Objective Based on dual channel melting curve analysis-based assay,we developed a method to rapidly detect the drug-resistant mutations in Mycobacterium tuberculosis through real-time PCR.Methods According to the common first-line drug-resistant mutations of Mycobacterium tuberculosis,we designed six dual-labeled fluorescence probes to rapidly detect the drug-resistant mutations through realtime PCR melting curve after amplifications of drug-resistant related gene region of DNA.The targets include rpoB 81 bp core region,katG315,inhA promoter,ahpC promoter and embB306.To validate the sensitivity and specificity of our method,we performed real-time PCR assays to detect drug-resistant mutations in 76 clinical MDR-TB samples,which were collected by Shanghai CDC in 2008.Results In the validation,this method successfully detected drug-resistant mutations in all 76 clinical MDR-TB samples.The △Tm of mutations were from 1.8 to 14.4 ℃.Comparing with the sequencing data,all mutations covered by the six probes were detected with 100％ sensitivity and 100％ specificity (rpoB,80/80; inhA,7/7 ; katG315,59/ 59;ahpC,8/8;embB306,27/27).This method can successfully detect drug-resistant mutations from 100 copies/μl DNA samples.Conclusions A widely applicable real-time PCR assay to detect first line drug-resistant mutations of Mycobacterium tuberculosis has benn developed.This method has proven to have the advantages of high sensitivity,specificity and low risk of contamination.It can be used in rapid diagnosis of clinical drug-resistant tuberculosis and the evaluation of laboratory drug sensitivity test.

Objective:To study the effect of invigorating the kidney and spleen complex prescription on deltamethrin intoxicated pregnant rats. Methods:Pregnant rats were divided into 5 groups. Except the blank group,other groups were orally fed 1/20LD50(6.93mg/kg)DM daily from day 1 to 15 when proved pregrancy. 4 Hours later,rats in model group were given physiological saline,other 3 groups were given high/ median /low dose of Zhuyun Formula. Half pregnant rats were killed on D15. The rest was kept until labour. The abortion rate,survival rate,delivery condition,the level of estradiol,progestogen were observed. Results:With respect to abortion rate,model group showed statistical signifi cance when compared with other groups. Model group also showed low progesterone level. With the treatment Zhuyun Ⅲ,it had desirable effect in decreasing abortion rate,and increasing the level of progesterone. The labour time of all groups was within the normal range. Conclusion:DM had toxicity on reproduction,it could lead to abortion which was related to endocrine function of pregnant rats. Zhuyun Ⅲ could regulate the above condition and relief DM toxicity.

Objective To evaluate the saline and ethanol for effects of disinfecting penicillin skin test.Methods Searching CNKI,VIP,CBM and Wanfang Data to collect case-control studies suiting enrolled conditions.Meta-analysis was conducted using Revman 5.0.0 software.Results Five case-control studies with 9 481 patients were enrolled.Meta-analysis demonstrated there was significant difference between saline and ethanol in penicillin skin test disinfection.Conclusions Saline has lower positive results than ethanol,and can better represent the result of penicillin skin test.

Objective To understand the changes of positive rates of IHA detections of outpatients in schistosomiasis clinic. Results The data of IHA detections of outpatients in schistosomiasis clinic in Hubei Provincial Center for Disease Control and Prevention were collected and analyzed statistically from 2005 to 2014. Results A total of 7 113 outpatients were detected by IHA test,and 547 of them were positives with a positive rate of 7.69%. The positive rate of IHA test was on an upward slope be?fore 2008,and the rate reached 14.85% in 2008,which was significantly higher than that in 2005(5.81%)( χ2 = 47.40,P<0.01),then it was on a declined stage after 2008,and the positive rate decreased to 3.76 in 2014,which was significantly lower than that in 2008( χ2 = 12.29,P<0.01). The positive rate of outpatients in the 10~<30 years age group was higher than those in other age groups(all P < 0.012 5),and the male positives were more than the female ones. Conclusions The schisto?somiasis endemic situation has been significantly decreased in Hubei Province. The male and people in 10~<30 age group are the high risk groups,so the targeted health education should be strengthened.

Objective To grasp bilirubin in the quality control serum and calibration items in the same batch the variation law of the designed to ensure that the test quality and the use of reasonable quality control serum and calibration items ,control testing cost .Methods laboratories use The double level quality control serum and calibration items after dilution is divided into five groups ,with automatic biochemical analyzer test for 12 weeks and recorded results of TBIL ,DBIL .use SPSS 17 .0 to calculate the mean ,standard deviation ,coefficient of variation ,Normal distribution analysis and make the results trend line chart to observe any changes .Results RANDOX normal concentration quality control serum TBIL and DBIL values variation RCV% 26 .0% ,48 .2% , more than CLIA bilirubin projects allow 1/3 of the total error value(6 .67% ) .Normal distribution test ,P value was 0 .006 ,0 .012 , less than 0 .05 ,do not obey the normal distribution ,test results line chart is on the decline .LEADMAN high concentration quality control serum and bilirubin calibration items TBIL and DBIL variation RCV% <6 .67% ,normal distribution test all P>0 .05 ,obey the normal distribution ,test results no trend line chart .Conclusion Through the experiment observation to the quality control ser‐um and calibration items bilirubin in the variation law of 12 weeks .Experimenter can be used according to the actual situation to ad‐just ,not only to ensure the quality of the bilirubin test ,and to get the most out of the quality control serum and calibration items use efficiency ,reduce the test cost .

Objective To investigate the appropriate time of replacing drainage bags after trans-cervical resection of adhesion(TCRA) surgery.Methods Totally 156 patients underwent TCRA were randomly divided into three groups:Group A,the drainage bags were replaced daily;Group B,the drainage bags were replaced every 3 days;Group C,the drainage bags were never replaced.The drainage bags were removed on the 6th day after TCRA for all groups.Bacterial culture results from the balloon surface of drainage bag,outer cervical orifice,and the connector of drainage bag for 3 groups were observed.The positive rates of bacterial cultures were analyzed statistically.Results The bacterial culture results from the drainage bag connector of 3 groups indicated that the positive rate of Group C was significantly lower than that of other two groups(P＜0.05).The bacterial culture from the drainage bag balloon surface also indicated the same result,but the result did not showed statistically significant difference(P＞0.05).There was no statistically significant difference in the culture result firom outer cervical orifice and the volume of drainage fluid within three groups(P＞0.05).Conclusion It is indicated that the patient may enjoy a lower rate of bacterial infection without replacing any drainage bags connected to balloon drainage tubes placed after TCRA within 6 days.We suggest that the intrauterine drainage hags may be kept without replacement until direct removing the drainage tube on day 6 after TCRA.

Objective:To construct a RNAi lentiviral vector targeting rat CD80 gene and detect its effect of gene silencing in NRK and IEC6 cells.Methods:The effective sequence of siRNA targeting rat CD80 gene was confirmed in our previous work.Oligo-DNA fragment containing short hairpin frame was synthesized and reannealed,and then cloned into pGCSIL-GFP lentiviral expression vector.PCR and sequencing analysis were made for verifying the positive clones.The virus packaging plasmids were transfected into 293T cells to harvest shRNA lentivirus.After infection in NRK and IEC6 cells,Real-time PCR was performed to determine the expressing level of CD80.Results:PCR and sequencing revealed that shRNA plasmids was correctly constructed.Virus with a titer of 4×10~8 TU/ml was successfully packaged.CD80 expression in NRK and IEC6 cells could be knockdown by virus infection as characterized by 66.9% and 63.5% decrease of CD80 mRNA in NRK and IEC6 cells respectively,compared with negative control lentivirus.Conclusion:The recombinant lentiviral shRNA expressing vector targeting rat CD80 gene has been successfully constructed and packaged.CD80 mRNA could be down-regulated availably in NRK and IEC6 cells.

Objective To study the expression and clinical significance of phosphorylated AKT (pAKT) and phosphatase and tensin homolog (PTEN) in colorectal carcinoma tissues.Methods The expression of pAKT and PTEN were detected by immunohistochemical SP method in 112 case of colorectal carcinomas tissue.Results The positive rates of pAKT and PTEN expression were 79.5 ％ (89/112) and 47.3 ％ (53/112) in colorectal cancerous tissues,respectively,which showed a statistically significance when compared with those in adjacent normal and adenoma tissues.The positive rate of pAKT expression was closely related with the invasive depth,clinical staging and lymph node metastasis of colorectal carcinomas.The positive expression of PTEN was also closely related with the invasive depth,degree of differentiation,clinical staging and lymph node metastasis of colorectal carcinomas.The expressions of pAKT and PTEN were negative correlaed.Conclusion The results showed that the expression of pAKT and PTEN were closely related with progression and metastasis of colorectal carcinomas,which may provide a new therapeutic target for colorectal carcinomas by blocking PTEN/PI3K/AKT signaling pathway.

Objective: To explore automatic breast full volume imaging (ABVS) and color doppler ultrasound (CDFI) for breast cancer neoadjuvant chemotherapy (NCT) in the evaluation of curative effect. Methods:The application of a CDFI and ABVS 42 cases of breast cancer patients receiving NCT lesions imaging characteristics before and after observation and analysis. Results:ABVS from coronal distribution and audio-visual change according to the primary tumor, and CDFI images showing the primary tumors and blood flow changes within all have significant difference in patients with complete remission after NCT (P<0.01);ABVS from coronal distri-bution and audio-visual change according to the primary tumor, and CDFI showed lesions in blood flow changes, etc. in patients with the absence of change and easing some lesions have no significant difference (P>0.05). Conclusion:We use ABVS and CDFI for breast cancer before and after the NCT curative effect evaluation of great clinical significance and value.

Objective To analyze the value of abnormal air bronchogram for predicting the invasiveness of lung adenocarcinoma with pure ground-glass nodule (pGGN).Methods From April 2014 to February 2016,157 patients with 165 pGGN lung adenocarcinomas confirmed by surgical pathology were selected.There were 30 pre-invasive lesions (AAH+AIS),39 minimally invasive adenocarcinoma (MIA),and 96 invasive adenocarcinoma (IAC).CT characteristics including lesion size,density,abnormal air bronchogram were recorded.All lesions were divided into two groups:normal group (no air bronchogram or normal air bronchogram) and abnormal air bronchogram group.Chi-square test was used to analyze the difference of pathological subtypes between the two groups.Mann-Whitney rank test was used to analyze the size difference of pGGN between the two groups.Two-independent samples t-test was used to analyze the lesion density difference of pGGN between the two groups.Results Of the 165 lesions,85 were found to have air bronchogram,there were 12 lesions in 30 pre-invasive lesions (AAH+AIS),17 in 39 MIAs,56 in 96 IACs.Abnormal air bronchogram were demonstrated in 61 lesions which was 1 in 30 pre-invasive lesions (IACs+AIS),13 in 39 MIAs and 47 in 96 IACs,significant differences were found between two groups (x2=25.943,P＜0.01).The mean size of the IACs were (10.8±4.2) mm for normal group,(17.0±6.7) mm for abnormal air bronchogram group,the mean density were (-519± 118) HU and (-518± 124) HU,respectively.There was a significant difference in lesion diameter between two groups (Z=-6.197,P＜0.01),but not in density (t=-0.042,P=-0.966).Conclusions Abnormal air bronchogram can be used to predict the invasiveness of pGGN lung adenocarcinoma,and is correlated with lesion size,but not with density.