[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
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
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.
