OBJECTIVE To search a method for identifying Clostridium perfringens and genotyping their toxin for gene diagnosis by multiplex PCR.METHODS The mutiplex PCR was developed with three sets of primers(designed) based on the sequences of three C.perfringens toxin genes(CP?,CP? and CPE) published in GenBank to identify C.perfringens and genotype their three toxin genes.RESULTS Three expected(sequences) were (obtained) successfully by multiplex PCR and identified by electrophoresis.CONCLUSIONS The(specific) sequences of C.perfringens could be amplified and their three genes of toxins could be identified by this multiplex PCR(system).Such method should be helpful for developing gene diagnosis well.

OBJECTIVE To discuss a highly effective method to immobilize probe on the surfaces of piezoelectric DNA sensors.METHODS Pseudomonas aeruginosa probe was immobilized on the gold surface of gene sensor(array) with routine self-assembly method(SAM)(non-reduction method) and SAM with deoxidized probe((reduction) method),respectively.The changes in frequency and time-cost were compared in reactions with(different) concentrations of probe.RESULTS Reduction method had the advantage of more probe immobilization;less time consumed in testing and higher changes in frequency during the reaction than non-reduction method.CONCLUSIONS Reduction method has a better ability to immobilize probe on the surfaces of piezoelectric DNA sensors.

OBJECTIVE To discuss the feasibility of signal amplification method with cationic conjugated polymers(liposome) applied during the detection of Pseudomonas aeruginosa using piezoelectric gene biosensors.(METHODS) Oligonucleotide probe for P.aeruginosa was immobilized on the surface of gene sensor array and(hybridized) by PCR production of P.aeruginosa.After hybridization,liposome was added.The frequency shifts were recorded and compared with those ones of the control groups.RESULTS The frequency shifts were(significantly) increasing when adding liposome and the compatible concentration of liposome was 0.8?g/?l.(CONCLUSIONS) Liposome signal amplification is proved to be an effective method to amplify the piezoelectric(signal).

OBJECTIVE To establish a SNP detection method by DNA piezoelectric biosensor and detect a SNP relative to HBV infection. METHODS To establish a model experiment with synthesis DNA sequences as target and find the lowest sensitivity. After extraction of genome DNA from inpatient blood sample, the SNP sites located in ESR1 gene region in samples were detected by SNP detecting method established. RESULTS The frequency shift of target-A was 416.0?21.5Hz, the frequency shift of target-G was 9.4?5.0Hz. And it could be detected that the lowest sensitivity of target-A was 2?10-11 mol/L. The three genotypes of blood samples, TT, TC and CC, had different frequency shifts, 109.4?13.4Hz, 52.0?11.4Hz and 7.2?4.5Hz, respectively. CONCLUSIONS SNP in blood sample could be detected specifically and sensitively by DNA piezoelectric biosensor.

Objective To develop a new type of piezoelectric quartz crystal microbalance DNA sensor based on lambda exonuclease to detect Staphylococcus aureus (S. aureus) and optimize the main detection conditions. Methods After the DNA was extracted from the stain of S. aureus and the target fragment was amplified with self-designed universal primers, the PCR products were treated with Lambda exonuclease and visualized by agarose gel electrophoresis with ethidium bromide followed by a gel documentation system analysis. Then the products with or without exonuclease digestion were added into our piezoelectric quartz crystal microbalance DNA sensor for hybridization. The temperature and time of hybridization were optimized respectively. The specificity and sensitivity of our detection system were evaluated. Results The optimal temperature for hybridization was 35℃ and the optimal time was 60 min. The sensitivity of the lambda exonuclease based DNA sensor was better than that of common DNA sensor. The lowest detection limit of this new type quartz crystal microbalance system was 1.0?104 CFU/ml. Conclusion The lambda exonuclease based quartz crystal microbalance system we designed has the advantages of high efficiency in hybridization, easy to operate, and good response performance. Thus it can be applied to detect S. aureus infections.

Humans ; Infant, Newborn ; Lupus Erythematosus, Systemic ; blood ; Male

OBJECTIVETo observe the effect of Bushen Tiaochong Recipe (BTR) on proliferation and secretion of ovarian granulocyte (OGC) cultured with excessive androgen by serum pharmacologic technique.

METHODSFollicular stimulating hormone (FSH) contained and BTR contained (high, moderate or low dose) serum prepared on 6-week-old Sprague Dawley rats by serum pharmacologic method were respectively added into cultured OGC cell-line after treatment with testosterone propionat (TP). Then the cells were incubated for 48 hrs. The quantity of 3 H-thymidine incorporated DNA was determined with a scintillation counter; the cell cycle distribution and proliferation index (PI) were estimated with flow cytometer (FCM); and the contents of estrodial and progestogen in culture fluid and intracellular cAMP were detected by radioimmuoassay.

RESULTSExcessive androgen obviously inhibited the proliferation and secretion of OGC, and reduced the intracellular cAMP. BTR contained serum could improve above-mentioned inhibition of androgen to promote the cell proliferation and secretion of E2 and P in dose-dependant manner.

CONCLUSIONBTR could alleviate the inhibitory action of excessive androgen on OGC, the ovarian function may be adjusted by BTR through promoting follicular development, increasing the levels of estrogen and progestogen, improving the ratio of estrogen/androgen.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Estradiol ; secretion ; Female ; Flow Cytometry ; Granulosa Cells ; cytology ; drug effects ; secretion ; Ovary ; cytology ; Rats ; Rats, Sprague-Dawley ; Serum ; Testosterone ; pharmacology

Objective To establish a rapid detection approach by visual interpretation directly for OprD2 resistance gene of Pseudomonas aeruginosa based on the Loop-mediated isothermal amplification (LAMP),and provide a quick and effective method for clinical monitoring of Pseudomonas aeruginosa strains.Methods Totally 47 strains of Pseudomonas aeruginosa collected from December 2011 to June 2012 in Southwest Hospital of microorganisms were prospectively studied.Four LAMP primers (two inner,two outer) were designed according to the six zones of the OprD2 gene of Pseudomonas aeruginosa.A positive reaction is indicated by the color change after adding an intercalating dye (hydroxy naphthol blue) to the reaction solution.This method was used to detect and analyze the distribution of OprD2 resistance gene in 47 strains of Pseudomonas aeruginosa and its coirelation with antibiotic resistance.Results The LAMP assays showed 100％ specificity for the OprD2 gene,and the sensitivity (with the lowest detection limits of 17.414 μg/L) was 10-fold higher than that of conventional PCR assays.The OprD2 gene was negative in 23 strains by both conventional PCR and LAMP.In OprD2 negative strains,the resistance rate of cefotaxime,levofloxacin,aztreonam,piperacillin,imipenem and meropenem was 100％ (23/23),57％ (13/23),48％ (11/23),48％ (11/23),48％ (11/23) and 43％ (10/23).Compared with the OprD2 positive strains,statistical analysis showed that the resistance rate of imipenem,levofloxacin and meropenem in OprD2 negative strains increased significantly (chisquare value is 9.155,4.846,4.037,P value was 0.002,0.028,0.045,and so there was significant difference).Conclusions The established LAMP approach in this study enables rapid,sensitive and specific detection of OprD2 gene in Pseudomonas aeruginosa by visual interpretation.Deficiency of OprD2 gene confers Pseudomonas aeruginosa a basal level of resistance to carbapenems especially to imipenem.The identification of OprD2 gene distribution in Pseudomonas aeruginosa is helpful to the selection of antimicrobial agents in the infection treatment.

OBJECTIVE To set-up safe alternatives to ethidium bromide(EB).METHODS Comparative analysis was performed on the DNA staining efficiencies of 4 fluorescent dyes including SYBR Gold,SYBR Green Ⅰ,(GoldView) and EB in preparative agarose gel electrophoresis.RESULTS Although both SYBR Gold and SYBR Green Ⅰ altered electrophoretic mobility and thus DNA size estimates,they were cost-effective alternatives to EB.SYBR Gold was more sensitive than SYBR Green Ⅰ at detecting short fragments,but 50 bp bands were clearly(visible) using either dye when visualized with a long integration time.CONCLUSIONS SYBR Gold or SYBR Green Ⅰ are sensitive and relatively safe alternatives to EB.

As an important part of the hierarchical medical system in China ,urban secondary hospitals play as a pivot between tertiary hospitals and community healthcare centers.By describing the overall development status of such hospitals in China ,the paper discussed the problems existing in the development of urban secondary hospitals and put forward strategies in the hope of providing reference for the transformation of these hospitals .