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| Development of Loop-mediated Isothermal Amplification for Rapid Detection of Mycoplasma bovis |
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Abstract Mycoplasma bovis is an important pathogen in cattle. In this research we developed a new and rapid test method for Mycoplasma bovis with loop-mediated isothermal amplification(LAMP). With the 5 specific primers targeting to a total of six distinct sequences on the conservative gene uvrC, the new method could be completed in 60 min under the isothermal condition of 58℃. With the LAMP method, we could detect 20 pg DNA of M. bovis, which was 100 times lower than using the method of PCR. In addition, using M. bovirhinis, M. agalactiae, M. arginini, Bovine parainfluenza virus(BPIV), Bovine adenoviruses(BADV) and Infectious Bovine rhinotracheitis virus(IBRV) to determine the specificity of LAMP and PCR methods, both the methods of LAMP and PCR displayed the high specificity results. The research had also applied the calcein, a fluorescence visualization reagent, and Mn2+ in LAMP, so as to visualize the result of the test. Additionally, clinical samples after boiling 10 min which was the best compared with DNA extraction and FTA Card could be directly used for LAMP. In the test for 167 clinical samples of nasal swabs, the positive rate using LAMP (26.95%) was higher than the one using PCR (19.16%), which proved that LAMP method, in comparison with PCR method, showed a more efficient ability in testing the clinic samples. In conclusion, LAMP method, featuring simplicity, high-efficiency, rapidity, sensitivity, specificity, and more economical, etc., possesses a potential for base laboratory.
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Received: 08 August 2011
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NI He-Jia, CHENG Xue, WANG Jing, GAO Ze-Ping, WEI Pan-Pan, TAN Jia-Li, SUN Jing, LI Hai-Tao, GAO Ji-Guo. Isolation and Screening of High Virulent Bacillus thuringiensis Strains Against Lepidopteran Pests in Some Areas of Liaoning Province[J]. 农业生物技术学报, 2019, 27(8): 1513-1520. |
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