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Objective To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on hemolysin gene(vvhA)that coding cytolysin.Method By using software Primer Express, the PCR primers and TaqMan probe,which located in the conserved region of vvhA gene sequence,were designed for establishment of a TaqMan real-time fluorescent quantitative PCR to detect 100 bp amplicon from V.vulnificus DNA.A recombinant plasmid pMD19-vvhA100 as a positive control during detection was constructed using gene cloning technique.Minimal amplification cycles(Ct value)and fluorescence intensity enhancement (△Rn value)were used as observing index to optimize the reaction conditions of the TaqMan real-time fluorescent quantitative PCR.The DNAs with different concentrations from V.vulnificus and other eight bacteria and pMD19- vvhA100 were applied as templates to determine the specificity,sensitivity and reappearance of the TaqMan real- time fluorescent quantitative PCR.ICR mice were intraperitoneally,subcutaneously and orally infected with V. vulnificus,respectively.The detection effect of the TaqMan real-time fluorescent quantitative PCR was measured using the specimens of peripheral blood,subcutaneous tissue and intestinal content collected from the infected mice.Results The established TaqMan real-time fluorescent quantitative PCR showed positive results only for V. vulnificus DNA and pMD19-vvhA100.The detection effectiveness of the TaqMan real-time fluorescent quantitative PCR was as high as 0.01 ng of V.vulnificus DNA or 103 copies of pMD19-vvhA100.The SD values of the detection results repeated for three times using pMD19-vvhA100 with different concentrations were lease than 0.79. The detection results of TaqMan real-time fluorescent quantitative PCR were positive for all the specimens of peripheral blood and subcutaneous tissue.Conclusions The TaqMan real-time fluorescent quantitative PCR established in this study for V.vulnificus vvhA gene detection has advantages such as quickness,stability, sensitivity and specificity,indicating this method can be used for clinical laboratory diagnosis of septicemia and wound infection caused by V.vulnificus.

8.The Effects of transcranial ultrasound stimulation on motor functioning and anti-oxidative capacity in mice with Parkinson's disease

Yong WANG ; Baixu REN ; Shufeng WU ; Qing ZHONG ; Xiaoli LI ; Chengbiao LU

Chinese Journal of Physical Medicine and Rehabilitation 2015;37(7):488-492

Objective To investigate the effects of transcranial ultrasound stimulation (TUS) on the motor functioning and anti-oxidative capacity of mice with Parkinson's disease (PD).Methods Thirty-two inbred C57BL male mice were randomized into a normal control group,a model group,a sham TUS group and a TUS group (n =8 for each group) according to a random number table.A PD model was induced in the mice of the model,sham TUS and TUS groups by injecting 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at 20 mg/kg intraperitoneally,while those in the normal control group were given saline.Low intensity (1 W) focused ultrasound (LIFU) at a frequency of 0.5 MHz was then applied to stimulate the nigra region,except for the mice in the sham TUS group,which were treated with the same procedure but with no ultrasound output.A pole climbing test was carried out before,2 weeks and 5 weeks after the injection of the MPTP.After 5 weeks the animals were sacrificed and the whole brain malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) content were measured.Results No significant differences in in pole climbing scores were observed among the four groups before the MPTP injections.However,the average value decreased significantly to (4.30 ± 1.19),(4.40 ± 0.23) and (4.80 ± 0.23) for the model,sham TUS and TUS groups respectively 2 weeks after the injection.It then rose to (5.12 ±0.83) and (5.51 ± 1.21)for the first two groups 3 weeks later,but was still lower that before the injection.After 5 weeks the TUS group's average score was significantly higher than 3 weeks earlier and than that of the model group and the sham TUS group.Compared with the control group,the other groups' average scores were all lower 2 weeks after MPTP injection,and those of the model and the sham TUS groups remained so 5 weeks after the injection.Five weeks after the injection,the average MDA content of the model group (10.2 ± 1.1 nmol/ml) and the sham TUS group (9.4 ±1.3 nmol/ml) were significantly higher than the normal control group (4.5 ± 0.8 nmol/ml),as well as the TUS group (6.8 ± 0.9 nmol/ml).However,GSH-Px enzyme activity in the model group (100 ± 35.4 U/mgprot) and the sham US group (444 ± 24.9 U/mgprot) was significantly lower than that in the normal control group (1262.5 ± 53 U/mgprot),together with the TUS group (1047.3 ± 77.8 U/mgprot).Conclusion TUS can improve motor function in PD,at least in mice.This may be due to its anti-oxidative capacity.