A field-ready colourimetric LAMP assay for detection of Mycoplasma gallisepticum using rapid DNA extraction.

Abstract

Mycoplasma gallisepticum (MG) is a major respiratory pathogen of poultry, and rapid point of care tests for routine on farm surveillance are currently unavailable. This study describes a user-friendly Loop Mediated Isothermal Amplification (LAMP) assay for detecting MG, used together with a rapid DNA extraction method that supports field deployment. The performance of the LAMP assay was compared with a conventional PCR assay using three sample sets, including MG cultures, tracheal swabs from vaccinated and unvaccinated chickens, and cloacal swabs from turkeys. DNA was extracted using a commercial kit, Ly 14 buffer, or Ly 14 buffer combined with Chelex 100. A positive LAMP result was based on a clear colour change from red to yellow. The LAMP assay detected MG in tracheal swabs with 100% agreement with PCR and showed 100% specificity when tested against DNA from 13 non-target bacterial species. Cloacal swabs produced strong agreement between LAMP and PCR, particularly when DNA templates were prepared using Ly 14 or Ly 14 plus Chelex 100 extraction procedures. When DNA was extracted using the commercial kit, minor discrepancies were noted, with the sensitivity, specificity, and accuracy of LAMP for detecting MG in cloacal samples being 100%, 97.4%, and 97.5%, respectively, when used with the rapid extraction method. The limit of detection was 1 pg/μl for PCR and 10 pg/μl for LAMP. Overall, the LAMP assay was simple to perform, produced rapid visual results, and demonstrated consistent diagnostic accuracy.