RESUMO
In order to establish a real-time RT-PCR based on SYBR Green Ⅱ for detection of hepatitis E virus (HEV),a pair of special primers was designed according to the conserved sequences of ORF2 in GenBank.Result showed that the standard curve of established SYBR Green Ⅱ real-time RT-PCR had a wide dynamic range from 4.10 × 102-4.10 × 108 copies/μL with a linear correlation(r2) of 0.996.The sensitivity could reach 1.00 × 102 copies/μL.The melting curve analysis using SYBR Green Ⅱ dye showed one specific peak with a melting temperature(Tm) of 84.0 C ±0.1 C.No amplification was detected from the RNA samples of porcine reproductive and respiratory syndrome virus,classial swine fever virus,transmissible gastroenteritis virus,porcine bocavirus,porcine epidemic dearrhoea virus porcine kobuvirus and porcine rotavirus by this PCR,respectively.Excellent reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay of 0.83 ％-0.94 ％ and inter-assay of 0.83％-0.94％.Further detection of 61 specimens showed that 9 of them were HEV positive,and the results of the quantitative RT-PCR were the same as that of the conventional RT-PCR.In conclusion,the real-time quantitative RT-PCR for HEV is feasible,the real-time RT-PCR established in this study will be useful for earlier rapid laboratory diagnosis and pathogenesis of HEV.
RESUMO
OBJECTIVE: The role of Ulinastatin in neuronal injury after cardiopulmonary resuscitation has not been elucidated. We aim to evaluate the effects of Ulinastatin on inflammation, oxidation, and neuronal injury in the cerebral cortex after cardiopulmonary resuscitation. METHODS: Ventricular fibrillation was induced in 76 adult male Wistar rats for 6 min, after which cardiopulmonary resuscitation was initiated. After spontaneous circulation returned, the rats were split into two groups: the Ulinastatin 100,000 unit/kg group or the PBS-treated control group. Blood and cerebral cortex samples were obtained and compared at 2, 4, and 8 h after return of spontaneous circulation. The protein levels of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) were assayed using an enzyme-linked immunosorbent assay, and mRNA levels were quantified via real-time polymerase chain reaction. Myeloperoxidase and Malondialdehyde were measured by spectrophotometry. The translocation of nuclear factor-κB p65 was assayed by Western blot. The viable and apoptotic neurons were detected by Nissl and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). RESULTS: Ulinastatin treatment decreased plasma levels of TNF-α and IL-6, expression of mRNA, and Myeloperoxidase and Malondialdehyde in the cerebral cortex. In addition, Ulinastatin attenuated the translocation of nuclear factor-κB p65 at 2, 4, and 8 hours after the return of spontaneous circulation. Ulinastatin increased the number of living neurons and decreased TUNEL-positive neuron numbers in the cortex at 72 h after the return of spontaneous circulation. CONCLUSIONS: Ulinastatin preserved neuronal survival and inhibited neuron apoptosis after the return of spontaneous circulation in Wistar rats via attenuation of the oxidative stress response and translocation of nuclear factor-κB p65 in the cortex. In addition, Ulinastatin decreased the production of TNF-α, ...