Objective:To obtain oligonucleotide aptamers which can specifically bind to Bacillus anthracis spores by in vitro selection protocol-SELEX (system evolution of ligands by exponential enrichment).Methods:An in vitro synthesized 78 mer random DNA library (≤1014-15types of different DNAs ) was subjected to 15 rounds of selection using SELEX method against spores of B.anthracis vaccine strain A.16R. Binding of the aptamers to spores was visualized by biotin-streptavidin-horseradish peroxidase system.Results:PCR amplification band pattern of the first round selection was different from that of the ninth round. The binding assay demonstrated that D absorbance at 450 nm of the fifteenth round pool increased 9 times as compared with that of the first round , and the D absorbance increased with the increment of aptamers′ quantity binding to spores. Conclusions: A set of aptamers with considerable binding affinity to B.anthracis spores were successfully selected from the initial random DNA pool.

Objective:To express and purify hemoglobin receptor(HgbA) and its partial fragment(HgbAF) from Haemophilus ducreyi and to develop a sandwich ELISA for the detection of H.ducreyi infection.Methods:The HgbA,a hemoglobin-binding outer membrane protein of H.ducreyi and its partial fragment(HgbAF) were expressed by cloning the genes of hgbA and its 705bp fragment into pET30a and pET28a respectively,and the expressing products were purified from E.coli BL21 with Ni-NTA-His affinity chromatography.The polyclonal antibodies were developed by immunizing rabbits with the rHgbA and rHgbAF.The anti-rHgbA IgG and anti-rHgbAF IgG were purified respectively by saturated amine sulphate precipitation,and their immunoactivity with rHgbA and rHgbAF was tested by Westen blot and ELISA analysis.A Sandwich ELISA was developed for the detection of chinical infection of H.ducreyi using the specific polyclonal antibodies.Results:The HgbA and its partial fragment,HgbAF of H.ducreyi,were expressed and purified successfully by cloning their genes respectively.The results obtained by Western blot analysis showed that each of the antibodies could react with both antigens,rHgbA and rHgbAF.The results of the ELISA analysis showed that H.ducreyi strain was strongly positive,and all other bacteria,including H.influenzae and the bacteria known to relate to genital ulcers were negative.The results of the ELISA analysis showed that the minimum amount of rHgbA detected was 1.56 ng/ml and the minimum number of CFU of H.ducreyi detected was 2?105 cfu/ml in buffer and 1?106 cfu/ml in pus.Conclusion:HgbA and its partial fragment,HgbAF of H.ducreyi are expressed and purified successfully.The polyclonal antibodies developed by immunizing rabbits using rHgbA and rHgbAF could react not only with rHgbA and rHgbAF,but also with H.ducreyi specifically.They do not react with other bacteria,including H.influenzae and the bacteria known to relate to genital ulcers.So the ELISA based on the polyclonal antibodies was specific for the detection of H.ducreyi.Although the level of sensitivity of the ELISA may not be sufficient to detect H.ducreyi in all clinical specimens,further work to increase the sensitivity could potentially make this assay a valuable tool in areas where chancroid is endemic.

A gas chromatographic analysis method was employed to determine the cellular fatty acids (CFAs)profiles of the spores of some aerobic endospore forming bacilli.Purified spore cultures of 51 experimentas strains were processed to acquire whole cell fatty acids methyl esters for the subsequent gas chromatographic analysis,and the corresponding vegetative cells were set as control.The reproducibility study of spore fatty acids revealed that,the fatty acids components of spores were stable enough for research purpose,provided under standardized experimentas procedure.The dendrograms obtained by cluster analysis provided some meaningful taxonomic information of the experimental strains.The fatty acids analysis of spores seemed to be a promising supplementary tool for the chemotaxonomic research of aerobic endospore-forming bacilli.

A gas chromatographic analysis method was employed to determine the cellular fatty acids (CFAs)profiles of the spores of some aerobic endospore4orming bacilli. Purified spore cultures of 51 experimentas strains were processed to acquire whole cell fatty acids methyl esters for the subsequent gas chromatographic analysis,and the corresponding vegetative cells were set as control. The reproducibility study of spore fatty acids revealed that,the fatty acids components of spores were stable enough for research purpose,provided under standardized experimentas procedure. The dendrograms obtained by cluster analysis provided some meaningful taxonomic information of the experimental strains. The fatty acids analysis of spores seemed to be a promising supplementary tool for the chemotaxonomic research of aerobic endospore-forming bacilli.

OBJECTIVETo evaluate the etiology of Shiga like toxin producing Escherichia coli (SLTEC) in children with diarrhea.

METHODSWe 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).

RESULTSOne 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.

CONCLUSIONShiga-like toxin E. coli has been identified as a major etiologic agent of children with diarrhea in Taiyuan, China.

Child ; Diarrhea ; microbiology ; Escherichia coli ; classification ; isolation & purification ; pathogenicity ; Feces ; microbiology ; Humans ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Shiga Toxins ; genetics