基于DNA染料EMA的RT-PCR技术定量检测海产品中病原性副溶血弧菌活细胞

将一种DNA染料叠氮溴化乙锭(ethidium bromide monoazide,EMA)与实时定量聚合酶链式反应(RT-PCR)技术相结合,建立一种能选择性定量检测牡蛎中trh阳性副溶血弧菌活细胞的新方法。结果表明：使EMA成功插入死细胞DNA并且光解溶液中游离EMA的最佳曝光时间为20min;不抑制副溶血弧菌活细胞DNA扩增的最大EMA质量浓度为2.0μg/mL;完全抑制热致死细胞DNA扩增的最小EMA质量浓度为1.0μg/mL;人工污染牡蛎样品,不经过富集,在2.0×103～2.0×107CFU范围内细胞数的常用对数值与Ct值之间呈严格的负相关性,并且纯培养与人工污染牡蛎样品的RT-PCR检测限均为2×103CFU,即人工污染牡蛎样品的RT-PCR检测灵敏度为每克牡蛎样品400个活细胞;冻融实验表明,在温度低于55℃的水浴中对冷冻海产品进行解冻时,冻融过程对副溶血弧菌活细胞几乎没有影响。该方法是一种快速、灵敏且能有效鉴别并定量检测病原活细胞的新方法。     

不同破壁方法提取绍兴黄酒麦曲中的微生物总DNA

分别采用超声波、Yatalase酶法、氯化苄法对黄酒麦曲样品进行破壁,提取微生物总DNA,并通过细胞裂解率、DNA的纯度、片段的分布情况、ITS(内转录间隔区)序列扩增产物的多态性来评估不同的破壁方法对麦曲样品总DNA提取效果的影响。实验结果表明,采用氯化苄法能裂解86%的孢子,OD260/OD280在1.5左右,所得DNA片段分布在100bp～20kbI,TS-PCR扩增出的条带最多,而且简单、经济、提取效果好,可以作为麦曲中微生物总DNA的提取方法。     

The Role of Natural Polymorphic Variants of DNA Polymerase β in DNA Repair

DNA polymerase β (Polβ) is considered the main repair DNA polymerase involved in the base excision repair (BER) pathway, which plays an important part in the repair of damaged DNA bases usually resulting from alkylation or oxidation. In general, BER involves consecutive actions of DNA glycosylases, AP endonucleases, DNA polymerases, and DNA ligases. It is known that protein–protein interactions of Polβ with enzymes from the BER pathway increase the efficiency of damaged base repair in DNA. However natural single-nucleotide polymorphisms can lead to a substitution of functionally significant amino acid residues and therefore affect the catalytic activity of the enzyme and the accuracy of Polβ action. Up-to-date databases contain information about more than 8000 SNPs in the gene of Polβ. This review summarizes data on the in silico prediction of the effects of Polβ SNPs on DNA repair efficacy; available data on cancers associated with SNPs of Polβ; and experimentally tested variants of Polβ. Analysis of the literature indicates that amino acid substitutions could be important for the maintenance of the native structure of Polβ and contacts with DNA; others affect the catalytic activity of the enzyme or play a part in the precise and correct attachment of the required nucleotide triphosphate. Moreover, the amino acid substitutions in Polβ can disturb interactions with enzymes involved in BER, while the enzymatic activity of the polymorphic variant may not differ significantly from that of the wild-type enzyme. Therefore, investigation regarding the effect of Polβ natural variants occurring in the human population on enzymatic activity and protein–protein interactions is an urgent scientific task.     

Application of IS900 PCR for detection of Mycobacterium avium subsp. paratuberculosis directly from raw milk

A polymerase chain reaction (PCR)-based assay was developed for detection of insertion sequence 900 (IS900) of Mycobacterium avium subsp. paratuberculosis in raw milk. This IS900 PCR assay included DNA extraction and PCR assay using commercially available kits. The DNA extraction and PCR assay were optimized to detect the IS900 sequence directly from raw milk. The IS900 PCR assay was evaluated by inoculating raw bulk milk and Middlebrook's 7H9 broth with 0 to 10(8) cfu/ml of each of four American Type Culture Collection strains of M. paratuberculosis. Under experimental conditions, both milk culture on Herrold's egg yolk medium slants, and IS900 PCR could detect 10 to 100 cfu/ml of M. paratuberculosis. Detection of M. paratuberculosis by IS900 PCR was consistent (24/24 PCR assays) when about 100 cfu/ml were present, whereas detection was variable (12/24 PCR assays) at concentrations as low as 10 cfu/ml. Based on the findings of the experimental study, IS900 PCR was further evaluated with pooled quarter milk samples from 211 cows from five herds with known history of Johne's disease. Out of 211 animals examined, nine (4%) and 69 (33%) were positive for M. paratuberculosis by milk culture and IS900 PCR from milk, respectively. A total of 20 bulk tank milk sample aliquots (one sample, four aliquots from each herd) were also examined, of which 10 (50%) were positive for M. paratuberculosis by IS900 PCR. By contrast, only one out of 20 (5%) bulk tank milk sample aliquots was positive by culture. The IS900 PCR amplified product of 229-bp obtained on testing of quarter milk and bulk tank milk samples was confirmed to be the IS900 of M. paratuberculosis by DNA sequence analysis. The results of this study suggest that M. paratuberculosis can be detected directly from quarter milk and bulk tank milk by IS900 PCR.     

籼型杂交水稻保持系IR58025B pin2转基因植株

杂交水稻技术在中国及其他国家已作为发展水稻生产,满足不断增加的人口对粮食需要的一条关键途径．而另一方面,据估计因螟虫危害造成水稻年均损失为5％～10％,有时高达60％～95％．应用马铃薯蛋白酶抑制剂基因(potato proteinase inhibitor Ⅱor pin2)进行粳稻的遗传转化实验表明,其转基因粳稻植株对螟虫的抗性显著提高,且该转基因能在后代中稳定遗传(Duan et al．,1996)．为了改良杂交水稻对螟虫的抗性,以大面积应用的籼型杂交稻保持系IR58025B成熟胚为外植体材料、以pin为目的基因、通过基因枪法高效获得了大量转基因植株．PCR检测结果表明转基因pin2成功地整合于植株基因组中．三化螟幼虫饲喂试验结果初步证实了该转基因在转基因当代植株中的表达．     

New modules for PCR-based gene targeting in Candida albicans: rapid and efficient gene targeting using 100 bp of flanking homology region

The use of PCR-based techniques for directed gene alterations has become a standard tool in Saccharomyces cerevisiae. In our efforts to increase the speed of functional analysis of Candida albicans genes, we constructed a modular system of plasmid vectors and successfully applied PCR-amplified functional analysis (FA)-cassettes in the transformation of C. albicans. These cassettes facilitate: (a) gene disruptions; (b) tagging of 3'-ends of genes with green fluorescent protein (GFP); and (c) replacements of endogenous promoters to achieve regulated expression. The modules consists of a core of three selectable marker genes, CaURA3, CaHIS1 and CaARG4. Modules for C-terminal GFP-tagging were generated by adding GFP-sequences flanked at the 5'-end by a (Gly-Ala)3-linker and at the 3'-end by the S. cerevisiae URA3-terminator to these selection markers. Promoter exchange modules consist of the respective marker genes followed by the regulatable CaMAL2 or CaMET3 promoters at their 3'-ends. In order to ensure a reliably high rate of homologous gene targeting, the flanking homology regions required a size of 100 bp of gene-specific sequences, which were provided with the oligonucleotide primers. The use of shorter flanking homology regions produced unsatisfactory results with C. albicans strain BWP17. With these new modules only a minimal set of primers is required to achieve the functional analysis of C. albicans genes and, therefore, provides a basic tool to increase the number of functionally characterized C. albicans genes of this human pathogen in the near future.     

Constitutive promoter modules for PCR-based gene modification in Saccharomyces cerevisiae

Initial steps in investigating gene function often include deleting and overexpressing the gene of interest and identifying the subcellular location of the gene product. To facilitate these procedures, a number of new PCR modules, which contain selectable markers and in some cases other genetic elements (e.g promoter elements, epitope tags, and reporter genes) have been developed. These modules are used as PCR substrates to create products that can be targeted to specified locations in the yeast genome, thus modifying that genomic locus. We describe here a series of plasmids that contain a truncated version of the strong ADH1 promoter with and without amino-terminal 3HA and GST tags. Because these plasmids contain the same vector sequences as the GAL1 promoter plasmids, a constitutive and an inducible promoter can now be integrated with a minimal number of primers.     

同时检测速冻食品中鸡、鸭、猪和牛源性成分实时荧光聚合酶链式反应方法的建立及应用

以鸡转化生长因子β-3(transforming growth factor beta-3,TGFB3)基因、猪朊蛋白(prion protein,PRNP)基因和鸭、牛生长激素(growth hormone,GH)基因为靶基因,设计合成特异性引物和TaqMan探针,通过对实时荧光聚合酶链式反应(polymerase chain reaction,PCR)体系和反应条件的优化,建立同时检测速冻食品中鸡、鸭、猪和牛源性成分的实时荧光PCR检测方法。结果表明:本研究所建立的方法只在鸡、鸭、猪或牛源性成分存在时才产生特异性扩增曲线,表明该方法具有良好的特异性;且对鸡、鸭、猪和牛源性成分的最低检测质量浓度分别可达到10.0、1.0、10.0、10.0 pg/μL,具有良好的灵敏性;应用建立的检测方法对市售34份速冻食品鸡、鸭、猪和牛源性成分进行同时检测,能够实现对速冻食品中4种动物源性成分的有效检测,进一步分析发现,15份速冻食品源性成分测定结果与外包装标注成分不一致。本研究所建立的同时检测速冻食品中鸡、鸭、猪和牛源性成分的实时荧光PCR检测方法具有良好的特异性和灵敏性,且操作简单、适用范围广,能够满足日常检测需求。