Objective To evaluate the feasibility of testing the high methylation of ppENK gene in stool with methylation-specific polymerase chain reaction (MSP) assay in pancreatic cancer diagnosis.Methods Twenty-four fresh stool samples of pancreatic cancer patients and six healthy control samples were collected.The methylation status of ppENK gene in all the stool samples was detected by MSP assay.The positive rate of wild-type ppENK gene in all the stool samples was determined by polymerase chain reaction (PCR).And 10 non wild-type ppENK gene negative pancreatic cancer samples were collected,and K-ras gene mutation was detected by PCR-restriction fragment length polymorphism (RFLP).The single cell suspension of pancreatic cancer PC3 cell line was added into stool sample from the same healthy individual,the positive rate of ppENK gene methylation was detected by MSP assay.The minimum number of pancreatic cancer cell was calculated when methylation was positive.Results The rate of methylation detection in 30 samples was 0 (0/30); and the rate of non-methylation detection was 10％ (3/30).The rate of wild-type ppENK detection was 6.7％ (2/30).By PCR-RFLP assay,eight were successfully amplified and seven had mutation in 12th code of K-ras gene in 10 selected wild-type ppENK gene negative pancreatic cancer samples.The minimum number of pancreatic cancer cells needed for ppENK methylation band positive detected by MSP was 50 cell/ml.Conclusion Detecting ppENK gene methylation status in stool samples of pancreatic cancer patients by MSP assay has not yet become the method of pancreatic cancer screening and diagnosis.

Objective To design a real-time oxygen generation and supply device for ambulances and to carry out finite element simulated analysis and performance evaluation .Methods Based on the working principles of pressure swing adsorption (PSA) and oxygen pneumatic compression technology ,the structural components and technological processes of an oxygen generation and compression unit as well as an oxygen filling and supply unit were built .The integrated structure of the device was designed by Solidworks .The static structure of the oxygen generation and compression unit was analyzed by ANSYS.Modal analysis of the circuit board was also conducted .The performance of the prototype was evaluated .Results Wtih a stable structure and reliable circuitry , the device could produce and fill oxygen automatically at the flow of 5.0 L/min, concentration of 94.0%, and pressure of 13.0 MPa.Conclusion This device can produce , fill and supply oxygen automatically,and the goal of design is achieved .

Objective To establish a Real-time quantitative PCR method (qPCR) for the detection of diatom UPA barcoding genes and evaluate its application in the drowning diagnosis. Methods The homologous sequences of diatoms UPA gene was obtained by Blast from GeneBank, based on which the universal primers for diatoms were designed. DNA were extracted from 2 common human symbiotic bacteria (Escherichia coli, Bifidobacterium longum), 3 species of planktonic bacteria, 15 species of planktonic algae, tissue samples (lung, liver and kidney) from human cadavers (28 drowning victims, 1 victims by non-drowning in the water, 3 victims deaths on land) in 32 cases. The specificity, sensitivity and repeatability of the designed primers were tested. The positive rates of diatoms detection in the drowning cases were calculated. The results of the real-time quantitative method were evaluated comparatively by Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy (MD-VF-Auto SEM) and PCR-Capillary Electrophoresis (PCR-CE). Results The results showed that the primers UPA99 had strong specificity for the diatomaceae (Synedra radians, Navicula sp., Melosira varians, Cyclotella sp. and Nitzschia sp.) DNA. The melting curve of the amplified product was smooth; the peak was narrow; the melting temperature was (87±1)℃. The sensitivity of qPCR method was 1.56×10-5ng/μL with the detection range of 1.56×102ng/mL~1.56×10-5ng/μL, in contrast with the PCR-CE method (1.56×10-3ng/μL). This real-time PCR method showed high repeatability and stability with the coefficient of variation less than 2%. The detection rate of lung, liver and kidney was 89.3%, 71.4% and 64.3% respectively. Conclusion The established qPCR method, based on the universal primers designed for diatom UPA gene, has high specificity, high sensitivity and good repeatability. With a promising prospect for application, qPCR is suitable for drowning diagnosis.