Development and Clinical Validation of a DNA Gyrase Subunit B Gene Based Loop-Mediated Isothermal Amplification Method for Detection of Melissococcus plutonius
2012
Nguyen, Phu Van, Kyonggi University, Suwon, Republic of Korea | Lee, B.R., Kyonggi University, Suwon, Republic of Korea | Yoo, M.S., Kyonggi University, Suwon, Republic of Korea | Yoon, B.S., Kyonggi University, Suwon, Republic of Korea
A novel loop-mediated isothermal amplification (LAMP) method was developed for the specific detection of Melissococcus plutonius, which is the causative agent of European foulbrood (EFB), a bacterial disease affecting honeybee larvae. The present LAMP method was performed at 62℃ for 30 min with no need for thermal cycling, using primers designed on the basis of the DNA gyrase subunit B gene (GyrB). The specificity of EFB-LAMP was confirmed by successful amplification of the bee genome infected with M. plutonius and through EcoRI restriction enzyme analysis of LAMP products. This method detected as little as 2 fg of DNA plasmid. Visual inspection of LAMP products was carried out using DNA intercalating dyes, a metal indicator, and pH indicators. Using Gene Finder, this method showed the greatest potential for clinical diagnoses of M. plutonius due to its exhibition of distinct and different colors in daylight between positive and negative reactions. Therefore, this rapid, sensitive, specific, and low-cost detection method can be used to facilitate development of apiaries.
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