[Evaluation of a self-prepared anti-WNV-IgG diagnostic ELISA kit with a panel of serum samples collected from the people from areas in which West Nile fever is endemic].

In view of that there is no report of west Nile virus infection cases in our country, evaluation the self-prepared anti-WNV-IgG diagnostic ELISA kit should be employed with the establishment of the serum sample panel collected from the entry personnel. All individuals of entry personnel were traveled from epidemic area of infectious west Nile disease. In our study, the serum samples were both detected by self-prepared anti-WNV-IgG diagnostic ELISA kit and the FDA certified kits ,which are FOCUS West Nile Virus IgG Dxselect and Panbio Dengue IgG Capture ELISA kits. The self-prepared kit and FDA certified kits were compared and assessed simultaneously. Furthermore, the specificity, repeatability and stability of the kits were also evaluated. The results indicated that no significant difference of detective rates (35. 6% for self-prepared kit vs. 32.5% for FOCUS kit, χ2 = 3. 05, P > 0.05) and good consistency (Kappa = 0.8372) between the self-prepared kit and FDA certified kits. Also, the positive coincidence rate, the negative coincidence rate and the total coincidence rate were calculated as 91.18%, 95.34% and 92.66%, respectively. The laboratory self-developed kit presented similar quality as the counterpart kits with FDA certificate. The development of our self-prepared anti-WNV-IgG diagnostic ELISA kit will provide technical support for the prevention and control of west Nile virus entry.

Complete Genome Sequence of Wohlfahrtiimonas chitiniclastica Strain SH04, Isolated from Chrysomya megacephala Collected from Pudong International Airport in China.

Wohlfahrtiimonas chitiniclastica bacilli that live in the larvae of a parasitic fly were recently isolated and are speculated to be the cause of fulminant sepsis. Here we report and analyze the complete genome sequence of Wohlfahrtiimonas chitiniclastica strain SH04. No complete genome sequence of a Wohlfahrtiimonas chitiniclastica isolate has been documented previously.

[Multiplex real-time PCR method for rapid detection of Marburg virus and Ebola virus].

OBJECTIVE: Marburg virus and Ebola virus are acute infections with high case fatality rates. A rapid, sensitive detection method was established to detect Marburg virus and Ebola virus by multiplex real-time fluorescence quantitative PCR. METHODS: Designing primers and Taqman probes from highly conserved sequences of Marburg virus and Ebola virus through whole genome sequences alignment, Taqman probes labeled by FAM and Texas Red, the sensitivity of the multiplex real-time quantitative PCR assay was optimized by evaluating the different concentrations of primers and Probes. RESULTS: We have developed a real-time PCR method with the sensitivity of 30.5 copies/microl for Marburg virus positive plasmid and 28.6 copies/microl for Ebola virus positive plasmids, Japanese encephalitis virus, Yellow fever virus, Dengue virus were using to examine the specificity. CONCLUSIONS: The Multiplex real-time PCR assays provide a sensitive, reliable and efficient method to detect Marburg virus and Ebola virus simultaneously.

Complete genome sequence of an amur virus isolated from Apodemus peninsulae in Northeastern China.

Amur virus was recently identified as the causative agent of hemorrhagic fever with renal syndrome. Here we report the complete genome sequence of an Amur virus isolated from Apodemus peninsulae in Northeastern China. The sequence information provided here is critical for the molecular epidemiology and evolution of Amur virus in China.

Complete genome sequence of Seoul virus isolated from Rattus norvegicus in the Democratic People's Republic of Korea.

Seoul virus (SEOV) is responsible for 25% of cases of hemorrhagic fever with renal syndrome in Asia. Here we report the complete genome of strain DPRK08. The sequence information provided here is useful for understanding the molecular character of SEOV in the Democratic People's Republic of Korea (DPRK) and the circulation of SEOV in East Asia.

[Removal of oral Prevotella intermedia Endotoxin by octyl phenyl polyoxyethylene ether extraction method].

OBJECTIVE: To investigate an effective purification method for removing endotoxin from Prevotella intermedia. METHODS: The main protein ingredients of bacteria prepared from ammonium sulfate precipitation were further treated with octyl phenyl polyoxyethylene ether (Triton X-114), and then processed at 4°C, 37°C and 25°C. The obtained aqueous phase after at least two more cycle repeated operations was assayed for endotoxin by Western blotting, LAL-clotting method, in vitro cell stimulation and in vivo animal experiments. RESULTS: Western blotting and LAL-clotting method demonstrated that the reduction in endotoxin level was greater than 99.99% and recovery of the proteins after endotoxin removal was greater than 90% with Triton X-114 treatment for 3 cycles. The cytokines expression level was lower in both in vitro cell stimulation and in vivo animal experiments than in untreated group (P < 0.05). CONCLUSIONS: The extraction method provides a new choice for endotoxin removal from large volumes of the oral Prevotella intermedia.

[Expression, purification and identification of St. Louis encephalitis virus-like particles in eukaryotic cells].

OBJECTIVE: To express St. Louis encephalitis virus-like particles in mammalian cells, it will provide a prerequisite for further immunological diagnostic studies. METHODS: 293T-cell were transiently transfected with recombinant PreM-E plasmid. Expression and antigenicity of the purified protein were determined by transmission electron microscope (TEM), Western-Blot, immunofluorescence assay and ELISA. RESULTS: Recombinant subviral particles, about 50 nm in diameter, were observed by TEM in the supernatant of transfected cells. The results of Western-Blot, IFA and ELISA showed the recombinant proteins retained immunoreactivity similar to those of native virus proteins. CONCLUSION: St. Louis encephalitis virus-like particles has good antigenicity and physical appearance. It will provide a prerequisite for further immune diagnostic reagent.

[Transmission electron microscopy investigation of the denudation of the envelope of influenza virus treated with Nonidet P-40 and the binding influence of antibody].

OBJECTIVE: Through observing the morphological changes of the prepared influenza viruses (H1N1) treated with the different concentration of Nonidet P-40 solutions and added with antibody against the influenza virus using transmission electron microscopy (TEM), to explore the principle of application of the internal antibody for immunoassay of influenza virus. METHODS: Through treating the virus samples with serial diluted Nonidet P-40 solutions from 0.01% to 0.2% and/or not adding antibody against the influenza virus, then investigating the samples by TEM to obtain the morphological changes of the virions. RESULTS: The serial images show that the denudation degree of the virions is proportional with the rise of NP-40 concentration, and partly denuded virion image appeared at 0.1% NP-40 treatment, also under this condition no influence was observed on antibody binding to the virus. CONCLUSION: This work demonstrated the interaction between influenza virion and its antibody under nonionic surfactants existing, which supports the advantages of sample preparation for immunoassay enveloped virus using internal antibody theoretically.

[Development of a multiplex PCR-suspension array for simultaneous detection of five bioterrorism bacteria].

OBJECTIVE: To develop a rapid, high-throughput screening method of gene suspension array technique to simultaneously detect five bioterrorism bacteria: Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp. and Burkholderia pseudomallei. METHODS: Highly validated specific primers were used to amplify diagnostic regions unique to each pathogen. Biotin labelled PCR products were hybridized to corresponding probes coupling on the unique sets of fluorescent beads. The hybridized beads were processed through the Bio-plex, which identified the presence of PCR products. RESULTS: Multiplex PCR-suspension array can detect five bioterrorism bacteria correctly with high specificity and high sensitivity, the results suggest the utility of suspension array system for high-throughput screening of bioterrorism samples. CONCLUSION: A multiplex PCR-suspension array for rapid detection of five bioterrorism bacteria was established.

[Development of a universal primers PCR-coupled liquid bead array to detect biothreat bacteria].

OBJECTIVE: To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria. METHODS: 16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp.and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B, the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed. RESULTS: After PCR amplification by 16 S rDNA primers and specific probe hybridization, the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/microl (Burkholderia pseudomallei), 20 pg/microl (Brucella spp.), 7 pg/microl (Bacillus anthracis), 0.1 pg/microl (Francisella tularensis), and 1.1 pg/microl (Yersinia pestis), respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%, it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples. CONCLUSION: The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.

[Study on the morphology of influenza virus A by atomic force microscopy].

The aim of the study is through observing the morphology of the prepared influenza virus (H1N1) with transmission electron microscopy (TEM) and atomic force microscopy (AFM) to explore the application of AFM on the research of the external character of viruses and provide a new, simple and efficient technique for the study of the viral morphology. TEM image was obtained by negatively stained influenza virus with 1% Phosphotungstic Acid; AFM image applied the tapping mode to influenza virus without any further treatment in air at room temperature, and the morphology parameters, including length (diameter), Ra and Rq are calculated by sectional analysis. The shapes of influenza virus A are spherical, filamentous or other pleomorphous particles observed by both AFM and TEM. TEM image of influenza virus A is two-dimensional image, and viral surface has visible spikes, while AFM exhibits the three-dimensional image that can be described with several quantifiable indexes through sectional analysis. AFM phase images show viral surface clearly which is characterized by rugged feature and gear-like protuberance. As compared with TEM, AFM is a new research tool for viral morphology study with the advantages of simple sample preparing, visible interface and is intuitionistic for researchers. The surface characteristic parameters of viruses provided by AFM can be served as the main quantifiable indexes for viral morphological study.

[Atomic force microscopy investigation of the denudation of the envelope of influenza virus treated with Nonidet P-40].

OBJECTIVE: Through observing the morphology and topography of the prepared influenza viruses (H1N1) treated with the different Nonidet P-40 solutions using atomic force microscopy (AFM), to explore the application of AFM on the research of the internal character of viral morphology and structural virology. METHODS: The virus samples were treated with serial diluted Nonidet P-40 solutions from 0.05% to 0.20% and then investigated by AFM with the tapping mode in air at room temperature to obtain the morphology and topography changes including height data,amplitude data and phase data for both spherical and filamentous influenza virus A. RESULTS: The serial AFM images show that the erosion degree of the virions is proportional with the improvement of NP-40 concentration,and partly denuded virion image appeared at 0.05% NP-40 treatment, which was revealed clearly on both amplitude images and phase images. CONCLUSION: This work demonstrated for the first time that the internal topography of influenza virion could be revealed by AFM via suitable nonionic surfactants chemical dissection.

[Development of a gold-immunochromatography test for rapid detecting E. coli O157].

OBJECTIVE: To develop a method for rapid detecting Escherichia coli (E. coli) O157 on site. METHODS: A colloidal gold immunochromatography test based on double-antibody sandwich assay for detecting E. coli O157 was developed. Its sensitivity and specificity were then evaluated, and its feasibility of screening food samples were evaluated by analyzing various samples added with E. coli O157. RESULTS: Typical detecting time is less than 15 minutes per sample. The sensitivity of the test is 1 x 10(5) cfu/ml. No any cross-reaction with 30 strains of 24 species in Enterobacteriaceae (including non-O157 E. coli, Salmonella, Shigella, Proteus, Citrobacter, Enterobacter, Serratia and Yersinia), Staphylococus, Listeria, Aeromonas and Vibrios was found. The test could be used to detect E. coli O157 in various samples such as milk powder, flour, starch, coffee, biscuit, cake, jelly and juice. CONCLUSION: The gold-immunochromatography test appears to be a rapid, convenient, specific and sensitive test for detecting E. coli O157: H7 on site.

[Analysis and evaluation for cosmetics allergy].

With the fast development of cosmetics research, the adverse skin reactions induced by cosmetics allergy has attracted more and more attention of researchers. The article briefly introduces the causes and classifications of cosmetics allergy, and presents in detail the internal and international development of cosmetics allergy analysis and evaluation methods, including in vivo patch test, in vitro epidermis equivalents test, skin stratum hydration test, skin transepidermal water loss (TEWL) test, skin redness test, etc. Also, the future developing trend for cosmetics allergy prevention and cure is discussed here. The article will provide a technical reference for the future healthy development of cosmetics in China.