Objective To explore the method of preparing urine quality control samples in metabonomics research. Methods Urine samples collected from healthy volunteers and cadmium exposure individuals, and quality control samples were analyzed by LC/MS techniques. Then, the total ion currents, the content of endogenous metabolites and principal components were compared. Results Study samples consisted of 40 morning urine samples from female healthy volunteers, 40 morning urine samples from cadmium exposure females, and 18 quality control samples prepared by mixing 80 urine research samples at equal volume from 40 morning urine samples of female healthy volunteers and 40 morning urine samples of cadmium exposure females. The total ion currents of 6 samples randomly selected from healthy volunteers and 6 morning urine samples randomly from cadmium exposure females were disordered, but 6 quality control samples nearly overlapped in EST+/- conditions. The inter-quartile range of endogenous metabolites indicated that contents of quality control samples were significantly different from those of healthy volunteers and cadmium exposure females (both P＜0.05). The score plots of principal component analysis (PCA) showed that urine samples of healthy volunteers and cadmium exposure females distributed dispersedly, but quality control samples concentrated. Except five quality control samples used in balance system, the deviation of the other thirteen quality control samples was kept in 2SD.Conclusion Quality control samples prepared in this study could reflect the basic information of samples and the status of system control, and the method of preparing quality control samples in urine metabonomics research was established.

Objective To construct virulence gene recombinant plasmid vectors in five food-borne pathogenic microorganisms of Escherichia coli O157∶H7, vibrio parahaemolyticus, staphylococcal aureus, salmonella, and shigella, identify their expression products, and explore the feasibility of directly detecting virulence genes of pathogenic microorganism by marking the corresponding antibody with high throughput magnetic fluorescent nanoparticles.Methods Specific virulence genes were selected for primer design from pathogenic islands in five food-borne pathogenic microorganisms, respectively. Firstly, DNA fragments were extracted from five target bacteria and gene fragments were obtained by using PCR and electrophoresis. Then, respective recombinant plasmids, constructed by pET-23a, pET-28a and pET-32a with the gene fragments, were transformed into E?coli DH5α to extract plasmid vectors for enzyme and sequencing identification and transformed into E?coliBL21(DE3). At last, the expression of target proteins were induced by 0.1 mmol/L IPTG and analyzed by SDS-PAGE. Results Recombinant plasmid vectors pET-23a-eaeA, pET-28a-tdh, pET-28a-enterotoxin B, pET-32a-invA and pET-28a-ipaH of E?coli O157:H7, vibrio parahaemolyticus, staphylococcal, salmonella, and shigella were successfully constructed. The eaeA gene fragment of 969 bp, tdh gene fragment of 567 bp, enterotoxin B gene fragment of 798 bp, invA gene fragment of 1 041 bp, and ipaH gene fragment of 771 bp were cloned. The result showed that recombinant plasmid vectors were consistent with target gene sequence. Plasmid expressing proteins 37.5 kDa (eaeA), 26 kDa(tdh), 34.5 kDa (enterotoxin B), 41 kDa (invA), and 32 kDa (ipaH) were detected in E?coliBL21 (DE3) strain. Conclusion The virulence gene recombinant plasmids were successfully constructed in five food-borne pathogenic microorganisms and efficiently expressed in prokaryotic cells, which laid foundation for obtaining corresponding virulence genes of antibodies and diagnostic reagent application.

Objective To develop a duplex fluorescence RT-PCR assay for detecting four pathogenic subtypes of Ebola virus, including the subtypes of Sudan, Zaire, Bendi bujiao and Ivory Coast.Methods Based on the conserved sequence of nucleoprotein (NP) gene of four subtypes of Ebola virus, two sets of primers and probes were designed corresponding to the Sudan/Zaire subtype and to Bendi bujiao/Ivory Coast subtype. Using the Ebola virus RNA of four subtypes transcribed in vitro as templates, the reaction condition were optimized and the specificity, sensitivity, and reproducibility were tested. Then a duplex fluorescence RT-PCR assay was developed. Results The specificity of the method was high on detection the Ebola virus nucleic acid. There were no cross reactions with hemorrhagic fever with renal syndrome virus, Dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus and Chikungunya virus. Detection sensitivities of two sets of primers and probes can reach a minimum of 50 copies/μL. Duplicate detections for three times had better repeatability at four dilutions (2×108, 2×106, 2×104, 2×102 copies/μL) of positive control RNA templates of subtypes of Sudan and Bendi bujiao. Conclusion The duplex fluorescence RT-PCR assay can simultaneously detect the four subtypes of Ebola virus with high sensitivity and specificity, and can be used for detection on suspected cases of Ebola virus infection.

Objective To establish a method for determination of Be, Cr, Cu, As, Cd, Sb, Ba, Hg, Tl, and Pb in hemodialysis water by inductively coupled plasma mass spectrometry (ICP-MS) with octopole reaction system (ORS) for practical requirement. Methods Contents of Be, Cr, Cu, As, Cd, Sb, Ba, Hg, Tl, and Pb in samples of hemodialysis water were determined by ICP-MS, using 45Sc, 89Y, 103Rh, and 175Lu as the internal standard elements. Results Linear relationships of the ten elements were optimal in the range of 0.0-50.0 μg/L. The correlation coefficients were 0.999 5-1.000 0, the limits of detection were between 0.002-0.069 μg/L, the determination limits were 0.007-0.230 μg/L, the relative standard deviation were 0.57%-10.76%, and the spike recoveries were 85.0%-106.9%. Conclusion The method was quick and accurate to determine Be, Cr, Cu, As, Cd, Sb, Ba, Hg, Tl, and Pb in hemodialysis water by ICP-MS with ORS.

Objective To develop an efficient and accurate method for simultaneous determination of multi-residues of 9 β-agonists in meat and liver by using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to investigate the residues in porcine, bovine, and ovine muscles and livers collected from local markets in Shenzhen. Methods Samples (10.0 g) were hydrolyzed by β-glucuronidase /sulfatase at 37 ℃ overnight to free bound β-agonists, extracted by perchloric acid, purified by HLB and MCX cartridges, and reconstituted with a methanol-0.1% formic acid (10+90).The residues of β-agonists were determined by electrospray ionization-trandem mass spectrometry, and quantified by internal standard method by d6-ractopamine, d9-clenbuterol and d3-salbutarnol. Results The linear relationships of the nine target β-agonists were optimal in the range of 0-50 μg/L and the linear correlation coefficient was >0.995. The limits of quantification were 0.5 μg/kg for 9 β-agonists in porcine, bovine, and ovine muscles and livers. Average recoveries were 66.2%-119.4% with relative standard deviations of 0.36%-10.02%. Nine of 720 samples detected by this method were positive, with a detection rate of 1.25%, of which the detection rates were 0.56% (4/720) for ractopamine, 0.42% (3/720) for clenbuterol, and 0.28% (2/720) for phenylethanolamine A. Conclusion This method proved to be quick and accurate for determination of the residues of 9 β-agonists in porcine, bovine and ovine muscle and liver specimens.

Objective To establish solvent desorption gas chromatographic method for determination of methyl methacrylate in the air of workplaces. Methods Air samples containing methyl methacrylate in the workplace were collected with activated carbon tube and desorbed with CS2 or with silica gel tube and desorbed with acetone, and then separated with FFAP capillary columns and detected with flame ionization detector. Results The linear ranges were 0.6-561.6 mg/L for samples collected with activated carbon tube and desorbed with CS2 and 0.6-1 497.6 mg/L for samples collected with silica gel tube and desorbed with acetone. The limits of detection were both 0.6 mg/L. The determination had a good reproducibility. Their relative standard deviations were 1.2%-4.0% and 0.5%-2.5%, respectively. The average desorption efficiencies were 94.2%-97.5% and 97.0%-98.3%. Absorption efficiencies were 99.8%-100.0% and 94.1%-100.0%. The breakthrough volume was above 28.6 mg in 100 mg activated carbon and 2.7 mg in 300 mg silica gel. Samples collected by the two solid adsorbent tubes could at least store 7 days at room temperature. Conclusion The two Methods proved to be suitable for the simultaneous determination of methyl methacrylate in the air of workplaces.

Objective To explore the feasibility of applying zinc protoporphyrin (ZPP) as a screening index of exposure to environmental lead pollution. Methods Blood samples (2-3 mL) were collected from each residents living nearby a battery factory in Zijin County, Guangdong Province. Lead in blood samples was detected by graphite furnace atomic absorption spectrometer and ZPP in blood, by ZPP hematofluorometer. Blood lead levels (BLLs) ≥100 μg/L and ≥400 μg/L were taken as the diagnostic criteria of chronic lead poisoning for children and adults, respectively. Results A total of 946 residents, 174 children aged 1-13 years and 772 adults aged 16-87, were surveyed. The over standard rate of children's BLL was 14.9% (26/174), and the medians of ZPP and BLL of children were 0.780 μmol/L and 47.675 μg/L, respectively. ZPP contents in blood of children showed no correlation with their BLLs (P>0.05). The over standard rate of BLL of adults was 9.5%, and the medians of ZPP and BLL of adults were 0.740 μmol/L and 69.572 μg/L, respectively. ZPP contents in blood of adults showed a weak correlation with their BLLs (r=0.344, P<0.05). ZPP contents of children with high BLL (≥100 μg/L) correlated with their BLLs (r=0.530，P<0.05), while ZPP contents of children with low BLL (<100 μg/L) showed no correlation with their BLLs (P>0.05). ZPP contents of adults with both high BLLs (≥400 μg/L) and low BLLs (<400 μg/L) showed positive correlations with their BLLs (r=0.566 and 0.142, respectively; P<0.05 for both). Conclusion ZPP could be used as a screening index of exposure to environmental lead population for adults or children with high BLL.

Objective To explore pathogenic characteristics of Vibrio parahaemolyticus in Dongguan City, to provide experimental basis for disease control and prevention. Methods According to GB 4789.7-2013, forty-three strains of Vibrio parahaemolyticus, isolated from food monitoring, patients with food poisoning and sporadic patients in 2012 and 2013, were tested for biochemistry and serological identification. The drug resistance to 12 antibiotics was tested by K-B method. tdh and trh genes of the strains were detected by real-time PCR and digested by restriction endonuclease Not I. Then, molecular typing was performed by the pulsed field gel electrophoresis (PFGE). Results The main serotype of the strains isolated from patients was O3∶K6, accounting for 83.3% of all patients (15/18), while serotype of 25 strains isolated from food showed diversification, without predominant serotype. The drug resistance of strains had low sensitivity to ampicillin (sensitive rate, 0.0%, 0/43) and keflin (sensitive rate, 20.9%, 9/43), while high sensitivity to ciprofloxacin, guanimycin and imipenem (sensitive rate, 100.0%, 43/43). Strains isolated from patients carried tdh gene, while the strains isolated from food had neither tdh nor trh gene. The result of PFGE indicated that the bands of strains isolated from patients were relatively concentrated, while the bands of the strains from food were relatively disperse. Strains of the same serotype were highly homologous and PFGE type was found to be consistent with different patients with food poisonings. Conclusion The strains isolated from food had disperse bands and low pathogenicity and those from patients had high homology and pathogenicity.

Objective To characterize O3∶K6 serotype of V. parahaemolyticus isolated from cases of food poisoning during 2006-2012 in Nanshan District, Shenzhen. Methods Strains of V. parahaemolyticus serotype O3∶K6 were collected. Multi-PCR, pulsed field gel electrophoresis (PFGE), and Kirby-Bauer were performed. Results A total of 44 strains of V. parahaemolyticus (O3∶K6) obtained from diarrhea patients with food poisoning were collected. All strains were tdh gene positive and trh gene negative. PFGE indicated that the 44 strains could be divided into 2 clone complexes. Clone 1 (C1) had 19 strains, with 17-20 bands for each strain, while clone 2 (C2) had 25 strains, with 17-18 bands for each one. Differences between C1 and C2 were found in position of 200, 190, and 100 Kb, while the other bands were similar. All the 44 strains (100.00%) were sensitive to cefrazidime, cefepime，gentamicin，levoflocacin，tetracycline，chloramphenicol, and sulphamethoxazole, 72.73% (32/44), 18.18% (8/44), and 18.18% (8/44) of strains were resistant to ampicillin, kanamycin, and streptomycin, and 9.09% (4/44), 65.91% (29/44), and 65.91% (29/44) of strains were moderately sensitive to the three antibiotics, respectively. Conclusion The main PFGE clone complexes of strains of V.parahaemolyticus serotype O3∶K6 isolated in the local area were C1 and C2, with tdh gene positive and trh gene negative.