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- 저자 : ( Heejun Yook ) (검색결과 2 건)
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( Heejun Yook ),( Woonsung Na ),( Minjoo Yeom ),( Aram Kang ),( Daesub Song ) 대한인수공통전염병학회 2017 창립총회 및 학술대회 초록집 Vol.2017 No.1
Introduction: Canine infectious respiratory disease (CIRD) is one of the most common contagious disease and spread worldwide in dog population. Numerous bacteria species and viruses contribute CIRD with synergistic co-infection in host leading clinical symptoms of flu-like illnesses such as dry coughing and sneezing, which alone limits the differential diagnosis. To provide an appropriate treatment by differential diagnosis, it is required that high sensitive and specific diagnostic system to detect various pathogens simultaneously. In this study, we have designed novel multiplex PCR(mPCR) for differential diagnosis of threatening pathogens that cause CIRD: Bordetella bronchiseptica, canine distemper virus(CDV), canine influenza virus H3N8 (cH3N8), canine influenza H3N2 (cH3N2), and pandemic influenza virus H1N1 (pH1N1). Methods: Dual priming oligonucleodtide(DPO) system was used, in which contains stabilizing priming region, determining region and polydeoxyinosine linker, to enhance sensitivity and specificity by reducing non-specific amplicon. Initial sequence analysis and securing the conservancy for the sequences were conducted using Bio Edit ver. 7.0, and primers for mPCR were designed using Primer-BLAST (NCBI) Results: The five different pathogens of CIRD were discriminated by 100bp size difference of each PCR product according to their target sequence size in gel electrophoresis, which indicates specific primer pairs amplifies target template, respectively. Various annealing temperature were applied and the optimal reaction was determined to 57℃. Conclusion: In this study, we have developed a DPO based novel multiplex PCR that discriminate five CIRD pathogens concurrently. This provides exact diagnosis of CIRD and supports an appropriate treatment to infected dogs. Especially, the mPCR can differentiate three kinds of influenza viruses to which dogs are susceptible, and this could be useful tool for surveillance study of influenza viruses in dog population. Therefore, this multiplex PCR can offer powerful diagnosis tool and effectively help both treatment and surveillance of CIRD.
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( Aram Kang ),( Woonsung Na ),( Minjoo Yeom ),( Hyekwon Kim ),( Sun-woo Yoon ),( Heejun Yook ),( Dae-gwin Jeong ),( Daesub Song ) 대한인수공통전염병학회 2017 창립총회 및 학술대회 초록집 Vol.2017 No.1
Introduction: Middle East respiratory syndrome coronavirus (MERS-CoV), a member of the family of betacoronavirus, was first identified in Saudi Arabia in 2012. MERS-CoV has ability to cross the host species from camel to human causing severe acute respiratory illnesses, and spread by contact in human population. For diagnosis of MERS-CoV in camels, the immunochromatographic assay (ICA) has been used due to its rapid decision and prompt triage of infected animal for the early quarantine. However, when the ICA is applied to an expectorated sputum in which antigens are present, the viscosity of sputum interferes with the migration of the antigens on the test strip. To overcome this limitation, it is necessary to use a mucolytic agent without affecting the antigens. In this study, we have developed a sputum pre-treatment method by testing specimens of the sputa spiked with alphacorona virus and MERS-CoV. Methods: Two mucolytic agents were used: Tris(2-carboxyethyl) Phosphine (TCEP) and N-acetyl-L-cysteine (NALC) and prepared at various concentration to treat with sputum. Bovine serum albumin (BSA) was used as a blocking agent and protease inhibitor cocktail (PI) was used to inhibit the cleavage of the antigens. After treating the compound to sputum, the mixture was applied to colloidal gold-based immunochromatographic test strip for rapid detection of MERS-CoV or alpha coronavirus (BIONOTE Inc., South Korea). The intensities of colloidal gold were measured by MEDISENSOR Gold reader (SD BIOSENSOR Ltd., South Korea). Results: Intensity of test line was higher when the sputa spiked with alpha coronavirus was processed with TCEP, BSA and PI than with TCEP alone. In the case of the sputa spiked with inactivated MERS-CoV, mixture of TCEP and BSA presented higher intensities, while it decreased in the addition of PI. This was reproduced when compound of NALC and BSA was treated to the specimens. Conclusion: In this study, we demonstrated that mixture of the mucolytics, blocking agent and protease inhibitor together effectively dissolve the viscosity of sputum minimizing the effect on antigens, which is more suitable for use in flow immunochromatographic test kit than when sputum or mucolytics alone were used.
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