ObjectiveTo facilitate the establishment of fully automatic clinical diagnostic nucleic acid assay,a study was carried out for developing a fast and convenient method of extracting highly purified viral nucleic acid from human blood with magnetic beads.Method Five KHB (Shanghai Kehua Biological Engineering Company Limited) internal strong positive HBV serum samples were used in the process,for the method development,screening and selecting the magnetic beads based on magnetism,suspension power and extraction effects of HBV DNA,the optimization of the selected key components of extraction reagents were performed,which including the quantity of magnetic beads( 1 - 1.75 μl/ml),the buffer pH of lysis (4 -9),washing conditions with or without alcohol,elution for proliferation with or without magnetic beads,and etc.The evaluation for the extraction method was carried out including linearity,precision,sensitivity,specificity,anti-interference and resistance to contamination.The magnetic beads method was compared with domestic mature boiling method in a quantitative test with 100 HBV serum samples,the magnetic beads method was also compared with the interntional Gold standard Roche COBAS Tagman test method ( Roche method) in a quantitative test with 50 HBV serum samples and the results were statistically compared for their linear regression.ResultsOptimization experiment result showed superparamagnetic beads A was the best among the candidates ( C,B,D) in terms of their physical properties and extraction effects of DNA.The optimum condition of the assay were as follow:magnetic beads best used at 1.25 μl/ml,buffer pH 7 - 8 of lysis,ethanol was eliminated from washing buffer,proliferation can be carried out with magnetic beads.The parallel comparison experiments with five KHB internal strong positive HBV serum sample and gradient diluted serum sample between beads method and boiling method were carried out.The results showed that the linear range of beads method was 4 × 101 -4 × 108 IU/ml and that of boiling method was 4 × 102 -4 × 108 IU/ml.The sensitivity for beads method was 30 IU/ml and that of boiling method was 100 IU/ml.The precision CV of beads method was 6.0％ and boiling method was 11.9％.The false positive rate was 0 for beads method vs 3.1％ for boiling method.The anti-interference ability experiment showed that when sample contained sodium heparin concentration exceed 62.5 U/ml, there was significant drop of HBV DNA detection with boiling method and no HBV DNA was detected when sodium heparin concentration in the sample reached 1000 U/ml.However,sample sodium heparin concentration had no effect on HBV DNA detection with beads method.The comparison study between beads method and boiling method was carried out by use of 100 clinical HBV positive serum samples.The result showed that the sensitivity for beads method was 86％ vs boiling method 74％.The correlation between the two method was R2 =0.948,P ＜ 0.05.The comparison study between beads method and Roche method was carried use of 50 clinical HBV DNA positive serum samples and the result showed that sensitivity for beads method was 95％ (49/50) vs Roche method was 100％ (50/50).The correlation was R2 =0.963,P ＜ 0.05.ConclusionsThe established method for automatic nucleic acid extraction with magnetic bead displayed characteristic of high yield nucleic acid extraction,broader testing range,accurate quantification and convenient in operation.This can be one choice for nucleic acid extraction and applicable for clinical automatic diagnostic system.( Chin J Lab Med,2012,35:843 -850 )

OBJECTIVE To evaluate the distribution of pathogens causing infection and current status of antimicrobial resistance in order to offer experimental data for clinical administration.METHODS The resistance tests were performed using K-B method.WHONET5.2 and EXCEL were used to analyze bacteria distribution and resistance.RESULTS From 119 cases of patients were earned the routine bacteria culture results,the tested rate was 96.75%,the positive specimens were 379 and the postitive rate was 83.85%.The strains of Pseudomonas aeruginosa were the most frequent organisms isolated then were the strains of Streptococcus pyogenes,coagulase-negative Staphylococcus,Nesseria spp,and Stenotrophomonas maltophilia.CONCLUSIONS It is important to strengthen antibiotics administration,bacteria isolation and resistance surveillance.

Objective To investigate the role of TLR9 in the glomerulonephritis through observing the changes of Toll-like receptor 9 in the glomerulo nephritis kidney tissue with or without CpG-ODN stimulation .Methods Wistar male rats were randomly divided into normal group(N),nephritis model group(M),CpG-ODN group(CpG) and GpC-ODN group(GpC).The urine samples were collected at 2,4,6 and 8 weeks after treatment,respectively.Blood samples were collected at the end of the last urine sample was collected,and the kidney tissue was collected,then the rats were killed.24 h urine protein was measured by Coomassie light blue technique.Serum album and renal function were determined by serological method.The pathologic changes of kidney were observed by light microscope and NF-?B p65 expression was detected using immunohistochemystry,RT-PCR was performed to detect the expressions of TLR9,INF-? and IL-6 mRNA.Results The expression of TLR9 was lighter in group M,and significantly increased after CpG-ODN stimulation compared with group M.Furthermore,24 h urine protein excretion was markedly increased,serum album was markedly decreased.The histopathologic changes of kidney were more severe.The mesangial cells(MCs) proliferated diffusifully in midrange and wide range,some of the glomeruli formatted cellularity crescent,micrangium loop was limitted,mononuclear macrophile cells were seen in the mesangial region.Conclusion Inflammatory factors mediated by TLR9 can deteriorate the biochemical and histopathologic changes.The immunologic reaction mediated by TLR9 is one of the mechanisms for the glomerulonephtitis' progression.

Objective To investigate the effect of heart rate variability (HRV) biofeedback training on patients with three types of insom-nia disorder. Methods From June, 2016 to March, 2017, 17 patients in simple insomnia group, 19 patients in insomnia with anxiety group, and 19 patients in insomnia with depression group were included. All the patients received HRV biofeedback training and same medication (zolpidem tartrate, 10 mg every night). They were assessed with Pittsburgh Sleep Quality Index (PSQI), Symptom Checklist 90 (SCL-90), Hamilton Anxiety Scale (HAMA) and Hamilton Depression Scale (HAMD), and HRV parameters were tracked before and after training. Re-sults After training, the scores of PSQI, SCL-90, HAMA , HAMD and the ratio of low frequency (LF) and high frequency (HF) (LF/HF) de-creased in all groups (t>1.446, P<0.05). There was significant difference in PSQI among three groups (F=3.537, P=0.038). The D-values of the PSQI score and LF/HF before and after training were more in the insomnia with anxiety group and the insomnia with depression group than in the simple insomnia group (P<0.05), however, no significant difference was observed between the insomnia with anxiety group and the insomnia with depression group (P>0.05). Conclusion HRV biofeedback training could improve the symptoms of patients with three types of insomnia disorder, especially for those with anxiety or depression.

Objective To investigate the reason for hidden hemorrhage of hip fracture in elder.Methods All of 94 elder patients,who were diagnosed with intertrochanteric fracture or femoral neck fracture and received treatment in our department from October,2013 to September,2015,were included in this study.The time between injuries to admission was less than 4 hours of the two groups of patients.And the patients whose hemoglobin was less than 100 g/L were removed when admission,in order to avoid the interference of primary anemia.All information,including height,weight,and the value of hemoglobin (Hb) and hematocrit (Hct),were collected.Blood tests were performed immediately after admission,at daily morning preoperatively,and at the morning of the day of surgery.Preoperative blood loss (hidden hemorrhage) was recorded.With respect to blood loss of hidden hemorrhage,statistical analysis was performed at different times (immediate time after admission,and day 1,2,and 3 postoperatively)in the group of intertrochanteric fracture or in the group of femoral neck fracture,and subsequently performed between the two groups.Results The blood loss in the group of intertrochanteric fracture was 196.3 ml,310.1 ml and 418.3 ml in the 1st day,the 2nd day and the third day after admission.There was a significant difference among different time with respect to blood loss.The blood loss was 39.8 ml,65.7 ml and 82.9 ml in the 1st day,the 2nd day and the third day after admission in the group of femoral neck fracture.There was also a significant difference among different time with respect to blood loss.In experimental group,mean blood loss was 418.3 ml and mean Hb decreased by 23.7 g/L at day 3 postoperatively.In control group,mean blood loss was 82.9 ml and mean Hb decreased by 6.7 g/L at day 3 postoperatively.A significant difference was observed between the two groups.The blood loss in patients with intertrochanteric fracture was higher than that in patients with femoral neck fracture.Conclusion The blood loss was gradually increased in elder patients with intertrochanteric fracture over time.There was a significant difference in different time with respect to blood loss.Moreover,a significant difference was found in blood loss of hidden hemorrhage between intertrochanteric fracture and femoral neck fracture.

Objective To investigate the role of OMA1 in acute kidney injury (AKI) induced by lipopolysaccharide (LPS).Methods OMA1 wild-type and knocked out mice (8 week old) were injected with 10 mg/kg body weight of LPS.The model was confirmed by testing mouse serum creatinine and blood urea nitrogen.The apoptosis in mouse kidney cortex was examined by TUNEL staining and cleaved caspase 3.In vitro,in humam kidney proximal tubular cells (HK2) were knocked down OMA1 by transfecting OMA1 shRNA,with the scramble shRNA being used as negative control of transfection.HK2 cells were cultured with 5 μg/ml of LPS for 24 hours to induce apoptosis.DAPI staining of cells and caspase-3 activity were applied to test apoptosis.The images of mitochondria in cells were obtained by transfection of mito-green plasmid and OMA1 shRNA.Western blotting was used to exam the OMA1 and Cytochrome C expressions.Resudts Compared with OMA1 KO mice,LPS induced more severe AKI of WT mice with higher Scr [(97.2±26.5) μmol/L vs (53.0±17.7) μmol/L,P ＜ 0.05] and BUN [(43.3± 13.7) mmol/L vs (29.7±7.7) mmol/L,P ＜ 0.05].Moreover,there were more apoptosis cells in kidney cortex in WT mice than in OMA1 KO mice [(75.4± 26.1)/ram2 vs (38.3± 14.4)/mm2,P＜ 0.05].About 46％ of OMA1 expressions in HK2 cells were inhibited by OMA1 shRNA transfection (P ＜ 0.05).Further,OMA1 shRNA cells with LPS stimulation had decreased mitochondria fragmentation [(29.8±10.9)％ vs (43.2±6.8)％,P ＜ 0.05],Cytochrome C release [(37.0±12.3)％ vs (76.0±26.2)％,P ＜ 0.05],and cell apoptosis [(13.2±3.9)％ vs (25.0±7.1)％,P ＜ 0.05] as compared with control cells.Conclusion Knockdown of OMA1 alleviated septic AKI through inhibition of cell apoptosis,mitochondria fragmentation,and Cytochrome C release.

Objectives To explore the humoral immunologic mechanisms of the susceptibility to invasive pneumococcal diseases (IPD) in asthmatic children. Methods Plasma samples were collected from 43 asthmatic and 20 non-asthmatic chil-dren. Anit-pneumococcal capsular polysaccharide (PPS)-IgG concentrations were measured by enzyme-linked immunosorbent assays. Results The mean concentrations of anti-PPS 14, 19A and 23F-IgG were signiifcantly higher in asthmatic children than in non-asthmatic children (P<0.05). The ratios of the asthmatic children who had anti-PPS 14, 19A and 23F-IgG concentrations higher than the protective antibody level (≥0.2 μg/ml ) were 100%for all the serotypes. Conclusions The immune responses of producing anti-PPS IgG to defense IPD were normal in asthmatic children. Asthmatic children may be more susceptive to pneumococcal infection or colonization than non-asthmatic children.

Objective:To compare the effect of fluorescence staining and periodic acid-Schiff staining in the diagnosis of fungal keratitis (FK).Methods:A total of 147 corneal specimens from 147 FK patients treated in Eye Hospital of Shandong First Medical University from January 2017 to May 2019 with positive corneal scraping or fungal culture were collected.Among them, there were 84 cases with penetrating keratoplasty (PKP), 42 cases with lamellar keratoplasty (LKP) and 21 cases with lesion resection.Another 11 cases with herpes simplex virus keratitis served as negative control.The corneal tissue specimens were performed with fungal fluorescence staining and periodic acid-Schiff staining, respectively.The stained sections were placed under fluorescence microscope and optical microscope to observe fungal hyphae or spores, respectively.The positive rates of the two staining methods were compared, and the positive cases of the diagnosis of FK in corneal tissue samples obtained by different surgical methods and corneal infection caused by different strains of the two staining methods were compared.Written informed consent was obtained from each patient.The study protocol adhered to the Declaration of Helsinki and was approved by the Ethics Committee of Shandong Eye Hospital (No.SDSYKYY-2016012).Results:The positive rate of periodic acid-Schiff staining and fungal fluorescence staining was 60.5% (89/147) and 79.6% (117/147), respectively.The positive rate of fluorescence staining in the diagnosis of fungal keratitis was significantly higher than that of periodic acid-Schiff staining ( χ2=28.00, P<0.01), and both the specificity of the two staining methods was 100%.The positive rate of specimens obtained by PKP with fluorescent staining was 85.7% (72/84), and the positive rate with periodic acid-Schiff staining was 65.5% (55/84), and the difference was statistically significant ( χ2=17.00, P<0.01). The positive rate of specimens obtained by LKP with fluorescent staining was 71.4% (30/42), and the positive rate with periodic acid-Schiff staining was 52.4% (22/42), and the difference was statistically significant ( χ2=8.00, P<0.01). The positive rate of resected foci specimens with fluorescent staining was 71.4% (15/21), and the positive rate with periodic acid-Schiff staining was 57.1% (12/21), and the difference was not statistically significant ( χ2=1.30, P=0.25). The positive cases of two kinds of staining were different among different fungal strains.Among them, the positive cases of Fusarium solani complex, Pythium insidiosum, Aspergillus fumigatus complex, Candida guilliermondii, Trichoderma and Nigrograna mackinnonii with fluorescence staining were 19, 5, 5, 1, 1 and 1, and the positive cases of periodic acid-Schiff staining were 11, 0, 3, 0, 0 and 0, respectively.The staining results of the 11 negative controls were negative. Conclusions:Fluorescence staining is more sensitive than periodic acid-Schiff staining in the detection of fungal components in paraffin-embedded corneal tissues, and it can significantly improve the fungal detection rates.

Objective To investigate the relationship between noninvasive brachial artery blood pressure (B) and radial artery blood pressure (R) of the right arm.Methods Two hundred and ninetyfive patients with 149 males and age of (47 ± 16) years were studied.The height of patients was 163 ± 8 cm,and weight of patients was (61.2 ± 7.8) kg.The patients with peripheral vascular disease,wounds of arm skin or subcutaneous tissue infection were all excluded.Their B (with adult cuffs) and R (with infant cuffs) of the right arm were measured and analyzed after the patients under general anesthesia and stable hemodynamics.The relationships between B and R were analyzed by linear regression,the differences between B and R of each interval were compared using one-way ANOVA and then followed by SNK procedure.Results Right brachial artery blood pressure was significantly lower than radial artery blood pressure.The differences between the two varied from 13 to 18 mmHg in systolic BP (SBP),diastolic BP (DBP) and mean blood pressure (MAP).And linear regression was most applicable to describe their correlation [r=0.841 (SBP),0.808 (DBP),0.833 (MAP),all P＜0.01].Conclusions Radial artery blood pressure measured with infant cuffs can well reflect the variation of brachial artery blood pressure.