[Objective] This study aimed to establish a real-time PCR method for de- tecting stx2 gene in Shiga toxin-producing E. coli （STEC）. [Method] According to the known STEC stx2 gene sequences published in GenBank, PCR ...[Objective] This study aimed to establish a real-time PCR method for de- tecting stx2 gene in Shiga toxin-producing E. coli （STEC）. [Method] According to the known STEC stx2 gene sequences published in GenBank, PCR primers and probes were designed based on the conserved region to construct recombinant plasmid as a positive template, thus optimizing the reaction conditions and establishing the real- time PCR method. [Result] A standard curve was established based on the opti- mized real-time PCR system, indicting a good linear correlation between the initial template concentration and Ct value, with the correlation coefficient F^e of above 0.995. The established method had a good specificity, without non-specific amplifica- tion for 10 non-STEC intestinal bacterial strains; the detection limit of initial template was 1.0x102 copies/μI, indicating a high sensitivity; furthermore, the coefficients of variation within and among batches were lower than 1% and 5% respectively, sug- gesting a good repeatability. [Conclusion] In this study, a real-time PCR method was successfully established for detecting STEC stx2 gene, which provided technical means for rapid detection of STEC in samples.展开更多
AIM： To investigate the selective cytotoxic effect of constructed hybrid protein on cells expressing granulocyte macrophage colony stimulating factor （GM-CSF） receptor. METHODS： HepG2 （human hepatoma） and LS174T...AIM： To investigate the selective cytotoxic effect of constructed hybrid protein on cells expressing granulocyte macrophage colony stimulating factor （GM-CSF） receptor. METHODS： HepG2 （human hepatoma） and LS174T （colon carcinoma） were used in this study. The fused gene was induced with 0.02% of arabinose for 4 h and the expressed protein was detected by Western blotting. The chimeric protein expressed in E..coli was checked for its cytotoxic activity on these cells and apoptosis was measured by comet assay and nudear staining. RESULTS： The chimeric protein was found to be cytotoxic to the colon cancer cell line expressing GM-CSFRs, but not to HepG2 lacking these receptors. Maximum activity was observed at the concentration of 40 ng/mL after 24 h incubation. The IC50 was 20 ± 3.5 ng/mL. CONCLUSION： Selective cytotoxic effect of the hybrid protein on the colon cancer cell line expressing GMCSF receptors （GM-CSFRs） receptor and apoptosis can be observed in this cell line. The hybrid protein can be considered as a therapeutic agent.展开更多
Non-O157 STEC has been shown to have a diverse ecological distribution among food-animals. It has been associated with both outbreaks and individual cases of severe illness. This group of the organisms is now consider...Non-O157 STEC has been shown to have a diverse ecological distribution among food-animals. It has been associated with both outbreaks and individual cases of severe illness. This group of the organisms is now considered as a major contributor to human disease. The clinical description of the diseases caused by these organisms is reviewed. The host specificity of these pathogens is described and discussed. These organisms appear widespread among food animals like cattle and sheep, and can therefore affect a range of foods directly from the meat and excretions of these animals being used in farming practices. This article reviews the origins, diversity and pathogenesis of non-O157 STEC.展开更多
Isolation and biochemical and molecular identification of 303 strains of Escherichia coli obtained from diarrheic and healthy young alpacas of Puno-Peru, were realized. PCR amplification for 7 virulence factor genes a...Isolation and biochemical and molecular identification of 303 strains of Escherichia coli obtained from diarrheic and healthy young alpacas of Puno-Peru, were realized. PCR amplification for 7 virulence factor genes associated with STEC, STEC O157:H7, EPEC: sxt1, sxt2, rfbO157, fliCH7, hlyA, eae y bfp were determined. A total of 39 strains (12.88%) showed amplification for one or more of these genes. Twenty three strains (59%) were classified as STEC and 16 strains (41%) as EPEC. An 88.18% (34/39) of STEC and EPEC strains were obtained from healthy alpacas and only 11.82% (5/39) from diarrheic alpacas considering this specie as potential zoonotic reservoir of STEC and EPEC.展开更多
Objective To evaluate the etiology of Shiga-like toxin-producing Escherichia coli (SLTEC) in children with diarrhea. Methods We designed and synthesized 3 pairs of primers located in the SLT1, SLT2, and eaeA genes of ...Objective To evaluate the etiology of Shiga-like toxin-producing Escherichia coli (SLTEC) in children with diarrhea. Methods We designed and synthesized 3 pairs of primers located in the SLT1, SLT2, and eaeA genes of enterohaemorrhagic Escherichia coli (EHEC), while the virulent genes SLT1, SLT2, and eaeA from E.coli species were amplified by polymerase chain reaction (PCR). Results One strain of EHEC with SLT1, SLT2, and eaeA in 29 reference strains of diarrhea-causing E.coli (DCEC) and 10 strains of other enterobacteria detected by PCR had positive reactions, while all other DCEC and enterobacteria were negative. Of 474 strains of E. coli isolated from 1032 children with diarrhea and detected by PCR, 20 strains of SLT1 producing E. coli (4.2%) positive, and 7 strains of SLT2 producing E.coli (1.5%) positive; while of 74 strains of entero-SLTs-producing and invasive Escherichia coli (ESIEC), 15 strains of SLT1 (20.3%) and 5 strains of SLT2 (6.8%) were positive. Conclusion Shiga-like toxin E. coli has been identified as a major etiologic agent of children with diarrhea in Taiyuan, China.展开更多
The inhibitory effects of five berberines alkaloids (BAs) from rhizoma ofCoptis chinensis Franch,a traditional Chinese medicinal (TCM) herb,on Bacillus shigae(B.shigae)growth were investigated by microcalorimetry.The ...The inhibitory effects of five berberines alkaloids (BAs) from rhizoma ofCoptis chinensis Franch,a traditional Chinese medicinal (TCM) herb,on Bacillus shigae(B.shigae)growth were investigated by microcalorimetry.The power-time curves of B.shigae with and without BAswere acquired;meanwhile,the extent and duration of inhibitory effects on the metabolism wereevaluated by growth rate constants (k1,k2),half inhibitory ratio (IC50),maximum heat output(P_(max)),and peak time (t_p).The values of k1 and k2 of a shigae in the presence of the five BAsdecreased with the increasing concentrations of BAs.Moreover,P_(max) was reduced,and the value oft_p increased with increasing concentrations of the five drugs.The inhibitory activity varied withdifferent drugs.IC50 of the five BAs was respectively 75 mu g/mL for berberine,90 ng/mL forcoptisine,115 mu g/mL for palmatine,220 mu g/mL for epiberberine,and 400 mu g/mL forjatrorrhizine.The sequence of antimicrobial activity of the five BAs:berberine 】 coptisine 】palmatine 】 epiberberine 】 jatrorrhizine.The functional groups methylenedioxy at C2 and C3 on phenylring improve antimicrobial activity more strongly than methoxyl at C2 and C3 on phenylring.However,the effect of bacteriostasis is not significant with methylenedioxy or methoxyl at C9and C10 on phenyl ring.展开更多
The bacteriophage clones which can bind with shiga toxin B subunit (StxB) and inhibit cytotoxicity of shiga toxin were obtained by using antibody capturing method from a 15-mer random peptide library displayed on the ...The bacteriophage clones which can bind with shiga toxin B subunit (StxB) and inhibit cytotoxicity of shiga toxin were obtained by using antibody capturing method from a 15-mer random peptide library displayed on the surface of bacteriophage fd. Among them, one peptide encoded by the random DNA region of a selected bacteriophage (A12) was synthesized and tested in vitro and in vivo, where the peptide competed with the receptor of shiga toxin to bind StxB, and inhibited the cytotoxicity and enterotoxicity of shiga toxin. The peptide can also block other apparently unrelated StxB binding bacteriophage (A3), which suggests that there are overlapping StxB interaction sites for those ligands with different sequences. The results provide a demonstration of bacteriophage display to screen peptide ligands for a small and/or unable biotinylated molecule by antibodies-capturing strategy, and take the lead for the development of receptor antagonists for shiga toxin.展开更多
The three parts(Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the ...The three parts(Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the three parts of Shiga toxin B subunit could be expressed as the Lamb fusion proteins in E. coli. Indirect immunofluorescence and immunoelectron microscopy analyses showed fusion proteins LamB/Stx17B and LamB/Stx27B could be expressed at cell surface in E. coli, but fusion protein LamB/StxB could not be expressed at cell surface; it was aggregated in cytoplasm and was toxic to host. This expression system provided a new way to construct an oral live vaccine against Shigella dysenteriae 1.展开更多
In 2011, Shiga toxin-producing Escherichia coli O104 : H4 resulted in a large outbreak of bloody diarrhea and hemolytic uremic syndrome (HUS) in Germany and 15 other countries in Europe and North America. This event r...In 2011, Shiga toxin-producing Escherichia coli O104 : H4 resulted in a large outbreak of bloody diarrhea and hemolytic uremic syndrome (HUS) in Germany and 15 other countries in Europe and North America. This event raised a serious public health crisis and caused more than two billion US dollars in economic losses. In this review, we describe the classification of E. coli, the Germany outbreak, and the characteristics and epidemical source-tracing of the causative agent. We also discuss the genomics analysis of the outbreak organism and propose an open-source genomics analysis as a new strategy in combating the emerging infectious diseases.展开更多
基金Supported by Agricultural Science and Technology Support Program(Social Development)of Jiangsu Province(BE2011771)~~
文摘[Objective] This study aimed to establish a real-time PCR method for de- tecting stx2 gene in Shiga toxin-producing E. coli （STEC）. [Method] According to the known STEC stx2 gene sequences published in GenBank, PCR primers and probes were designed based on the conserved region to construct recombinant plasmid as a positive template, thus optimizing the reaction conditions and establishing the real- time PCR method. [Result] A standard curve was established based on the opti- mized real-time PCR system, indicting a good linear correlation between the initial template concentration and Ct value, with the correlation coefficient F^e of above 0.995. The established method had a good specificity, without non-specific amplifica- tion for 10 non-STEC intestinal bacterial strains; the detection limit of initial template was 1.0x102 copies/μI, indicating a high sensitivity; furthermore, the coefficients of variation within and among batches were lower than 1% and 5% respectively, sug- gesting a good repeatability. [Conclusion] In this study, a real-time PCR method was successfully established for detecting STEC stx2 gene, which provided technical means for rapid detection of STEC in samples.
文摘AIM： To investigate the selective cytotoxic effect of constructed hybrid protein on cells expressing granulocyte macrophage colony stimulating factor （GM-CSF） receptor. METHODS： HepG2 （human hepatoma） and LS174T （colon carcinoma） were used in this study. The fused gene was induced with 0.02% of arabinose for 4 h and the expressed protein was detected by Western blotting. The chimeric protein expressed in E..coli was checked for its cytotoxic activity on these cells and apoptosis was measured by comet assay and nudear staining. RESULTS： The chimeric protein was found to be cytotoxic to the colon cancer cell line expressing GM-CSFRs, but not to HepG2 lacking these receptors. Maximum activity was observed at the concentration of 40 ng/mL after 24 h incubation. The IC50 was 20 ± 3.5 ng/mL. CONCLUSION： Selective cytotoxic effect of the hybrid protein on the colon cancer cell line expressing GMCSF receptors （GM-CSFRs） receptor and apoptosis can be observed in this cell line. The hybrid protein can be considered as a therapeutic agent.
文摘Non-O157 STEC has been shown to have a diverse ecological distribution among food-animals. It has been associated with both outbreaks and individual cases of severe illness. This group of the organisms is now considered as a major contributor to human disease. The clinical description of the diseases caused by these organisms is reviewed. The host specificity of these pathogens is described and discussed. These organisms appear widespread among food animals like cattle and sheep, and can therefore affect a range of foods directly from the meat and excretions of these animals being used in farming practices. This article reviews the origins, diversity and pathogenesis of non-O157 STEC.
文摘Isolation and biochemical and molecular identification of 303 strains of Escherichia coli obtained from diarrheic and healthy young alpacas of Puno-Peru, were realized. PCR amplification for 7 virulence factor genes associated with STEC, STEC O157:H7, EPEC: sxt1, sxt2, rfbO157, fliCH7, hlyA, eae y bfp were determined. A total of 39 strains (12.88%) showed amplification for one or more of these genes. Twenty three strains (59%) were classified as STEC and 16 strains (41%) as EPEC. An 88.18% (34/39) of STEC and EPEC strains were obtained from healthy alpacas and only 11.82% (5/39) from diarrheic alpacas considering this specie as potential zoonotic reservoir of STEC and EPEC.
文摘Objective To evaluate the etiology of Shiga-like toxin-producing Escherichia coli (SLTEC) in children with diarrhea. Methods We designed and synthesized 3 pairs of primers located in the SLT1, SLT2, and eaeA genes of enterohaemorrhagic Escherichia coli (EHEC), while the virulent genes SLT1, SLT2, and eaeA from E.coli species were amplified by polymerase chain reaction (PCR). Results One strain of EHEC with SLT1, SLT2, and eaeA in 29 reference strains of diarrhea-causing E.coli (DCEC) and 10 strains of other enterobacteria detected by PCR had positive reactions, while all other DCEC and enterobacteria were negative. Of 474 strains of E. coli isolated from 1032 children with diarrhea and detected by PCR, 20 strains of SLT1 producing E. coli (4.2%) positive, and 7 strains of SLT2 producing E.coli (1.5%) positive; while of 74 strains of entero-SLTs-producing and invasive Escherichia coli (ESIEC), 15 strains of SLT1 (20.3%) and 5 strains of SLT2 (6.8%) were positive. Conclusion Shiga-like toxin E. coli has been identified as a major etiologic agent of children with diarrhea in Taiyuan, China.
基金Supported by the National Natural Science Foundation of China (Grant No. 39970911)
文摘The inhibitory effects of five berberines alkaloids (BAs) from rhizoma ofCoptis chinensis Franch,a traditional Chinese medicinal (TCM) herb,on Bacillus shigae(B.shigae)growth were investigated by microcalorimetry.The power-time curves of B.shigae with and without BAswere acquired;meanwhile,the extent and duration of inhibitory effects on the metabolism wereevaluated by growth rate constants (k1,k2),half inhibitory ratio (IC50),maximum heat output(P_(max)),and peak time (t_p).The values of k1 and k2 of a shigae in the presence of the five BAsdecreased with the increasing concentrations of BAs.Moreover,P_(max) was reduced,and the value oft_p increased with increasing concentrations of the five drugs.The inhibitory activity varied withdifferent drugs.IC50 of the five BAs was respectively 75 mu g/mL for berberine,90 ng/mL forcoptisine,115 mu g/mL for palmatine,220 mu g/mL for epiberberine,and 400 mu g/mL forjatrorrhizine.The sequence of antimicrobial activity of the five BAs:berberine 】 coptisine 】palmatine 】 epiberberine 】 jatrorrhizine.The functional groups methylenedioxy at C2 and C3 on phenylring improve antimicrobial activity more strongly than methoxyl at C2 and C3 on phenylring.However,the effect of bacteriostasis is not significant with methylenedioxy or methoxyl at C9and C10 on phenyl ring.
文摘The bacteriophage clones which can bind with shiga toxin B subunit (StxB) and inhibit cytotoxicity of shiga toxin were obtained by using antibody capturing method from a 15-mer random peptide library displayed on the surface of bacteriophage fd. Among them, one peptide encoded by the random DNA region of a selected bacteriophage (A12) was synthesized and tested in vitro and in vivo, where the peptide competed with the receptor of shiga toxin to bind StxB, and inhibited the cytotoxicity and enterotoxicity of shiga toxin. The peptide can also block other apparently unrelated StxB binding bacteriophage (A3), which suggests that there are overlapping StxB interaction sites for those ligands with different sequences. The results provide a demonstration of bacteriophage display to screen peptide ligands for a small and/or unable biotinylated molecule by antibodies-capturing strategy, and take the lead for the development of receptor antagonists for shiga toxin.
文摘The three parts(Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the three parts of Shiga toxin B subunit could be expressed as the Lamb fusion proteins in E. coli. Indirect immunofluorescence and immunoelectron microscopy analyses showed fusion proteins LamB/Stx17B and LamB/Stx27B could be expressed at cell surface in E. coli, but fusion protein LamB/StxB could not be expressed at cell surface; it was aggregated in cytoplasm and was toxic to host. This expression system provided a new way to construct an oral live vaccine against Shigella dysenteriae 1.
基金supported by the National Basic Research Program of China(2009CB522600)Shenzhen Biological Industry Development Special Foundation-Basic Research Key Projects (JC201005250088A)
文摘In 2011, Shiga toxin-producing Escherichia coli O104 : H4 resulted in a large outbreak of bloody diarrhea and hemolytic uremic syndrome (HUS) in Germany and 15 other countries in Europe and North America. This event raised a serious public health crisis and caused more than two billion US dollars in economic losses. In this review, we describe the classification of E. coli, the Germany outbreak, and the characteristics and epidemical source-tracing of the causative agent. We also discuss the genomics analysis of the outbreak organism and propose an open-source genomics analysis as a new strategy in combating the emerging infectious diseases.
