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Wang J.,Centers for Disease Control and Prevention | Xu Z.,Centers for Disease Control and Prevention | Niu P.,Centers for Disease Control and Prevention | Zhang C.,Centers for Disease Control and Prevention | And 5 more authors.
BioMed Research International | Year: 2014

Diarrhea caused by viral and bacterial infections is a major health problem in developing countries. The purpose of this study is to develop a two-tube multiplex PCR assay using automatic electrophoresis for simultaneous detection of 13 diarrhea-causative viruses or bacteria, with an intended application in provincial Centers for Diseases Control and Prevention, China. The assay was designed to detect rotavirus A, norovirus genogroups GI and GII, human astrovirus, enteric adenoviruses, and human bocavirus (tube 1), and Salmonella, Vibrio parahaemolyticus, diarrheagenic Escherichia coli, Campylobacter jejuni, Shigella, Yersinia, and Vibrio cholera (tube 2). The analytical specificity was examined with positive controls for each pathogen. The analytical sensitivity was evaluated by performing the assay on serial tenfold dilutions of in vitro transcribed RNA, recombinant plasmids, or bacterial culture. A total of 122 stool samples were tested by this two-tube assay and the results were compared with those obtained from reference methods. The two-tube assay achieved a sensitivity of 20-200 copies for a single virus and 102-10 3 CFU/mL for bacteria. The clinical performance demonstrated that the two-tube assay had comparable sensitivity and specificity to those of reference methods. In conclusion, the two-tube assay is a rapid, cost-effective, sensitive, specific, and high throughput method for the simultaneous detection of enteric bacteria and virus. © 2014 Ji Wang et al.


Luo P.J.,China National Institute for Nutrition and Food Safety | Jiang W.X.,China Agricultural University | Chen X.,National Institute for Infectious Disease Control and Prevention | Shen J.Z.,China Agricultural University | Wu Y.N.,China National Institute for Nutrition and Food Safety
Journal of Animal Science | Year: 2011

One polyclonal antibody against florfenicol and thiamphenicol was produced and a competitive ELISA was developed for the detection of florfenicol and thiamphenicol in swine feed. The ELISA gave a 50% inhibiting concentration of 1.02 ng/mL for florfenicol. For swine feed fortified with 0.05 to 3.0 mg/ kg, the interassay recoveries of florfenicol and thiamphenicol ranged from 86.4 to 118.6%, whereas intraassay recoveries of both drug ranged from 90.1 to 126.5% with less than 15% CV. Results obtained from HPLCtandem mass spectrometry indicated this ELISA procedure could be used as a convenient method for rapid screening of florfenicol and thiamphenicol in swine feed. © 2011 American Society of Animal Science.


Sun L.,National Institute for Infectious Disease Control and Prevention | Shen X.,Chinese National Institute for Viral Disease Control and Prevention | Liu Y.,Capital Medical University | Zhang G.,Xi'an Jiaotong University | And 5 more authors.
Acta Biochimica et Biophysica Sinica | Year: 2010

The mechanism underlining human papillomaviruses (HPVs) causing cancer has been studied extensively, and it was concluded that the high-risk HPVs' E6 targeted and degraded tumor suppressor protein p53, leading to infected cells malignant transformation. In contrast, the low-risk HPVs only cause proliferative but non-invasive lesions of infected epithelia. Therefore, we hypothesized that low-risk HPVs' E6 might interact with p53 in a different pattern. We used a mammalian green fluorescent protein (GFP) expression system to express HPV-18E6 and HPV-6E6 fusion proteins in wild-type (wt) p53 cell lines, 293T and HEK293 cells, to investigate the traffic and location of E6s and p53. The results indicated GFP-18E6 was mainly expressed in nucleus, whereas GFP-6E6 was expressed exclusively in cytoplasm. Endogenous wt p53 was shown to be localized in the nuclei of cells transfected with GFP-18E6. Interestingly, for the first time, we observed that p53 was trapped in the cytoplasm and never translocated into the cell nuclei transfected with GFP-6E6. In conclusion, HPV-6E6 was responsible for the cytoplasmic localization of p53. Therefore, our experiments provide a new insight into the pathogenesis of HPV.


Zhang G.,Beijing Haidian Hospital | Liu Y.,Capital Medical University | Yu L.,Beijing Haidian Hospital | Sun L.,National Institute for Infectious Disease Control and Prevention
Oncology Reports | Year: 2012

Since mucosal high-risk human papillomavirus (HPV) E6 can target and degrade the tumor suppressor p53, it is recognized as a major causative agent of cervical cancer. However, to date the distribution of high-risk HPV-E6 protein remains elusive. Thus, in the present study we used a mammalian green fluorescent protein (GFP) expression system to express a GFP/HPV-16E6 fusion protein (GFP-16E6) in wildtype (wt) p53 cells, such as MCF-7 and 293T cells to investigate the trafficking and localization of E6 and p53. Following transfection, we observed that the overexpressed GFP-16E6 was a nuclear protein, and that endogenous wt p53 localized to the nucleus together with GFP-16E6. Strikingly, p53 levels were not decreased but increased in 24 h transfected with pGFP-16E6. Furthermore, we observed significant apoptosis induced by GFP-16E6, which proved to be dependent on p53 expression.


Sun L.-N.,National Institute for Infectious Disease Control and Prevention | Shen X.-X.,Chinese National Institute for Viral Disease Control and Prevention | Wei J.-C.,National Institute for Infectious Disease Control and Prevention | Zhang H.-J.,National Institute for Infectious Disease Control and Prevention | And 2 more authors.
Chinese Journal of Cancer Prevention and Treatment | Year: 2010

OBJECTIVE: To investigate the difference of biological behavior between high-risk HPV-16E6 and low-risk HPV-11E6 proteins. METHODS: Eeukaryotic expression Plasmids named pGFP-16E6 and pGFP-116 were constructed, then tranfected to CHO cell line and the E6's location was observed by a fluorescent microscope dynamically. RESULTS: GFP reached the highest expression level at 21 h post-transfection. After transfecting, GFP-16E6 mainly expressed in the nuclei of the three cells used, but GFP-11E6 exclusively in the cytoplasm of all the cells used. The results indicated that GFP and GFP-16E6 proteins were expressed from 6 h post-transfection (54. 12,48. 27). While GFP-11E6 protein was not detected at 6 h post-transfection, it expressed from 12 h post-transfection (85. 64). The expressions of the three proteins increased gradually, and reached their maximum expression levels at 21 h (132. 19, 121. 94, 127. 91,P<0.05). Then, they decreased gradually. CONCLUSION: HPV-16E6 mainly expresses in the nuclei, but HPV-11E6 exclusively in the cytoplasm, and this may be one of the reasons why only the high-risk HPV E6 is associated with cancer.


Zhang C.,Chinese National Institute for Viral Disease Control and Prevention | Niu P.,Chinese National Institute for Viral Disease Control and Prevention | Hong Y.,Capital Medical University | Wang J.,Chinese National Institute for Viral Disease Control and Prevention | And 2 more authors.
Journal of Microbiological Methods | Year: 2015

Objectives: We aim to develop a multiplex real-time PCR assay to detect the most common pathogens causing community outbreaks of diarrhea. Methods: Four reaction systems of fluorescence dye-based real-time PCR assay were performed to amplify genes of norovirus, sapovirus, rotavirus, astrovirus, adenovirus, Campylobacter jejuni, Yersinia enterocolitica, Vibrio parahaemolyticus, Salmonella spp., Escherichia coli, and Shigella spp. PCR products of each pathogen were identified by characteristic peaks in melting curves. Results: The assay was able to achieve detection limit of 50 copies/reaction for each individual virus target, and 140-500. CFU/mL for each individual bacterium target. A total of 122 clinical specimens from hospitalized children with acute diarrhea were used to evaluate the assay. The clinical sensitivity was very similar to that of reference methods. Norovirus genogroup II revealed the highest detectable rate (45/122, 36.9%). Coinfection was found in 28 out of 122 (23%) clinical specimens. Conclusion: This assay proved to be a cost-effective, sensitive and reliable method for simultaneous detection of enteric viruses and bacteria. © 2015 Elsevier B.V.


PubMed | Capital Medical University, Chinese National Institute for Viral Disease Control and Prevention and National Institute for Infectious Disease Control and Prevention
Type: | Journal: Journal of microbiological methods | Year: 2015

We aim to develop a multiplex real-time PCR assay to detect the most common pathogens causing community outbreaks of diarrhea.Four reaction systems of fluorescence dye-based real-time PCR assay were performed to amplify genes of norovirus, sapovirus, rotavirus, astrovirus, adenovirus, Campylobacter jejuni, Yersinia enterocolitica, Vibrio parahaemolyticus, Salmonella spp., Escherichia coli, and Shigella spp. PCR products of each pathogen were identified by characteristic peaks in melting curves.The assay was able to achieve detection limit of 50 copies/reaction for each individual virus target, and 140-500CFU/mL for each individual bacterium target. A total of 122 clinical specimens from hospitalized children with acute diarrhea were used to evaluate the assay. The clinical sensitivity was very similar to that of reference methods. Norovirus genogroup II revealed the highest detectable rate (45/122, 36.9%). Coinfection was found in 28 out of 122 (23%) clinical specimens.This assay proved to be a cost-effective, sensitive and reliable method for simultaneous detection of enteric viruses and bacteria.

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