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Objective To establish a rapid method for the clinical detection and identification of fungi in clinical urine samples.Methods DNA was extracted from clinically collected urine sample,and the fungal ribosomal internal transcribed spacer was amplified by polymerase chain reaction (PCR) and followed by pyrosequencing.The fungal species were identified by sequence alignment.Results The identification results were compared between PCR-pyrosequencing and conventional culture method.Among the 1320 urine samples,180 were detected positive by conventional method with the positive rate of 13.6％,while 192 were positive by the pyrosequencing based method with the positive rate of 14.5％.The overall coincidence rate of the two methods was 99.09 ％,with the positive coincidence rate of 100 ％ and the negative coincidence rate of 98.95 ％.The Kappa value was 0.963,suggesting a good consistency.The results of 13 standard strains were consistent with the actual results.Conclusions A rapid culturefree method for the detection of fungi in urine sample has been successfully established.This method is based on PCR-pyrosequencing technology with highly accuracy,sensitivity and reproducibility.It is highly automated,cost effective and with high throughput (96 samples per run).The fungal pathogen in urine is identified by single step test within 3 hours without conventional culture.Thus,it is applicable in the clinical laboratory.
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Objective To develop a method for rapid and accurate detection and identification of bacterial pathogens directly from body fluid specimens and to evaluate its application in clinical laboratory.Methods Bacteria DNA was extracted from 205 body fluid specimens with column-based kit,and the high variable V1 and V3 regions of bacterial 16S rRNA gene were amplified with broad-range primers.Amplicons were analyzed by pyrosequencing and the generated sequences were searched in the bacterial identification database.Traditional culture-biochemical method was also used for these specimens and the results were taken as the golden standard.SPSS 11.0 was used to calculate the sensitivity,specificity,false positive/negative rate,positive/negative predictive value and positive/negative likelihood rate of pyrosequencing method.Results The positive rate of bacteria culture was 39.5％ (81/205),among which 71 were infected with single bacterium,and 10 were infected with two species of bacteria.Compared with the culture identification results,pyrosequencing had a 100.0％ (71/71) concordance when applied to detect and identify bacterial pathogens from specimens with single specie bacterium infected.To specimens with two species bacteria infected,7 out of 10 specimens were in concordance with the culture identification results.Besides,pyrosequencing detected 10 positive specimens and identified bacterial pathogens infected in the 124 culture-negative specimens.Taken bacteria culture as the standard method,the sensitivity of pyrosequencing for identifying bacterial pathogen in body fluid was 100.0％,and with a specificity of 91.9％,the false positive rate was 8.1％,the false negative rate was 0.0％,the positive predictive value was 89.0％,the negative predictive value was 100.0％,and the positive and the negative likelihood rate were 12.4 and 0,respectively.Conclusion Pyrosequencing can be used to detect and identify bacterial pathogens directly from body fluid specimens with the advantages of rapidity,high sensitivity,high accuracy and high throughput.
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Objective To discover biological markers of male infertility.Methods Two-dimensional electrophoresis and Mass-Spectrum techniques(MS+MS/MS) were used for analyzing the seminal plasma from idiopathic male infertility and the control.Results Serum albumin and prostatic acid phosphatase were reduced in seminal plasma from idiopathic male infertility,while Cathepsin B and Zn-alpha-2-glycoprotein were increased.ConclusionIdiopathic male infertility was potentially associated with disorder of sperm capacitation and seminal immune function.
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Objective: To search for the optimizing parameters and distribution pattern of NF ?B decoy oligodeoxynucleotides in transfecting J774.1 cells mediated by lipofectin. Methods: With the change of ODNs/lipofectin ratio and transfection time, the uptake rate and mean fluorescence intensity of NF ?B decoy oligodeoxynucleotides in J774.1 cells were measured by flow cytometry to evaluate transfection efficiencies. Intracellular distribution of NF ?B decoy oligodeoxynucleotides was determined with fluorescence microscopy. The lactate dehydrogenase (LDH) activity in the supernatant was assayed to assess the cytotoxicity. Results: The uptake of NF ?B decoy oligodeoxynucleotides into J774.1 cells was significantly improved by lipofectin. In 24 well culture plate, 1 ?g ODNs with 5?g lipofectin ( W/W =1∶5) resulted in the highest transfection efficiency and lower cytotoxicity. The NF ?B decoy oligodeoxynucleotides localized in the nucleus as well as in the cytoplasm following an incubation of 6 h with lipofectin. While NF ?B decoy oligodeoxynucleotides had faint fluorescences in cytoplasm in the absence of lipofectin. Conclusion: lipofectin can enhance the cellular uptake of NF ?B decoy oligodeoxynucleotides in J774.1 cells and alter intracellular distribution of NF ?B decoy oligodeoxynucleotides. Efficiency of transfection is the highest when the ratio of ODNs/lipofectin is 1∶5 for incubation of 6 h.
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AIM: To study the effect of NF-?B "decoy" oligodeoxynucleotides on TNF-? and IL-6 expression in LPS-induced mouse macrophages. METHODS: Mouse macrophage cell line J774.1 cells were cultured with LPS and liposome-mediated oligodeoxynucleotides, and the levels of TNF-? and IL-6 measured in the different culture supernatant by enzyme linked immunosorbent assay. RNA was extracted from macrophages, and the mRNA expression of TNF-? and IL-6 in macrophages was observed by RT-PCR. RESULTS: NF-?B "decoy" oligodeoxynucleotides decreased the expression of TNF-? and IL-6 in LPS-induced macrophages and inhibited generation of TNF-? and IL-6. The level of TNF-? and IL-6 did not change in control group. CONCLUSIONS: NF-?B "decoy" oligodeoxynucleotides inhibit the expression of TNF-? and IL-6 in LPS-induced macrophages, which is probably due to the specific inhibition of activated NF-?B binding sites .
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AIM: To study the effects of nuclear factor-kappa B decoy oligodeoxynucleotides on generation of nitric oxide and activated oxygen in lipopolysaccharide-induced mouse macrophages.METHODS: Mouse macrophage cell lines J774.1 cells were cultured with lipopolysaccharide and liposome-mediated oligodeoxynucleotides, and the level of nitric oxide, activated oxygen and inducible nitric oxide synthase were measured. RESULTS: Nuclear factor-kappa B “decoy”oligodeoxynucleotides decreased the activity of inducible nitric oxide synthase in lipopolysaccharide-induced macrophage, inhibited generation of nitric oxide and reactive oxygen species. The level of nitric oxide, activated oxygen and inducible nitric oxide synthase did not change in control group.CONCLUSION: Nuclear factor-kappa B “decoy”oligodeoxynucleotides decrease the generation of nitric oxide and activated oxygen in lipopolysaccharide-induced macrophages, which is probably due to inhibiting the binding sites of activated nuclear factor-kappa B specially.
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AIM: To investigate the expression profile of peripheral-type benzodiazepine receptor(PBR) involved in mitochondrial permeability transition(PT) regulation,and to observe the binding dynamic of the mitochondrial PBR with specificity ligand during rat live regeneration.METHODS: Liver regeneration model was produced by 70% partial hepatectomy(PH) performed in male SD rats.The animals of sham groups underwent the same surgical operations as PH groups did,but the liver lobes were not resected.The animals in the PH groups and corresponding sham groups were sacrificed at 3,6,12,24,48,72,120 and 168 hours after the operation.The livers were removed,weighted and processed for isolation of mitochondria.Semi-quantitative RT-PCR was performed to examine the expression level of PBR in 70% hepatectomized rat livers during the whole regeneration process and compared to that in the sham and normal groups.Compared with healthy rats,the kinetic parameters of PBR was evaluated by using a specific radioligand -PK11195.RESULTS: Compared with healthy rats,the expression of PBR was unchanged.Meanwhile,the results obtained in the present experiments by scatchard analysis,Bmax of PK11195 for PBR significantly decreased,returned to normal level in 168 h after PH.Kd of PK11195 for PBR significantly decreased at 72 h and 168 h after PH of rat liver regeneration(P