A fluorometric method for direct detection of inorganic polyphosphate in enterohemorrhagic O157:H7 / 南方医科大学学报

OBJECTIVE@#To establish a quantitative fluorescent detection method using DAPI for detecting inorganic polyphosphate (polyP) in enterohemorrhagic Escherichia coli (EHEC) O157:H7.@*METHODS@#The DNA of wild-type strain of EHEC O157:H7 was extracted and purified. DAPI was combined with the extracted DNA and polyP45 standards for measurement of the emission spectra at 360 nm and 415 nm fluorescence spectrophotometry. The fluorescence of DAPI-DNA and DAPI-polyP complexes was detected by fluorescence confocal microscopy to verify the feasibility of DAPI for detecting polyP. To determine the optimal pretreatment protocol for improving the cell membrane permeability, the effects of 6 pretreatments of the cells (namely snap-freezing in liquid nitrogen, freezing at -80 ℃, and freezing at -20 ℃, all followed by thawing at room temperature; heating at 60 ℃ for 10 min; treatment with Triton x-100; and placement at room temperature) were tested on the survival of EHEC O157:H7. The fluorescence values of the treated bacteria were then measured after DAPI staining. A standard calibration curve of polyP standard was established for calculation of the content of polyP in the live cells of wildtype EHEC strain and two mutant strains.@*RESULTS@#At the excitation wavelength of 360 nm, the maximum emission wavelength of DAPI-DNA was 460 nm, and the maximum emission wavelength of DAPI-polyP was 550 nm at the excitation wavelength of 415 nm. The results of confocal microscopy showed that 405 nm excitation elicited blue fluorescence from DAPIDNA complex with the emission wavelength of 425-475 nm; excitation at 488 nm elicited green fluorescence from the DAPIpolyP complex with the emission wavelength of 500-560 nm of. Snap-freezing of cells at -80 ℃ followed by thawing at room temperature was the optimal pretreatment to promote DAPI penetration into the live cells. The standard calibration curve was =1849+127.5 (R=0.991) was used for determining polyP content in the EHEC strains. The experimental results showed that wild-type strain had significantly higher polyP content than the mutant strains with deletion.@*CONCLUSIONS@#We established a convenient quantitative method for direct and reliable detection polyP content to facilitate further study of polyP and its catalytic enzymes in EHEC O157:H7.

The practice and topic selection of innovative experiment competition of preventive medicine / 中华医学教育探索杂志

Innovative Experiment Competition of Preventive Medicine is a significant constitution of Guangdong Undergraduate Technique Competition of Preventive Medicine. Without the constriction of majors, students participate in group with 3 people, design their own experiments around the hot spots and practical problems in public health area and are awarded according to the grades of the experiment design and thesis defence. The most topics in the first and the second competitions are about environmental hygiene and occupational hygiene, and the infectious diseases are attracting increasing attention. More and more topics combine the method of experiment with investigation. Some tactics are also suggested that innovation, view of general population, autonomy and investigation should be included in topic selection for this contest.

"Analysis of the Utilization of Essential Medicines in a Community Health Service Center in Foshan before and after the""New Medical Reform""" / 中国药房

OBJECTIVE:To provide reference for the further promotion ofNew Medical Reformand the further implementa-tion and improvement of National Essential Medicine System. METHODS:By systematic sampling,1728 outpatient prescriptions were randomly selected from a community health service center in Foshan during 2012 to 2015,and then analyzed and compared statistically in respects of the amount of drugs,rational drug use indexes,prescription cost,main disease composition,treatment cost. The change rules and characteristics of all the above mentioned information were all studied. RESULTS:The average number of medicines per prescription and the number of essential medicines decreased year by year,Comparied with 2012,the proportion of antimicrobial agents,the proportion of two or more antimicrobial agents,the proportion of injection prescription and the propor-tion of transfusion prescription were significantly reduced,the cost of medical treatment of patients decreased significantly;medi-cine cost and medicare reimbursement amount had increased to different extents,there was statistical significance(P<0.05). Multi-ple linear regression analysis(stepwise)showed that the cost of medical treatment was closely related to rationality of prescriptions. Respiratory diseases,cardiovascular and cerebrovascular diseases,gastrointestinal diseases were the top 3 of the major diseases, the proportion of prescriptions for respiratory diseases,gastrointestinal diseases decreased year by year,while that for cardiovascu-lar and cerebrovascular diseases increased year by year. CONCLUSIONS:The promotion ofNew Medical Reformand the imple-mentation of National Essential Medicine System play a positive role in promoting rational drug use and the quality of medical ser-vice in community health service center. However,there are still many problems in the community health service centers,such as too many medicines each prescription,the lack of diagnosis and treatment ability,the defects of medicine selection and administra-tion.

Construction of enterohemorrhagic Escherichia coli O157:H7 strains with espF gene deletion and complementation / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains with delection espF gene and its nucleotide fragment and with espF gene complementation.</p><p><b>METHODS</b>A pair of homologous arm primers was designed to amplify the gene fragment of kanamycin resistance, which was transformed into EHEC O157:H7 EDL933w strain via the PKD46 plasmid by electroporation. The replacement of the espF gene by kanamycin resistance gene through the PKD46-mediated red recombination system was confirmed by PCR and sequencing. The entire coding region of espF along with its nucleotide fragment was amplified by PCR and cloned into pBAD33 plasmid, which was transformed into a mutant strain to construct the strain with espF complementation. RT-PCR was used to verify the transcription of espF and its nucleotide fragment in the complemented mutant strain.</p><p><b>RESULTS AND CONCLUSION</b>We established EHEC O157:H7 EDL933w strains with espF gene deletion and with espF gene complementation. Both espF and its nucleotide fragment were transcribed in the complemented mutant strain. The two strains provide a basis for further study of the regulatory mechanism of espF.</p>

Construction and characterization of enterohemorrhagic Escherichia coli O157:H7 ppk- deleted strain / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics.</p><p><b>METHODS</b>The gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5' and 3' ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157: H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD600 value and Giemsa staining.</p><p><b>RESULTS AND CONCLUSION</b>We established a ppk-deleted EHEC O157:H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.</p>

Origin and the recombinant model of H7N9 virus prevailing in China / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the origin and the recombinant model of H7N9 virus prevailing in China by sequence analysis.</p><p><b>METHODS</b>The sequences of H7N9 virus were collected and analyzed with the software BLAST and MEGA 5.0. The phylogenetic trees were constructed after multiple sequences alignment. The homologous sequences of H7N9 segments were determined and the model was inferred according to the origin of H7N9 segments.</p><p><b>RESULTS</b>The most relevant sequences of HA, NA, NS and PB2 segments were located at one branch of the phylogenetic tree, while the closest relevant sequences of PB1, PA, NP and MP contained two H9N2 virus origins. According to the analysis of sequence origin, H7N9 viruses might be divided into 5 genotypes: namely A, B, A/Shanghai/1/2013-H7N9, A/Pigeon/Shanghai/S1069-H7N9 and A/Zhejiang/HZ1/2013-H7N9, and the genotype A consisted of A1 and A2 subtypes.</p><p><b>CONCLUSION</b>The prevailing H7N9 virus might be derived from 5 different viruses after 4 times of recombination, which resulted in the two major types of A and B. The subtypes of A1 and A2 were two different derivatives from one reassortant. The A/Pigeon/Shanghai/S1069-H7N9 strain might be the recombinant of type A H7N9 virus with a local H9N2 virus during the H7N9 epidemics. The A/Zhejiang/HZ1/2013-H7N9 strain could be the re-arrangement of subtype A2 with type B H7N9 virus.</p>

Study and Practice of Emergency Talent Training Model in Public Health / 中华医学教育探索杂志

Emergency in public health is one major issue relating to the national economy and the people's livelihood.In dealing with the key technologies of detects,diagnosis,disinfection,prevention and control,it is wise to syncretize the correlated subjects in nucleus,chemistry and biology,improve the support condition of teaching,strengthen the reform of teaching contents,add some lessons of emergency treatment,make emergency equipments,and form the ability of rapid diagnosis on the spot and precise identifying in lab,for cultivating the students'capability of spot handling,organization and command,and furthermore exploring and practicing the training pattern of the public heath talent with high quality and emergency ability in our country.

Current situation and preliminary exploration of specialty construction of eight-year medical education of China / 中华医学教育探索杂志

Eight-year medical education has been run in some of the unversities in China,which has made some effective trials in personnel training mode according to the training purposes sent by Ministry of Education,but there still exists weaknesses in curriculum provision and reasearch training.This article briefly analyzed the major construction of the eight-year medical education and displayed several views.