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Optimization of Pulsed-field Gel Electrophoresis Procedure for Bacillus cereus
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作者 ZHANG Hui Juan PAN Zhuo +4 位作者 WEI Jian Chun ZHANG En Min CAI Hong LIANG Xu Dong LI Wei 《Biomedical and Environmental Sciences》 SCIE CAS CSCD 2016年第3期233-237,共5页
In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of... In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth (Luria-Bertani agar plates for 12-18 h) and lysis conditions (4 h incubation with 500 μg/mL lysozyme) produced sharp bands on the gel. 展开更多
关键词 PFGE Optimization of pulsed-field gel electrophoresis Procedure for Bacillus cereus
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Establishment and Comparison of Pulsed-field Gel Electrophoresis,Multiple-locus Variable Number Tandem Repeat Analysis and Automated Ribotyping Methods for Subtyping of Citrobacter Strains 被引量:1
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作者 ZHANG Xiao Ai BAI Xue Mei +2 位作者 YE Chang Yun REN Zhi Hong XU Jian Guo 《Biomedical and Environmental Sciences》 SCIE CAS CSCD 2012年第6期653-662,共10页
Objective To establish and compare the pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA) and automated ribotyping for subtyping of Citrobacter strains. Methods P... Objective To establish and compare the pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA) and automated ribotyping for subtyping of Citrobacter strains. Methods PFGE protocol was optimized in terms of plug preparation procedure, restriction enzymes and configuration of electrophoretic parameters. MLVA method was evaluated by finding variable number tandem repeats in two genomes of Citrobacter strains. The ribotyping was performed by using the automated RiboPrinter system. Results We optimized the plug preparation procedure, focused on the cell suspension concentration (turbidity of 2.5 to 3.5), SDS addition (no SDS needed) and lysis time (1 h), and selected the appropriate restriction enzyme (Xbal) and the electrophoretic parameters (1.0 s-20.0 s for 19 h) of PFGE. There was nearly no discriminatory power of MLVA between Citrobacter strains. For 51 Citrobacter strains, automated ribotyping gave a D-value of 0.9945, while PFGE gave a D-value of 0.9969. Both PFGE and automated ribotyping clustered strains from the same sources (with the same species from the same place at the same time identified as the same source) and divided strains from different sources (from different years, places and hosts) into different subtypes. Conclusion PFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping. 展开更多
关键词 CITROBACTER pulsed-field gel electrophoresis Multiple-locus variable number tandem repeatanalysis RIBOTYPING Molecular subtyping
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Prevalence and Study of the Clonality of Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Strains in Neonatology at the University Hospitals of Abidjan by the Pulsed Field Gel Electrophoresis and the Quantitative Antibiogram
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作者 Valérie M’Bengue Gbonon Sidjè Arlette Afran +6 位作者 Stanislas Assohoun Egomli Djeda Franck Arnaud Gnahoré Aboubakar Sylla N’Golo David Coulibaly Nathalie Guessennd Assanvo Simon-Pierre N’Guetta Mireille Dosso 《Advances in Microbiology》 CAS 2024年第9期416-429,共14页
Background: ESBL-producing strains of Klebsiella pneumoniae, one of the main causes of nosocomial and hospital-acquired infections, are commonly associated with therapeutic impasses. Surveillance of these multidrug-re... Background: ESBL-producing strains of Klebsiella pneumoniae, one of the main causes of nosocomial and hospital-acquired infections, are commonly associated with therapeutic impasses. Surveillance of these multidrug-resistant pathogens is a crucial tool for controlling and preventing infections. This surveillance involves the use of appropriate molecular and phenotypic typing techniques. The choice of techniques is based on criteria such as discriminatory power, intra- and inter-laboratory reproducibility, epidemiological concordance, ease of use and cost. The aim of our study was to identify clusters of Extended-Spectrum Beta-Lactamase-producing Klebsiella pneumoniae (ESBL-K. pneumoniae) strains circulating in neonatology using quantitative antibiogram (QA) and Pulsed Field Gel Electrophoresis (PFGE). Materials and Methods: This cross-sectional study included 55 K. pneumoniae strains isolated from a total of 513 samples. These various samples are taken from newborns, healthcare personnel, and the environment. K. pneumoniae identification followed standard bacteriological procedures and was confirmed using the Vitek® 2 (bioMérieux). The detection of the ESBL phenotype was performed using the synergy test. QA and PFGE were used to identify clonal relationships between the various strains isolated. Concordance between these two methods was assessed by calculating Cohen’s KAPPA coefficient and Simpson’s diversity index. Results: Among the 55 K. pneumoniae strains included in this study, 58.2% (32/55) were found to be Extended-Spectrum Beta-Lactamase (ESBL) producers. Most of these strains were isolated from neonatal samples (blood samples and rectal swabs). The quantitative antibiogram method applied to 28 out of the 32 ESBL-producing strains revealed that the isolates were grouped into 5 clusters. Pulsed Field Gel Electrophoresis performed on a total of 16 ESBL-producing strains showed the existence of four profiles. A perfect concordance was observed between the two methods. Conclusion: The results of this study highlighted the existence of clonal strains of various origins within neonatology units. 展开更多
关键词 Resistance-Clone-Klebsiella pneumoniae-Pulsed Field gel electrophoresis-Quantitative Antibiogram
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The Superiority of Like-rocket Immunoelectrophoresis Using in Single Cell Gel Electrophoresis Assay 被引量:1
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作者 穆淑梅 康现江 郭明申 《Agricultural Science & Technology》 CAS 2010年第7期17-19,共3页
[Objective] The like-rocket immunoelectrophoresis was used to explore a new feasible electrophoresis method for single cell gel electrophoresis assay (comet assay).[Method] The like-rocket immunoelectrophoresis was ... [Objective] The like-rocket immunoelectrophoresis was used to explore a new feasible electrophoresis method for single cell gel electrophoresis assay (comet assay).[Method] The like-rocket immunoelectrophoresis was used for single cell gel electrophoresis assay to detect DNA damage at single cell level,then it was compared with traditional electrophoresis method to analyze its advantage and disadvantages.[Result] Under cell DNA undamaged state,the results of two electrophoresis methods were consistent.When cell DNA was damaged,the comet tail divergence of some cells under traditional electrophoresis method were drifted,however,the comet tail image of like-rocket immunoelectrophoresis was concentrated and not shifted.[Conclusion] The like-rocket immunoelectrophoresis had some advantages. 展开更多
关键词 Single cell gel electrophoresis DNA damage Like-rocket immunoelectrophoresis
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Single Cell Gel Electrophoresis Assay of Porcine Leydig Cell DNA Damage Induced by Zearalenone 被引量:1
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作者 甄建伟 刘青 +5 位作者 顾建红 袁燕 刘学忠 王捍东 刘宗平 卞建春 《Agricultural Science & Technology》 CAS 2012年第7期1587-1590,1594,共5页
[Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD... [Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD50) of ZEN with tetrazolium-based colorimetric assay (MTT assay). Comet assay was carried out to detect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 μmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%, 52.00% and 64.67% under 0, 1, 5, 10 and 20 μmol/L of ZEN, respectively; the differences between the percentages of cell tail in various experimental groups had extremely significant statistical significance compared with the negative group (P<0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60±4.78, 57.75±6.25, 78.97±5.83, 100.50±6.94 and 146.83±12.31 μm, respectively; Tail DNA % in various groups was 21.29±2.25%, 22.24±2.43%, 31.21±6.27%, 37.45±4.33% and 60.68±9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 μmol/L showed significant differences (P<0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship. 展开更多
关键词 Leydig cells ZEARALENONE DNA damage Comet assay (Single cell gel electrophoresis assay)
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Optimization of SSR-PCR Non-denatured Polyacrylamide Gel Electrophoresis Conditions in Kernelled Apricot 被引量:1
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作者 艾鹏飞 方闪闪 +1 位作者 吴学敏 靳占忠 《Agricultural Science & Technology》 CAS 2010年第9期50-52,139,共4页
[Objective] The aim was to optimize the SSR-PCR non-denatured polyacrylamide gel electrophoresis conditions in kernelled apricot.[Method]25 accessions of kernelled apricot and three accessions of edible apricot were s... [Objective] The aim was to optimize the SSR-PCR non-denatured polyacrylamide gel electrophoresis conditions in kernelled apricot.[Method]25 accessions of kernelled apricot and three accessions of edible apricot were selected as experimental materials to screen the repeatable SSR loci with high polymorphism by the use of SSR markers combined with non-denatured polyacrylamide gel electrophoresis.And the effect of different factors on electrophoresis conditions was compared to explore the optimal SSR-PCR non-denatured polyacrylamide gel electrophoresis conditions in kernelled apricot.[Result]The optimal non-denatured polyacrylamide gel electrophoresis conditions for SSR-PCR were established as follows:polyacrylamide gel concentration 6%,the ratio of acrylamide to bisacrylamide 29∶1,electrophoresis at 1 000 V for 2-3 h,and staining for 15 min within 0.1% AgNO3.[Conclusion]The optimum electrophoresis system has provided some technical foundations to further study the phylogenetic relationship of kernelled apricots by SSR markers. 展开更多
关键词 Kernelled Apricot SSR markers Polyacrylamide gel electrophoresis Silver staining
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Optimization of Multiplex PCR and Multiplex Gel Electrophoresis in Sunflower SSR Analysis Using Infrared Fluorescence and Tailed Primers 被引量:3
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作者 张潞生 Vanessa BECQUET +1 位作者 李绍华 David ZHANG 《Acta Botanica Sinica》 CSCD 2003年第11期1312-1318,共7页
In an effort to simplify the procedure and to reduce the cost of fluorescence SSR analysis, the conditions of the multiplex PCR and the multiplex gel electrophoresis were optimized in the genetic analysis of sunflower... In an effort to simplify the procedure and to reduce the cost of fluorescence SSR analysis, the conditions of the multiplex PCR and the multiplex gel electrophoresis were optimized in the genetic analysis of sunflower (Helianthus annuus L.) inbred lines. Results indicated that factors for a successful multiplex PCR assay were related to the cycling touchdown annealing temperature, the balance of primer concentration at the various loci, the concentration of PCR buffer and the Taq DNA polymerase. Based on the optimization, a tailed primer strategy was outlined, and the effective ways were proposed to overcome the troubleshootings commonly encountered in the multiplex PCR and the multiplex gel electrophoresis. 展开更多
关键词 simple sequence repeat (SSR) tailed primer multiplex PCR multiplex gel electrophoresis SUNFLOWER
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Optimization of Two-Dimensional Gel Electrophoresis for Kenaf Leaf Proteins 被引量:8
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作者 CHEN Tao QI Jian-min XU Jian-tang CHEN Pin-pin TAO Ai-fen CHEN Fu-cheng CHEN Wei 《Agricultural Sciences in China》 CAS CSCD 2011年第12期1842-1850,共9页
To establish a suitable and effective protocol of protein extraction for two-dimensional gel electrophoresis (2-DE) analysis in kenaf leaf tissues, three extraction methods (trichloroacetic acid/acetone, urea/thiou... To establish a suitable and effective protocol of protein extraction for two-dimensional gel electrophoresis (2-DE) analysis in kenaf leaf tissues, three extraction methods (trichloroacetic acid/acetone, urea/thiourea, and phenol extraction methods) were applied to the extraction of kenaf leaf protein. The results were compared in regard to protein extraction efficiency, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and 2-DE gels. Furthermore, the 2-DE system was optimized for four aspects: the pH range of IPG (immobilized pH gradient) stripes, sampling methods, sample volumes, and concentration of polyacrylamide gels. The data presented showed that the phenol extraction method is the best method to perform 2-DE analysis of kenaf leaf protein. The protein extracted from phenol extraction method reached the purity of (26.40±0.859)%, showed (25.67±1.53) protein bands in one dimension SDS-PAGE gels, and (1 374±54.44) protein spots on 2-DE gels. The research also indicates that kenaf leaf protein spots were distributed mainly within the pH range of 4-8. More clear background with a better distribution effect and many protein spots could be obtained on 2-DE gels under the conditions of active rehydration loading, 24 cm IPG strips (linear pH gradient of 4-7), 1.4 mg samples, and 12% SDS-PAGE gels. 展开更多
关键词 KENAF protein extraction PROTEOME two-dimensional gel electrophoresis
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Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electrophoresis and mass spectrometry 被引量:9
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作者 Ting-Ting Liao Zhen Xiang Wen-Bing Zhu Li-Qing Fan 《Asian Journal of Andrology》 SCIE CAS CSCD 2009年第6期683-694,共12页
Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence o... Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa on semen analysis, and patients with this condition are absolutely infertile. The objective of this study was to investigate the differences in protein expression between human round- headed and normal spermatozoa. Two-dimensional (2-D) fluorescence difference gel electrophoresis (DIGE) coupled with mass spectrometry (MS) was used in this study. Over 61 protein spots were analysed in each paired normal/round-headed comparison, using DIGE technology along with an internal standard. In total, 35 protein spots identified by tandem mass spectrometry (MS/MS) exhibited significant changes (paired t-test, P 〈 0.05) in the expression level between normal and round-headed spermatozoa. A total of nine proteins were found to be upregulated and 26 proteins were found to be downregulated in round-headed spermatozoa compared with normal spermatozoa. The differentially expressed proteins that we identified may have important roles in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism and spermatozoa motility. 展开更多
关键词 differential protein GLOBOZOOSPERMIA mass spectrometry (MS) two-dimensional difference gel electrophoresis (2-D DIGE)
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Characterization of depth-related microbial communities in lake sediment by denaturing gradient gel electrophoresis of amplified 16S rRNA fragments 被引量:5
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作者 ZHAO Xingqing YANG Liuyan +4 位作者 YU Zhenyang PENG Naiying XIAO Lin YIN Daqiang QIN Boqiang 《Journal of Environmental Sciences》 SCIE EI CAS CSCD 2008年第2期224-230,共7页
The characterization of microbial communities of different depth sediment samples was examined by a culture-independent method and compared with physicochemical parameters, those are organic matter (OM), total nitro... The characterization of microbial communities of different depth sediment samples was examined by a culture-independent method and compared with physicochemical parameters, those are organic matter (OM), total nitrogen (TN), total phosphorus (TP), pH and redox potential (Eh). Total genomic DNA was extracted from samples derived from different depths. After they were amplified with the GC-341 f/907r primer sets of partial bacterial 16S rRNA genes, the products were separated by denaturing gradient gel electrophoresis (DGGE). The profile of DGGE fingerprints of different depth sediment samples revealed that the community structure remained relatively stable along the entire 45 cm sediment core, however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. The principle coordinates analysis suggested that the bacterial communities along the sediment core could be separated into two groups, which were located 0-20 cm and 21-45 cm, respectively. The sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belonged to Bacillus, Bacterium, Brevibacillus, Exiguobacterium, γ-Proteobacterium, Acinetobacter sp. and some uncultured or unidentified bacteria. The results indicated the existence of highly diverse bacterial community in the lake sediment core. 展开更多
关键词 16S rRNA denaturing gradient gel electrophoresis (DGGE) Lake Taihu microbial diversity SEDIMENT vertical distribution
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Studies on Human Immunoglobulin G from GBS Patient (Ⅲ)-The Determination of Molecular Weight of Human Immunoglobulin G by Capillary SDS Gel Electrophoresis 被引量:5
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作者 Qin Hua RU Yi Ming WANG Guo An LUO(Department of Chemistry. School of Life Science and Engineering,Tsinghua University. Beijing, 100084) 《Chinese Chemical Letters》 SCIE CAS CSCD 1999年第1期55-58,共4页
Guillain-Barre syndrome (GBS) is considered to be an autoimmune disorder of peripheralnervous system. In this paper. capillary SDS gel electrophoresis was performed on neutral coatedfused-silica capillary to determine... Guillain-Barre syndrome (GBS) is considered to be an autoimmune disorder of peripheralnervous system. In this paper. capillary SDS gel electrophoresis was performed on neutral coatedfused-silica capillary to determine the molecular weight of purified IgG samples from GBS patient. 展开更多
关键词 Immunoglobulin G Guillain-Barre syndrome Capillary SDS gel electrophoresis Molecular weight of protein
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Comparative Proteomic Analysis of B. henselae Houston and B. henselae Marseille by Two-dimensional Gel Electrophoresis 被引量:3
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作者 SU-QING ZHAO YAN-FEI CAI ZHEN-YU ZHU 《Biomedical and Environmental Sciences》 SCIE CAS CSCD 2005年第5期341-344,共4页
To compare the protein difference between B. henselae Houston and B. henselae Marseille by two-dimensional gel electrophoresis. Method Protein samples were prepared by vorterx, ultrasonic treatment, and centrifugation... To compare the protein difference between B. henselae Houston and B. henselae Marseille by two-dimensional gel electrophoresis. Method Protein samples were prepared by vorterx, ultrasonic treatment, and centrifugation. Protein concentrations were determined by Bradford method. Protein difference was compared by the first IEF and the second SDS-polyacrylamide gel electrophoresis. Results Protein concentrations in samples of Bartonella henselae Houston and Bartonella henselae Marseille were 2.117 μg/μL and 2.200 μg/μL respectively. Sample protein of 40 μg for IPG strips loading was perfect. The results of 2-DE in pH 4 to 7 IPG strips showed that the total protein spots of Bartonella henselae Houston and Bartonella henselae Marseille were 375 and 379 respectively, 95% of the spots were the same between the two strains of Bartonella henselae. Conclusion The procedure of 2-DE may prove successful for the proteomic analysis of Bartonella henselae. Bartonella henselae Houston and Bartonella henselae Marseille are different genotypes. 展开更多
关键词 PROTEIN B.henselae Two-dimensional gel electrophoresis
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Analysis of Sperm Membrane Protein Relevant to Antisperm Antibody by Two-Dimensional Gel Electrophoresis and Western Blotting 被引量:3
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作者 Hao-fei WANG 1, Zhu-qiong XIANG 2, Yi-xing WANG 2 1. Department of Urology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025,China 2. Department of Urology, Renji Hospital, Shanghai Second Medical University, Shanghai 200025,China 《Journal of Reproduction and Contraception》 CAS 2003年第3期147-156,共10页
Objective To identify the sperm membrane proteins that are associated with antisperm antibody Methods Using antisperm antibody positive serum through unidimensional polyacrylamide gel electrophoresis and 2-dimensi... Objective To identify the sperm membrane proteins that are associated with antisperm antibody Methods Using antisperm antibody positive serum through unidimensional polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis followed by Western blot analysis to determine the molecular weights (MW) and isoelectric points (pI) of sperm membrane proteins that are associated with antisperm antibody. Results Eight kinds of MW with more than ten sperm membrane proteins can be recognized by antisperm antibody positive serum, of which the MWs and pI were 23 kD, 31 kD, 32 kD, 34 kD, 41 kD, 51 kD, 60 kD, 78 kD and 5.3, 5.5,5.7, 5.0, 5.3, 5.8, 6.0, 5.5~6.2, 4.6,5.1,5.5~5.8 respectively. The identification ratios of the sperm membrane proteins on 78 kD (60.7%), 60 kD (71.4%), 51 kD (14.9%) and 23 kD (14.29%) were higher. Conclusion The sperm membrane proteins with MW of 78 kD, 60 kD, 51 kD and 23 kD were associated with antisperm antibody and immunological infertility. Two- dimensional gel electrophoresis and Western blotting can precisely identify the sperm membrane proteins that are associated with antisperm antibody. 展开更多
关键词 immunological infertility antisperm antibody sperm membrane protein 2-dimensional gel electrophoresis
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In Vitro Protein Expression Profile of Campylobacter jejuni Strain NCTC11168 by Two-dimensional Gel Electrophoresis and Mass Spectrometry 被引量:2
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作者 ZHANG Mao Jun GU Yi Xin +4 位作者 DI Xiao ZHAO Fei YOU Yuan Hai MENG Fan Liang ZHANG Jian Zhong 《Biomedical and Environmental Sciences》 SCIE CAS CSCD 2013年第1期48-53,共6页
Objective To investigate the protein expression profiles of the major food‐borne pathogen Campylobacter jejuni NCTC11168.Methods Membrane and soluble cellular proteins were extracted from the genome‐sequenced C.jeju... Objective To investigate the protein expression profiles of the major food‐borne pathogen Campylobacter jejuni NCTC11168.Methods Membrane and soluble cellular proteins were extracted from the genome‐sequenced C.jejuni strain NCTC11168.Protein expression profiles were determined using two‐dimensional gel electrophoresis(2‐DE).All the detected spots on the 2‐DE map were subjected to matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry(MALDI‐TOF/TOF) analysis.Results A total of 537 and 333 spots were detected from the whole cell and membrane‐associated proteins of C.jejuni NCTC11168 cultured on Columbia agar medium at 42 ℃ by 2‐DE and Coomassie Brilliant Blue staining,respectively.Analyses of whole cell and membrane‐associated proteins included 399 and 133 spots,respectively,which included 182 and 53 functional proteins identified by MALDI‐TOF/TOF analysis.Conclusion The comprehensive expression protein profiles of C.jeuni NCTC11168 obtained in this study will be useful for elucidating the roles of these proteins in further pathogenesis investigation. 展开更多
关键词 Campylobacter jejuni Two‐dimensional gel electrophoresis MALDI‐TOF Soluble cellular protein Membrane protein
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Optimization of Pulse-Field Gel Electrophoresis for Borrelia burgdorferi Subtyping 被引量:2
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作者 GENG Zhen HOU Xue Xia +3 位作者 HAO Qin ZHOU Hai Jian WANG Feng WAN Kang Lin 《Biomedical and Environmental Sciences》 SCIE CAS CSCD 2013年第7期584-591,共8页
Objective To optimize the performance of Pulsed-Field Gel Electrophoresis (PFGE) for the comparison of inter-laboratory results and information exchange of Borrelia burgdorferi subtypingo Methods A panel of 34 strai... Objective To optimize the performance of Pulsed-Field Gel Electrophoresis (PFGE) for the comparison of inter-laboratory results and information exchange of Borrelia burgdorferi subtypingo Methods A panel of 34 strains of B. burgdorferi were used to optimize PFGE for subtyping. In order to optimize the electrophoretic parameters (EPs), all 34 strains of B. burgdorferi were analyzed using four EPs, yielding different Simpson diversity index (D) values and the epidemiological concordance was also evaluated. Results The EP of a switch time of l s to 25 s for13 h and l s to10 s for 6 h produced the highest D value and was declared to be optimal for Mlul and 5mal PFGE of B. burgdorferi. Mlul and Smal were selected as the first and second restriction enzymes for PFGE subtyping of B. burgdorferi according to discrimination and consistency with epidemiological data. Conclusion PFGE can be used as a valuable test for routine genospecies identification of B burgdorferi. 展开更多
关键词 Molecular subtyping Pulse-Field gel electrophoresis Borrelia burgdor^eri
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Two-dimensional gel electrophoresis and surface-enhanced laser desorption ionization-time of flight-mass spectrometry for detection of protein expression profiles in the hippocampus following closed brain injury 被引量:2
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作者 Qingming Shu Zhiqiang Li +3 位作者 Shuwang Yang Lingzhi Li Xiao Bai Yongliang Zhang 《Neural Regeneration Research》 SCIE CAS CSCD 2010年第23期1795-1801,共7页
Gene expression profile changes in brain regions following traumatic brain injury at the gene level cannot sufficiently elucidate gene expression time, expression amount, protein post-translational processing or modif... Gene expression profile changes in brain regions following traumatic brain injury at the gene level cannot sufficiently elucidate gene expression time, expression amount, protein post-translational processing or modification. Therefore, it is necessary to quantitatively analyze the gene expression profile using proteomic techniques. In the present study, we established a rat model of closed brain injury using Marmarou's weight-drop device, and investigated hippocampal differential protein expression using two-dimensional gel electrophoresis and surface-enhanced laser desorption ionization-time of flight-mass spectrometry. A total of 364 protein peaks were detected on weak cation exchange-2 protein chips, including 37 differential protein peaks. 345 protein peaks were detected on immobilized metal affinity capture arrays-Cu, including 12 differential protein peaks Further examination of these differential proteins revealed that glucose-regulated protein and proteasome subunit alpha type 3 expression were significantly upregulated post-injury. These results indicate that brain injury can alter protein expression in the hippocampus, and that glucose-regulated protein and proteasome subunit alpha type 3 are closely associated with the occurrence and development of traumatic brain injury. 展开更多
关键词 surface-enhanced laser desorption ionization-time of flight-mass spectrometry two-dimensional gel electrophoresis HIPPOCAMPUS PROTEOMICS brain injury neural regeneration
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Analysis of interspecies adherence of oral bacteria using a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis profiling 被引量:1
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作者 Ren-ke Wang Xue-song He +4 位作者 Wei Hu Renate Lux Ji-yao Li Xue-dong Zhou Wen-yuan Shi 《International Journal of Oral Science》 SCIE CAS CSCD 2011年第2期90-97,共8页
Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise co... Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise coaggregation of known, cultivable species. In this study, we employed a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to systematically analyze the co-adherence profiles of oral bacterial species, and achieved a more profound knowledge beyond pairwise coaggregation. Two oral bacterial species were selected to serve as "bait": Fusobacterium nucleatum (F. nucleatum) whose ability to adhere to a multitude of oral bacterial species has been extensively studied for pairwise interactions and Streptococcus mutans (S. mutans) whose interacting partners are largely unknown. To enable screening of interacting partner species within bacterial mixtures, cells of the "bait" oral bacterium were immobilized on nitrocellulose membranes which were washed and blocked to prevent unspecific binding. The "prey" bacterial mixtures (including known species or natural saliva samples) were added, unbound ceils were washed off after the incubation period and the remaining cells were eluted using 0.2 mol.L1 glycine. Genomic DNA was extraeted, subjeeted to 16S rRNA PCR amplification and separation of the resulting PCR produets by DGGE. Selected bands were recovered from the gel, sequenced and identified via Nucleotide BLAST searches against different databases. While few bacterial species bound to S. mutans, consistent with previous findings F.. nucleatum adhered to a variety of bacterial species including uncultivable and uneharacterized onesl This new approach can more effectively analyze the co-adherence profiles of oral bacteria, and could facilitate the systematic study of interbacterial binding of oral microbial species. 展开更多
关键词 membrane binding assay polymerase chain reaction-denaturing gradient gel electrophoresis COAGGREGATION Fusobacterium nucleatum Streptococcus mutans
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Detection of Sperm DNA Damage in Workers Exposed to Benzene by Modified Single Cell Gel Electrophoresis 被引量:1
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作者 Bo SONG Zhi-ming CAI +3 位作者 Xin LI Li-xia DENG Qiao ZHANG Lu-kang ZHENG 《Journal of Reproduction and Contraception》 CAS 2005年第3期131-136,共6页
Objective To assess the effect of benzene on sperm DNA damage ;Methods Twenty-seven benzene-exposed workers were selected as exposed group and 35 normal sperm donors as control group. Air concentration of benzene seri... Objective To assess the effect of benzene on sperm DNA damage ;Methods Twenty-seven benzene-exposed workers were selected as exposed group and 35 normal sperm donors as control group. Air concentration of benzene series in workshop was determined by gas chromatography. As an internal exposure dose of benzene, the concentration of trans, trans-muconic acid (ttMA) was determined by high performance liquid chromatography. DNA was detected by modified single cell gel electrophoresis (SCGE). Results The air concentrations of benzene, toluene and xylene at the workplace were 86.49±2.83 mg/m^3, 97.20±3.52 mg/m^3 and 97.45± 2.10 mg/m^3, respectively. Urinary ttMA in exposed group (1.040 ± 0.617 mg/L) was significantly higher than that of control group (0.819 ± 0.157 mg/L). The percentage of head DNA, determined by modified SCGE method, significantly decreased in the exposed group (n=13, 70.18% ± 7.36%) compared with the control (n=16, 90.62% ± 2.94%)(P〈0.001). Conclusion The modified SCGE method can be used to investigate the damage of sperm DNA. As genotoxin and reprotoxins, benzene had direct effect on the germ cells during the spermatogenesiss. 展开更多
关键词 single cell gel electrophoresis (SCGE) SPERM DNA damage
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Establishment of two-dimensional gel electrophoresis for soybean protein isolate and its application 被引量:3
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作者 Xinkai Lu Yaoming Cui +6 位作者 Junjun Guan Xue Liu Hao Zhu Xuyang Ji Jianzhang Zheng Yunlong Cheng Xiaofei Fu 《Grain & Oil Science and Technology》 2020年第3期100-109,共10页
To optimize the conditions for the establishment of two-dimensional gel electrophoresis(2-DE)of soybean protein isolate(SPI),we investigated Ampholine mixture,anodic and cathodic electrolytes,loading amount of sample,... To optimize the conditions for the establishment of two-dimensional gel electrophoresis(2-DE)of soybean protein isolate(SPI),we investigated Ampholine mixture,anodic and cathodic electrolytes,loading amount of sample,acrylamide concentration,p H gradient and gel staining method in twodimensional gel electrophoresis to optimize the protein imaging conditions in two-dimensional gel.The results of mixed-level design experiments showed that Ampholine,loading amount and gel staining method had significant effect(P<0.05)on 2-DE of SPI.The optimal conditions were Ampholine mixture(pH 3–10+pH 5–7 or pH 4–6+pH 5–7),loading amount of 2 mg sample and silver staining.Although the acrylamide concentration of the gel,the p H gradient,the anodic and cathodic electrolyte solutions had significant statistical effects on the protein separation degree,the complexity of the protein composition and the visibility of the gel images were more inclined to the 12%gel,the 3–10 pH gradient and the H3PO4/NaOH electrolyte.According to the established conditions,the hydrolyzed products of SPI emulsion were determined by 2-DE,and the dynamic changes of protein in the process of enzymatic hydrolysis were described. 展开更多
关键词 Two-dimensional gel electrophoresis Soybean protein isolate Enzymatic hydrolysate Soybean protein isolate emulsion
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Specific protein expression in a rat model of early focal cerebral ischemia:Fluorescent two-dimensional difference gel electrophoresis
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作者 Xueling Ma Wei Yang +4 位作者 Xinmei Jiang Fuchun Li Xia Li Linlin Ye Kangding Liu 《Neural Regeneration Research》 SCIE CAS CSCD 2010年第3期209-213,共5页
BACKGROUND: The use of fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) has been shown to compensate for the shortcomings of conventional two-dimensional gel electrophoresis, such as poor repeat... BACKGROUND: The use of fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) has been shown to compensate for the shortcomings of conventional two-dimensional gel electrophoresis, such as poor repeatability and large systematic errors. However, little information is presently available regarding the use of 2D-DIGE to investigate mechanisms of ischemic cerebrovascular diseases. Plasma and body fluids have been utilized in proteomic technology to study ischemic cerebrovascular diseases. OBJECTIVE: To perform proteomic analysis of fresh rat brain tissue in peripheral ischemic regions using 2D-DIGE 6 hours after middle cerebral artery occlusion (MCAO), and to identify specific proteins closely associated with early ischemic cerebrovascular diseases. DESIGN, TIME AND SETTING: Proteomics-based, randomized, controlled, animal experiment was performed at the Laboratories of Neurology and Proteomics, Jilin University between January and April 2006. MATERIALS: 2, 3, 5-triphenyl tetrazolium chloride was purchased from Sigma, USA. Ettan DALTSix system, DeCyder DIA V5.0 differential analysis software, and Ettan matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) were purchased from Amersham Bioscience, Sweden. METHODS: Eight healthy, male, Wistar rats were randomized to experimental and control groups, with four rats in each group. In the experimental group, rat models of focal cerebral ischemia were established by MCAO. In the control group, the internal and external carotid arteries were exposed and then immediately sutured, and the remaining procedures were identical to the experimental group. MAIN OUTCOME MEASURES: At 6 hours after cerebral ischemia, protein expression in the peripheral ischemia region of the experimental group was compared with the control group using 2D-DIGE. Protein spots that exhibited statistical differences between experimental and control groups with 〉 1.4 attributable risk were screened using DeCyder DIA V5.0 differential analysis software. Differential proteins were identified using MALDI-TOF-MS. RESULTS: Triphenyl tetrazolium chloride staining results revealed pink, normal brain tissue and white, ischemic brain tissue, suggesting successful MCAO establishment. The average matching rate of four 2D-DIGE gels was 92.4%. There were (1 758 ± 43) protein spots on each gel, with similar distribution modes. At 6 hours after focal cerebral ischemia, 13 protein spots exhibited marked expression changes, including significantly increased (n = 7) and decreased (n = 6) expression (P 〈 0.05). MALDI-TOF-MS results revealed two differential protein spots: a-tubulin and heat shock protein 27, which were significantly decreased in the experimental group compared with the control group (P 〈 0.05). CONCLUSION: Thirteen protein spots with expression changes were revealed by 2D-DIGE proteomics technology. Of them, a-tubulin and heat shock protein 27 expressions were markedly decreased during the early stage of cerebral ischemia. These two proteins were presumed to be proteins associated with early ischemic cerebrovascular diseases. 展开更多
关键词 fluorescent two-dimensional difference gel electrophoresis cerebral ischemia mass spectrometry rats PROTEOMICS neural regeneration
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