The rRNA genetic locus is found in all prokaryotic organisms, and is highly conservative, although its relatively stable variations are found frequently in different bacteria. The utility of this locus as a taxonomic ...The rRNA genetic locus is found in all prokaryotic organisms, and is highly conservative, although its relatively stable variations are found frequently in different bacteria. The utility of this locus as a taxonomic and phylogenetic tool has been reported widely. This study, aimed at 16S rRNA gene (16S rDNA) and with the help of biomolecular methods, attempted to achieve the goal of rapid identification of common pathogens. In this study, 333 clinical isolated pathogenic bacteria were collected. Two pairs of primers were chosen and labeled with different fluorescent dyes and then used to amplify the genomic DNA extracted from bacteria. The PCR products were then detected by capillary electrophoresis-single strand conformation polymorphism (CE-SSCP). In order to pursue higher resolution and peak-separation effect, a high efficient separating medium, liner polyacrylamidedel (LPA), was put to use in this study. Finally, every bacteria colony generated distinct patterns from each other, which were easily to be used for identification. These results indicated that PCR-CE-SSCP was a rapid identification method for bacterial identification, with the aspects of high efficiency and high precision. Compared with traditional method, this technology is of great utility for clinical use especially for its high sensitivity.展开更多
Loop-mediated isothermal amplification(LAMP)is a novel nucleic acid amplification method.Compared with the widely utilized polymerase chain reaction(PCR),LAMP has higher speed and efficiency as well as lower requireme...Loop-mediated isothermal amplification(LAMP)is a novel nucleic acid amplification method.Compared with the widely utilized polymerase chain reaction(PCR),LAMP has higher speed and efficiency as well as lower requirement for system temperature control because the whole amplification process is isothermal and no efforts are needed to switch between different temperatures.In this paper,we designed and fabricated different kinds of polycarbonate(PC)microfluid chips,explored appropriate reaction condition for LAMP in microenvironment(1 nL→10μL),and developed a microfluidic isothermal amplification detection system.The DNA optimal amplification temperature is obtained;the starting time of exponential amplification of DNA is put forward farther.The optimal condition of DNA amplification in microenvironment,with a little reaction materials and early starting exponential amplification time of DNA are very important for clinic DNA detection and the application of Lab-on-a-Chip.展开更多
在分离Y染色体性别决定区(Sex determining region of the Y chromosome,SRY)、人11号染色体短臂β-珠蛋白基因部分序列(108/B)扩增产物和PBR322/HaeⅢ标准片段的混合样品时,SRY和108/B的迁移时间均产生较大的偏差,在尿素变性剂存在的...在分离Y染色体性别决定区(Sex determining region of the Y chromosome,SRY)、人11号染色体短臂β-珠蛋白基因部分序列(108/B)扩增产物和PBR322/HaeⅢ标准片段的混合样品时,SRY和108/B的迁移时间均产生较大的偏差,在尿素变性剂存在的无胶筛分体系中迁移时,这种迁移时间的偏差得以消除,这意味着尿素变性剂的存在消除了DNA空间结构对迁移的影响,DNA在此筛分介质中的迁移只与其片段长度有关.在变性无胶筛分体系中鉴别PCR产物,其结果的准确度明显提高.展开更多
文摘The rRNA genetic locus is found in all prokaryotic organisms, and is highly conservative, although its relatively stable variations are found frequently in different bacteria. The utility of this locus as a taxonomic and phylogenetic tool has been reported widely. This study, aimed at 16S rRNA gene (16S rDNA) and with the help of biomolecular methods, attempted to achieve the goal of rapid identification of common pathogens. In this study, 333 clinical isolated pathogenic bacteria were collected. Two pairs of primers were chosen and labeled with different fluorescent dyes and then used to amplify the genomic DNA extracted from bacteria. The PCR products were then detected by capillary electrophoresis-single strand conformation polymorphism (CE-SSCP). In order to pursue higher resolution and peak-separation effect, a high efficient separating medium, liner polyacrylamidedel (LPA), was put to use in this study. Finally, every bacteria colony generated distinct patterns from each other, which were easily to be used for identification. These results indicated that PCR-CE-SSCP was a rapid identification method for bacterial identification, with the aspects of high efficiency and high precision. Compared with traditional method, this technology is of great utility for clinical use especially for its high sensitivity.
基金supported by the National Foundation of High Technology of China(2006AA020701 and 2006AA020803)National Program on Key Basic Research Projects 973 of China(2006CB705700)+1 种基金the Nature Science Foundation of Zhejiang Province(2006C21G3210005)Tsinghua-Yuyuan Medicine Foundation(40000510B).
文摘Loop-mediated isothermal amplification(LAMP)is a novel nucleic acid amplification method.Compared with the widely utilized polymerase chain reaction(PCR),LAMP has higher speed and efficiency as well as lower requirement for system temperature control because the whole amplification process is isothermal and no efforts are needed to switch between different temperatures.In this paper,we designed and fabricated different kinds of polycarbonate(PC)microfluid chips,explored appropriate reaction condition for LAMP in microenvironment(1 nL→10μL),and developed a microfluidic isothermal amplification detection system.The DNA optimal amplification temperature is obtained;the starting time of exponential amplification of DNA is put forward farther.The optimal condition of DNA amplification in microenvironment,with a little reaction materials and early starting exponential amplification time of DNA are very important for clinic DNA detection and the application of Lab-on-a-Chip.
文摘在分离Y染色体性别决定区(Sex determining region of the Y chromosome,SRY)、人11号染色体短臂β-珠蛋白基因部分序列(108/B)扩增产物和PBR322/HaeⅢ标准片段的混合样品时,SRY和108/B的迁移时间均产生较大的偏差,在尿素变性剂存在的无胶筛分体系中迁移时,这种迁移时间的偏差得以消除,这意味着尿素变性剂的存在消除了DNA空间结构对迁移的影响,DNA在此筛分介质中的迁移只与其片段长度有关.在变性无胶筛分体系中鉴别PCR产物,其结果的准确度明显提高.
