2+浓度、Taq DNA聚合酶活、dNTPs浓度、引物和探针浓度以及模板DNA用量等进行了优化,确定了最佳的PCR扩增体系.同时,根据优化后的结果,对特异性进行进一步的测试,并使用建立的优化荧光PCR鉴别体系对混合样品和市售样品进行检测,确定整体检出限为0.1%(质量分数),并证明该鉴别体系的实际应用价值.     

Elucidating the identity of Anisakis larvae from a broad range of marine fishes from the Yellow Sea,China, using a combined electrophoretic‐sequencing approach

Larval nematodes were collected from marine fishes from the Yellow Sea, China. Specimens (n=1731) of Anisakis type I from 311 fishes (representing 40 species) were each identified based on morphological characters. From the genomic DNA from individual specimens, a region of nuclear ribosomal DNA was amplified by PCR, followed by digestion with restriction endonuclease HinfI, TaqI or HhaI. Subsequently, the ITS‐1 and ITS‐2 regions of selected samples were sequenced. The results revealed three species of Anisakis, namely Anisakis pegreffii (n=1709), A. typica (n=3) and a genotype (n=19) proposed, also based on comparison with previous studies, to be a “hybrid” between A. pegreffii and A. simplex sensu stricto. Thus, A. pegreffii was the dominant species, accounting for 98.7% of the total number of specimens examined herein. This is the first report of A. typica and the “hybrid” genotype from fishes from the Yellow Sea. This study provides important basic information on Anisakis in this region and suggests that the genus Anisakis has substantial host and geographical distributions.     

应用PCR技术对DMD做基因诊断的研究

杜氏肌营养不良症，是致死性Ｘ链锁隐性遗传病。     

Quantitation of PCR Products by Capillary Electrophoresis in a Single Run

IntroductionItisnecessarytoevaluatetheamountofDNAfragmentinsuchcasesasgeneexpressioninvestigati0n.HybridizationwithradioactivitytaggedoligonucleotidesfollowedbyphotograPhyanddensitometricscanisoftenemployedtomeettheneedsofsuchrequirementS,whichislaborintensive,timec0nsumingandunautomated-CaPillaryelectrophoresis(CE)tendst0beastrongtoolintheanalysis0fDNAfragmentSwiththeadvantagesofhighsensitivity,highres0luti0nandautomation.QuantitativeanalysisofDNAfragmentshasbeendonebysomeauthorsusingC…