[Development of a high-throughput suspension microarray technology for detection of three kinds of veterinary drug residues: chloramphenicol, clenbuterol and 17-beta-estradiol].

OBJECTIVE: To establish a novel suspension microarray technology for the detection of three kinds of veterinary drug residues: chloramphenicol, clenbuterol and 17-beta-estradiol (CAP, CL and E2). METHODS: The three conjugates that veterinary drug coupled with bovine serum albumin (BSA) were synthesized and identified by ultraviolet (UV) spectrophotometry and mass spectrum. The veterinary drug conjugates were immobilized on the polystyrene fluorescent microspheres/beads. There were competitive reactions between the veterinary drugs in the aqueous phase and that on the beads for combination with their specific biotinylated monoclonal antibodies. The optimum amount of the veterinary drug conjugates and the antibodies were optimized and selected. The detective standard curves were plotted. The specificity and the unknown samples were also determined by grouping according to different concentrations of the interferes and the samples. Meantime, the different microstructures of the surfaces of the beads were also observed by scanning electron microscope. RESULTS: Couplings were completed between small molecular veterinary drugs and BSA. The amounts of the three conjugates and the antibodies were optimized. The detective standard curves of the suspension array and their corresponding coefficients of determination (R2) were good (R2 > 0.99). The detection ranges of the three veterinary drugs were (40.00 - 6.25) x 10(5) ng/L, (50.00-7.81) x 10(5) ng/L and 1.00 x 10(3) - 7.29 x 10(5) ng/L respectively. Simultaneously, the specific detection of the suspension microarray was excellent and did not indicate significant cross-reactions. Errors between the found and the real are in the range of 8.09% - 17.03%. It can be considered that the relative standard deviations were relatively small. Successful couplings were also directly confirmed by the observation for microstructures of the surfaces of the beads by scanning of electron microscope and laid good foundation for the following responses. CONCLUSION: The high-throughput suspension microarray should provide a novel method for multi-analysis of the veterinary drugs and have a wide applicative prospects with simple operation, sensitive, rapid and low cost.

Oxidative damage to lung tissue and peripheral blood in endotracheal PM2.5-treated rats.

OBJECTIVE: To investigate the oxidative damage to lung tissue and peripherial blood in PM2.5-treated rats. METHODS: PM2.5 samples were collected using an auto-sampling instrument in summer and winter. Treated samples were endotracheally instilled into rats. Activity of reduced glutathione peroxidase (GSH-Px) and concentration of malondialdehyde (MDA) were used as oxidative damage biomarkers of lung tissue and peripheral blood detected with the biochemical method. DNA migration length (microm) and rate of tail were used as DNA damage biomarkers of lung tissue and peripheral blood detected with the biochemical method. RESULTS: The activity of GSH-Px and the concentration of MDA in lung tissue significantly decreased after exposure to PM2.5 for 7-14 days. In peripheral blood, the concentration of MDA decreased, but the activity of GSH-Px increased 7 and 14 days after experiments. The two indicators had a dose-effect relation and similar changing tendency in lung tissue and peripheral blood. The DNA migration length (microm) and rate of tail in lung tissue and peripheral blood significantly increased 7 and 14 days after exposure to PM2.5. The two indicators had a dose-effect relation and similar changing tendency in lung tissue and peripheral blood. CONCLUSION: PM2.5 has a definite oxidative effect on lung tissue and peripheral blood. The activity of GSH-Px and the concentration of MDA are valuable biomarkers of oxidative lung tissue damage induced by PM2.5. The DNA migration length (microm) and rate of tail are simple and valuable biomarkers of PM2.5-induced DNA damage in lung tissues and peripheral blood. The degree of DNA damage in peripheral blood can predict the degree of DNA damage in lung tissue.

Directed molecular evolution of nitrite oxido-reductase by DNA-shuffling.

OBJECTIVE: To develop directly molecular evolution of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremely slow and are unable to nitrify effectively inorganic nitrogen in wastewater treatment. METHODS: The norB gene coding the ndtrite oxido-reductase in nitrobacteria was cloned and sequenced. Then, directed molecular evolution of nitrite oxido-reductase was developed by DNA-shuffling of 15 norB genes from different nitrobacteria. RESULTS: After DNA-shuffling with sexual PCR and staggered extension process PCR, the sequence was different from its parental DNA fragments and the homology ranged from 98% to 99%. The maximum nitrification rate of the modified bacterium of X16 by DNA-shuffling was up to 42.9 mg/L x d, which was almost 10 times higher than that of its parental bacteria. Furthermore, the modified bacterium had the same characteristics of its parental bacteria of E. coli and could grow rapidly in normal cultures. CONCLUSION: DNA-shuffling was successfully used to engineer E. coli, which had norB gene and could degrade inorganic nitrogen effectively.

Development and application of an oligonucleotide microarray for the detection of food-borne bacterial pathogens.

The rapid and accurate detection and identification of food-borne pathogenic bacteria is critical for food safety. In this paper, we describe a rapid (<4 h) high-throughput detection and identification system that uses universal polymerase chain reaction (PCR) primers to amplify a variable region of bacterial the 16S rRNA gene, followed by reverse hybridization of the products to species-specific oligonucleotide probes on a chip. This procedure was successful in discriminating 204 strains of bacteria from pure culture belonging to 13 genera of bacteria. When this method was applied directly to 115 strains of bacteria isolated from foods, 112/115 (97.4%) were correctly identified; two strains were indistinguishable due to weak signal, while one failed to produce a PCR product. The array was used to detect and successfully identify two strains of bacteria from food poisoning outbreak samples, giving results through hybridization that were identical to those obtained by traditional methods. The sensitivity of the microarray assay was 10(2) CFU of bacteria. Thus, the oligonucleotide microarray is a powerful tool for the detection and identification of pathogens from foods.

Inhibition of hepatitis B virus by D-fraction from Grifola frondosa: synergistic effect of combination with interferon-alpha in HepG2 2.2.15.

In this study, D-fraction extracted from Grifola frondosa (GF-D) and its combination with human interferon alpha-2b (IFN) were investigated for the inhibitory effect on hepatitis B virus (HBV) in HepG2 2.2.15 cells (2.2.15 cells). HBV DNA and viral antigens were analyzed by a quantitative real-time polymerase chain reaction and end-point titration in radioimmunoassays, respectively. The results showed that GF-D or IFN alone could inhibit HBV DNA in 2.2.15 cells with the 50% inhibitory concentration (IC50) of 0.59 mg/ml and 1399 IU/ml, respectively. We further investigated the combination of GF-D and IFN for anti-HBV activity and found that they synergistically inhibited HBV replication in 2.2.15 cells. In combination with 0.45 mg/ml GF-D, the apparent IC50 value for IFN was 154 IU/ml. This 9-fold increase in antiviral activity of IFN suggested that GF-D could synergize with IFN. These results indicate that GF-D, in combination with IFN, might provide a potentially effective therapy against chronic HBV infections.

Effects of formaldehyde inhalation on lung of rats.

OBJECTIVE: To analyze protein changes in the lung of Wistar rats exposed to gaseous formaldehyde (FA) at 32-37 mg/m3 for 4 h/day for 15 days using proteomics technique. METHODS: Lung samples were solubilized and separated by two-dimensional electrophoresis (2-DE), and gel patterns were scanned and analyzed for detection of differently expressed protein spots. These protein spots were identified by MALDI-TOF-MS and NCBInr protein database searching. RESULTS: Four proteins were altered significantly in 32-37 mg/m3 FA group, with 3 proteins up-regulated, 1 protein down-regulated. The 4 proteins were identified as aldose reductase, LIM protein, glyceraldehyde-3-phosphate dehydrogenase, and chloride intracellular channel 3. CONCLUSION: The four proteins are related to cell proliferation induced by FA and defense reaction of anti-oxidation. Proteomics is a powerful tool in research of environmental health, and has prospects in search for protein markers for disease diagnosis and monitoring.

Excretion and detection of SARS coronavirus and its nucleic acid from digestive system.

AIM: To study whether severe acute respiratory syndrome coronavirus (SARS-CoV) could be excreted from digestive system. METHODS: Cell culture and semi-nested RT-PCR were used to detect SARS-CoV and its RNA from 21 stool and urine samples, and a kind of electropositive filter media particles was used to concentrate the virus in 10 sewage samples from two hospitals receiving SARS patients in Beijing in China. RESULTS: It was demonstrated that there was no live SARS-CoV in all samples collected, but the RNA of SARS-CoV could be detected in seven stool samples from SARS patients with any one of the symptoms of fever, malaise, cough, or dyspnea, in 10 sewage samples before disinfection and 3 samples after disinfection from the two hospitals. The RNA could not be detected in urine and stool samples from patients recovered from SARS. CONCLUSION: Nucleic acid of SARS-CoV can be excreted through the stool of patients into sewage system, and the possibility of SARS-CoV transmitting through digestive system cannot be excluded.

Concentration and detection of SARS coronavirus in sewage from Xiao Tang Shan Hospital and the 309th Hospital.

The transmission of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is associated with close contact to SARS patients and droplet secretions of those patients. The finding of positive RT-PCR results from stools of SARS patients suggests that stools of SARS patients or sewage containing stools of patients could transmit SARS-CoV. We used a novel style of electropositive filter media particle to concentrate the SARS-CoV from the sewage of two hospitals receiving SARS patients in Beijing. We also used cell culture, RT-PCR and gene sequencing to detect and identify the viruses from sewage. No infectious SARS-CoV contamination was found in any of the samples collected, but the nucleic acid of SARS-CoV could be detected in the sewage from the two hospitals before disinfection. While the RNA was only detected in three samples from the 309th Hospital, the others were negative after disinfection. These findings provide strong evidence that SARS-CoV can be excreted through the stool/urine of patients into sewage system, thus making the sewage system a possible route of transmission.

Preparation of immunomagnetic iron-dextran nanoparticles and application in rapid isolation of E.coli O157:H7 from foods.

AIM: To prepare a kind of magnetic iron-dextran nanoparticles that was coated with anti-E.coli O157:H7 IgG, analyze its application conditions, and try to use it to isolate E.coli O157:H7 from foods. METHODS: Magnetic iron-dextran nanoparticles were prepared by the reaction of a mixture of ferric and ferrous ions with dextran polymers under alkaline conditions. The particles were coated with antiserum against E.coli O157:H7 by the periodate oxidation-borohydride reduction procedure. The oxidation time, amount of antibody coating the particles, amount of nanoparticles, incubation time and isolation time were varied to determine their effects on recovery of the organisms. Finally, the optimum conditions for isolating E.coli O157:H7 from food samples were established. RESULTS: E.coli O157:H7 can be isolated from samples within 15 min with the sensitivity of 10(1) CFU/mL or even less. In the presence of 10(8) CFU/mL of other organisms, the sensitivity is 10(1)-10(2) CFU/mL. Nonspecific binding of other bacteria to the particles was not observed. Two and a half hours of enrichment is enough for the particles to detect the target from the food samples inoculated with 1 CFU/g. CONCLUSION: Isolation of target bacteria by immunomagnetic nanoparticles is an efficient method with high sensitivity and specificity. The technique is so simple that it can be operated in lab and field even by untrained personnel.

8-hydroxydeoxyguanosine as a biomarker of oxidative DNA damage induced by environmental tobacco side-stream smoke and its mechanism.

OBJECTIVE: To study the genotoxicity effect of environmental tobacco side-stream smokes (ETSS) on oxidative DNA damage and its molecular mechanism. METHODS: DNA adduct 8-hydroxydeoxyguanosine (8-OHdG) was used as a biomarker of oxidative DNA damage. The level of 8-OHdG in DNA exposed to ETSS was detected by high performance liquid chromatography with electrochemical detection. Organic and inorganic components in ETSS were analyzed by gas chromatography-mass spectrum and atomic absorption spectrum respectively. RESULTS: Particle matters (PMs) and volatile organic compounds (VOCs) in ETSS could directly induce oxidative DNA damage and formation of 8-OHdG. There were 123 and 84 kinds of organic components in PMs and VOCs respectively, and 7 kinds of inorganic components in ETSS. Some components, especially quinones and polyphenols in ETSS, could produce free radicals in vitro by auto-oxidation without any biological activity systems, and with the catalytic reaction of metals, the DNA adduct 8-OHdG was produced. CONCLUSION: ETSS have biological oxidative effect on DNA in vitro and in vivo, and expressed direct genotoxicity. 8-OHdG is a valuable biomarker of oxidative DNA damage.

Study on the resistance of severe acute respiratory syndrome-associated coronavirus.

In this study, the persistence of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) was observed in feces, urine and water. In addition, the inactivation of SARS-CoV in wastewater with sodium hypochlorite and chlorine dioxide was also studied. In vitro experiments demonstrated that the virus could only persist for 2 days in hospital wastewater, domestic sewage and dechlorinated tap water, while 3 days in feces, 14 days in PBS and 17 days in urine at 20 degrees C. However, at 4 degrees C, the SARS-CoV could persist for 14 days in wastewater and at least 17 days in feces or urine. SARS-CoV is more susceptible to disinfectants than Escherichia coli and f2 phage. Free chlorine was found to inactivate SARS-CoV better than chlorine dioxide. Free residue chlorine over 0.5 mg/L for chlorine or 2.19 mg/L for chlorine dioxide in wastewater ensures complete inactivation of SARS-CoV while it does not inactivate completely E. coli and f2 phage.

Detection and identification of intestinal pathogenic bacteria by hybridization to oligonucleotide microarrays.

AIM: To detect the common intestinal pathogenic bacteria quickly and accurately. METHODS: A rapid (<3 h) experimental procedure was set up based upon the gene chip technology. Target genes were amplified and hybridized by oligonucleotide microarrays. RESULTS: One hundred and seventy strains of bacteria in pure culture belonging to 11 genera were successfully discriminated under comparatively same conditions, and a series of specific hybridization maps corresponding to each kind of bacteria were obtained. When this method was applied to 26 divided cultures, 25 (96.2%) were identified. CONCLUSION: Salmonella sp., Escherichia coli, Shigella sp., Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Proteus sp., Bacillus cereus, Vibrio cholerae, Enterococcus faecalis, Yersinia enterocolitica, and Campylobacter jejuni can be detected and identified by our microarrays. The accuracy, range, and discrimination power of this assay can be continually improved by adding further oligonucleotides to the arrays without any significant increase of complexity or cost.

[Detection of RNA of SARS coronavirus in hospital sewage].

OBJECTIVE: In order to explore the existence of SARS coronavirus (Co-V) and/or its RNA in sewage of hospitals administered SARS patients. METHODS: A novel electropositive filter was used to concentrate the SARS-CoV from the sewage of two hospitals administered SARS patients in Beijing, including twelve 2,500 ml sewage samples from the hospitals before disinfection, and ten 25,000 ml samples after disinfection; as well as cell culture, RT-PCR and sequencing of gene to detect and identify the viruses from sewage. RESULTS: There was no live SARS-CoV detected in the sewage in this study. The nucleic acid of SARS-CoV had been found in the 12 sewage samples before disinfection from both hospitals by semi-nested PCR. After disinfection, SARS-CoV RNA could only be detected from the samples from the 309th Hospital, and the others were negative. CONCLUSION: It provides evidence that there is no live SARS-Cov in the sewage from hospitals with SARS patients though SARS-CoV RNA can be detected.

[The effects of DNA damage induced by acetaldehyde].

The effects of DNA damage induced by the typical environmental pollutant acetaldehyde were studied with single cell gel electrophoresis (SCGE) and high performance liquid chromatography with electrochemical detection (HPLC-EC). The results showed that acetaldehyde not only could cause DNA strand breakage but also DNA-DNA, DNA-protein crosslinks of lymphocytes of human peripheral blood. The reaction of acetaldehyde with DNA in vitro was weak, but the oxidative ability was enhanced and the reaction could produce a number of 8-OHdG adducts mediated by the Fe2+. The animal experiment shows that acetaldehyde can cause the oxidative DNA damage of rat lung tissues, which suggests that acetaldehyde have the potential genotoxicity and its chemical mechanism is relative to the crosslinks and oxidation with DNA.

A study on detecting and identifying enteric pathogens with PCR.

OBJECTIVE: To develop a rapid and definite diagnostic test of bacterial enteritis caused by pathogenic enterobacteria, the most frequent etiologic agent of infectious enteritis in the world. METHODS: A set of conventional PCR assays were applied to detect and identify salmonella, shigella, and E. coli O157:H7 directly from pure culture and fecal samples. The general primers of pathogenic enterobacteria were located on the uidA gene, which were found not only in E. coli nuclear acid, but also in shigella and salmonella genes. Shigella primer was from ipaH gene whose coded invasive plasmid relative antigen existed both in plasmid and in genome. The primers of salmonella were designed from the 16SrRNA sequence. The primer of E. coli O157:H7 was taken from eaeA gene. Five random primers were selected for RAPD. The detection system included common PCR, semi-nested PCR and RAPD. RESULTS: This method was more sensitive, specific and efficient and its processing was rapid and simple. For example, the method could be used to specifically detect and identify salmonella, shigella, and E. coli O157:H7, and its sensitivity ranged from 3 to 50 CFU, and its detection time was 4 hours. CONCLUSION: This PCR method, therefore, can serve as a routine and practical protocol for detecting and identifying pathogenic microorganisms from clinical samples.

Mechanisms of inactivation of hepatitis A virus in water by chlorine dioxide.

In this study, to elucidate the mechanisms of inactivation of hepatitis A virus (HAV) by chlorine dioxide, cell culture, enzyme-linked immunosorbent assay (ELISA), and long-overlapping RT-PCR were used to detect the infectivity, antigenicity, and entire genome of HAV before and after disinfection. The results revealed the complete inactivation of infectivity after a 10-min exposure to 7.5mg of chlorine dioxide per liter; and the highest level of sensitivity in the 5'non-translated regions (5'NTR) (the sequence from bp 1 to 671), inactivation of which took as much time as the inactivation of infectivity of HAV by chlorine dioxide; the complete destruction of antigenicity after a 10-min exposure to 7.5mg of chlorine dioxide per liter. It is suggested that the inactivation mechanism of HAV by chlorine dioxide was due to the loss of the 5'NTR and/or destruction of the antigenicity, which is not similar to that of chlorine (Appl Environ Microbiol 68: 4951).

[Attenuation of chronic stress-induced hippocampal damages following physical exercise].

The long-term potentiation (LTP) in the hippocampal dentate gyrus and the plasma glucocorticoids level were observed in rats to study the effects of physical exercise on chronic stress-induced hippocampal damages. Eight-week spontaneous wheel running exercise could attenuate the suppression of LTP induced by 21-day restraint stress, and maintain the normal plasma glucocorticoids levels. It is suggested that long-term physical exercise may protect the hippocampus from stress-induced damages.

Mechanisms of inactivation of hepatitis a virus by chlorine.

The study was intended to investigate the feasibility of reverse transcription-PCR (RT-PCR) for evaluation of the efficacy of inactivation of viruses in water and to elucidate the mechanisms of inactivation of hepatitis A virus (HAV) by chlorine. Cell culture, enzyme-linked immunosorbent assay, and long-overlap RT-PCR were used to detect the infectivity, antigenicity, and entire genome of HAV inactivated or destroyed by chlorine. The cell culture results revealed the complete inactivation of infectivity after 30 min of exposure to 10 or 20 mg of chlorine per liter and the highest level of sensitivity in the 5' nontranslated regions (5'NTR), inactivation of which took as much time as the inactivation of infectivity of HAV by chlorine. However, antigenicity was not completely destroyed under these conditions. Some fractions in the coding region were resistant to chlorine. To determine the specific region of the 5'NTR lost, three segments of primers were redesigned to monitor the region from bp 1 to 1023 across the entire genome. It was shown that the sequence from bp 1 to 671 was the region most sensitive to chlorine. The results suggested that the inactivation of HAV by chlorine was due to the loss of the 5'NTR. It is believed that PCR can be used to assess the efficacy of disinfection of HAV by chlorine as well as to research the mechanisms of inactivation of viruses by disinfectants.

Detection of enteroviruses and hepatitis A virus in water by consensus primer multiplex RT-PCR.

AIM: To develop a rapid detection method of enteroviruses and Hepatitis A virus (HAV). METHODS: A one-step, single-tube consensus primers multiplex RT-PCR was developed to simultaneously detect Poliovirus, Coxsackie virus, Echovirus and HAV. A general upstream primer and a HAV primer and four different sets of primers (5 primers) specific for Poliovirus, Coxsacki evirus, Echovirus and HAV cDNA were mixed in the PCR mixture to reverse transcript and amplify the target DNA. Four distinct amplified DNA segments representing Poliovirus, Coxsackie virus, Echovirus and HAV were identified by gel electrophoresis as 589-,671-, 1084-, and 1128bp sequences, respectively. Semi-nested PCR was used to confirm the amplified products for each enterovirus and HAV. RESULTS: All four kinds of viral genome RNA were detected, and producing four bands which could be differentiated by the band size on the gel. To confirm the specificity of the multiplex PCR products, semi-nested PCR was performed. For all the four strains tested gave positive results. The detection sensitivity of multiplex PCR was similar to that of monoplex RT-PCR which was 24 PFU for Poliovrus,21 PFU for Coxsackie virus,60 PFU for Echovirus and 105 TCID(50) for HAV. The minimum amount of enteric viral RNA detected by semi-nested PCR was equivalent to 2.4 PFU for Poliovrus, 2.1 PFU for Coxsackie virus, 6.0 PFU for Echovirus and 10.5 TCID(50) for HAV. CONCLUSION: The consensus primers multiplex RT-PCR has more advantages over monoplex RT-PCR for enteric viruses detection, namely, the rapid turnaround time and cost effectiveness.