Y-012. Loop-Mediated Isothermal Amplification Assay (LAMP) for the Rapid Detection of Bacillus anthracis

J. Narayanan, L. Rose, V. R. Hill;
CDC, Atlanta, GA.

Background: The development of rapid detection techniques for the identification of biothreat agents is an important research area in molecular biology. In particular, it is important to identify detection techniques that require minimum analytical equipment for use under field conditions. We report the specific detection of Bacillus anthracis using the loop mediated isothermal amplifications (LAMP) technique. Methods: To investigate the LAMP assay for B. anthracis, a set of eight primers (two outer, two accelerated, and four inner) was designed specifically to recognize the 222 bp fragment of the B. anthracis sequence of the plasmid pXO1, which encodes anthrax toxic proteins, edema factor, lethal factor, and protective antigen. The intercalating dye, SYTO9, was used for real-time monitoring of amplification in a 20 µL reaction volume. The reaction temperature and time for the LAMP assay were 62 °C for 60 minutes. Detection of the LAMP assay positive signals was investigated using different detection formats, including visual turbidity and different intercalating dyes [SYTO-9 (448 nm excitation), POPO-3 (570 nm excitation) and TO-PRO-3 (633 nm excitation)]. A transilluminator served as an excitation source for the fluorescent dyes. Results: Six previously characterized strains of B. anthracis (0267, 865, 871, Sterne, Vollum and Ames) were amplified. The specificity of LAMP primer set was investigated by testing versus Francisella tularensis, Yersinia enterocolitica, B. cereus, B. thuringiensis, Klebsiella pneumonia and Escherichia coli. The sensitivity of the LAMP assay was determined to be as low as 1.2 cfu per reaction, with detection signal observed in less than an hour. The detection limit of this LAMP assay was comparable to a real-time TaqMan assay targeting the same plasmid. SYTO9 was found to provide the best visual detection fluorescence. Conclusion: The results of this study show that the LAMP assay can potentially be used for rapid detection of B. anthracis in response to suspected bioterrorism threats and can be integrated to develop a portable field detection device.