Becton Dickinson and Company Limited

Deborah R Howard

Margaret S Dey

James G Nadeau

James A Down

Michael C Little

James L Schram

William E Keating

Cheryl H Dean

David J Wright

Adriann H Walters

Primers and methods for adapter-mediated multiplex amplification of the IS6110 insertion element of the Mycobacterium tuberculosis (M.tb) complex and a 16S rDNA target common to essentially all mycobacteria are described. In certain embodiments, the primers are optimized for efficient multiplex amplification in thermophilic SDA. The multiplex Strand Displacement Amplification methods of the invention are capable, in a single amplification reaction, of simultaneously identifying M. tuberculosis and providing a screen for substantially all of the clinically relevant species of mycobacteria. Also disclosed are internal control sequences designed for coamplification with the two targets, allowing assessment of amplification efficiency and/or quantitation of the targets.

Detection of mycobacteria by multiplex nucleic acid amplification

Methods for processing samples which may contain mycobacteria which are compatible with both conventional culturing techniques and nucleic acid analysis. It has been found that the harsh chemical treatment previously thought necessary to obtain efficient lysis can be eliminated without loss of lysis efficiency, thereby eliminating reagents which may inhibit subsequent nucleic acid-based reactions. Heat alone is sufficient to lyse mycobacteria, even in buffers which do not contain detergents, chelators, enzymes, etc. Residual reagents introduced by conventional sample processing methods for culture can be successfully removed from the processed pellet without significant loss of microorganisms by at least two washings with saline, water, or buffers compatible with nucleic acid analysis and appropriate centrifugation.

Sample processing method for Mycobacterium tuberculosis

Growing community of inventors

Durham, NC, United States of America

Deborah R Howard