SOLID-PHASE HYBRIDIZATION CAPTURE OF LOW-ABUNDANCE TARGET DNA-SEQUENCES - APPLICATION TO THE POLYMERASE CHAIN-REACTION DETECTION OF MYCOBACTERIUM-PARATUBERCULOSIS AND MYCOBACTERIUM-AVIUM SUBSP SILVATICUM

Polymerase chain reaction (PCR) has been widely applied to the detecti on of microorganisms. Overall sensitivity of PCR tests may be substant ially reduced due to a large excess of nontarget DNA and inhibitory su bstances in the sample. We used a 5'-biotinylated 513-bp probe from th e 3' region of the IS 900 element specific for Mycobacterium paratuber culosis (Mptb) to capture target Mptb DNA from crude sample DNA extrac ts. Captured target DNA was separated using streptavidin-coated magnet ic particles (Dynal). Since the IS 900 element shares homology over th is region with IS 902 in Mycobacterium avium subsp. silvaticum (Mavs), target DNA from this other pathogen was also retained. Highly specifi c PCR for the detection of either organism directed to the 5' regions of IS 900 or IS 902 was then performed directly on the solid phase. Hy bridization capture of target DNA using sequence adjacent to the desir ed specific PCR site applied to Mptb increased overall sensitivity of detection in tissue and fecal extracts 10- to 100-fold. False positive s due to contamination artifact were substantially excluded since the capture probe did not retain amplicons from the detection PCR. Develop ment of the method to involve covalent 5' immobilization of capture pr obes on heat-resistant polymers should, in the future, provide a simpl e system with broad potential applications. (C) 1995 Academic Press, I nc.