Development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the Mycoplasma bovis
Abstract Background Mycoplasma bovis (M. bovis) is a major etiological agent of bovine mycoplasmosis around the world. Point-of-care testing in the field is lacking owing to the requirement for a simple, robust field applicable test that does not require professional laboratory equipment. The recomb...
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doaj-8fd85ee9fdbf4247a12aecafac6ad5082020-11-25T01:50:10ZengBMCBMC Veterinary Research1746-61482018-12-0114111010.1186/s12917-018-1703-xDevelopment of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the Mycoplasma bovisGuimin Zhao0Peili Hou1Yanjun Huan2Chengqiang He3Hongmei Wang4Hongbin He5Key Laboratory of Animal Resistant Biology of Shandong, Ruminant Disease Research Center, College of Life Science Shandong Normal UniversityKey Laboratory of Animal Resistant Biology of Shandong, Ruminant Disease Research Center, College of Life Science Shandong Normal UniversityCollege of Animal Science and Technology, Qingdao Agricultural UniversityKey Laboratory of Animal Resistant Biology of Shandong, Ruminant Disease Research Center, College of Life Science Shandong Normal UniversityKey Laboratory of Animal Resistant Biology of Shandong, Ruminant Disease Research Center, College of Life Science Shandong Normal UniversityKey Laboratory of Animal Resistant Biology of Shandong, Ruminant Disease Research Center, College of Life Science Shandong Normal UniversityAbstract Background Mycoplasma bovis (M. bovis) is a major etiological agent of bovine mycoplasmosis around the world. Point-of-care testing in the field is lacking owing to the requirement for a simple, robust field applicable test that does not require professional laboratory equipment. The recombinase polymerase amplification (RPA) technique has become a promising isothermal DNA amplify assay for use in rapid and low-resource diagnostics. Results Here, a method for specific detection of M. bovis DNA was established, which was RPA combined with lateral flow dipstick (LFD). First, the analytical specificity and sensitivity of the RPA primer and LF-probe sets were evaluated. The assay successfully detected M. bovis DNA in 30 min at 39 °C, with detection limit of 20 copies per reaction, which it was compared the real-time quantitative PCR (qPCR) assay. This method was specific because it did not detect a selection of other bacterial pathogens in cattle. Both qPCR and RPA-LFD assays were used to detect M. bovis 442 field samples from 42 different dairy farms in Shandong Province of China, also the established RPA-LFD assay obtained 99.00% sensitivity, 95.61% specificity, and 0.902 kappa coefficient compared with the qPCR. Conclusions To the author’s knowledge, this is the first report using an RPA-FLD assay to visualise and detect M. bovis. Comparative analysis with qPCR indicates the potential of this assay for rapid diagnosis of bovine mycoplasmosis in resource limited settings.http://link.springer.com/article/10.1186/s12917-018-1703-xMycoplasma bovisLateral flow dipstickRecombinase polymerase amplificationIsothermal nucleic acid amplificationRapid and visual detection |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Guimin Zhao Peili Hou Yanjun Huan Chengqiang He Hongmei Wang Hongbin He |
spellingShingle |
Guimin Zhao Peili Hou Yanjun Huan Chengqiang He Hongmei Wang Hongbin He Development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the Mycoplasma bovis BMC Veterinary Research Mycoplasma bovis Lateral flow dipstick Recombinase polymerase amplification Isothermal nucleic acid amplification Rapid and visual detection |
author_facet |
Guimin Zhao Peili Hou Yanjun Huan Chengqiang He Hongmei Wang Hongbin He |
author_sort |
Guimin Zhao |
title |
Development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the Mycoplasma bovis |
title_short |
Development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the Mycoplasma bovis |
title_full |
Development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the Mycoplasma bovis |
title_fullStr |
Development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the Mycoplasma bovis |
title_full_unstemmed |
Development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the Mycoplasma bovis |
title_sort |
development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the mycoplasma bovis |
publisher |
BMC |
series |
BMC Veterinary Research |
issn |
1746-6148 |
publishDate |
2018-12-01 |
description |
Abstract Background Mycoplasma bovis (M. bovis) is a major etiological agent of bovine mycoplasmosis around the world. Point-of-care testing in the field is lacking owing to the requirement for a simple, robust field applicable test that does not require professional laboratory equipment. The recombinase polymerase amplification (RPA) technique has become a promising isothermal DNA amplify assay for use in rapid and low-resource diagnostics. Results Here, a method for specific detection of M. bovis DNA was established, which was RPA combined with lateral flow dipstick (LFD). First, the analytical specificity and sensitivity of the RPA primer and LF-probe sets were evaluated. The assay successfully detected M. bovis DNA in 30 min at 39 °C, with detection limit of 20 copies per reaction, which it was compared the real-time quantitative PCR (qPCR) assay. This method was specific because it did not detect a selection of other bacterial pathogens in cattle. Both qPCR and RPA-LFD assays were used to detect M. bovis 442 field samples from 42 different dairy farms in Shandong Province of China, also the established RPA-LFD assay obtained 99.00% sensitivity, 95.61% specificity, and 0.902 kappa coefficient compared with the qPCR. Conclusions To the author’s knowledge, this is the first report using an RPA-FLD assay to visualise and detect M. bovis. Comparative analysis with qPCR indicates the potential of this assay for rapid diagnosis of bovine mycoplasmosis in resource limited settings. |
topic |
Mycoplasma bovis Lateral flow dipstick Recombinase polymerase amplification Isothermal nucleic acid amplification Rapid and visual detection |
url |
http://link.springer.com/article/10.1186/s12917-018-1703-x |
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