SEQUENCE CAPTURE-PCR IMPROVES DETECTION OF MYCOBACTERIAL DNA IN CLINICAL SPECIMENS

The rapid identification of mycobacterial DNA in clinical samples by P CR can be useful in the diagnosis of tuberculous infections, but sever al large studies have found that the sensitivity of this approach is n ot better than that of culture, In order to improve the sensitivity of detection of mycobacterial DNA in clinical specimens from patients wi th paucibacillary forms of tuberculosis, we have developed a procedure permitting the specific capture of mycobacterial DNA in crude samples prior to amplification, thereby concentrating the target sequences an d removing irrelevant DNA and other potential inhibitors of the amplif ication reaction (sequence capture-PCR). By using this approach to cap ture and amplify two different sequences specific for organisms of the Mycobacterium tuberculosis complex (IS6110 and the direct repeat regi on), it was possible to detect as little as one genome of mycobacteria l DNA in samples containing up to 750 mu g of total DNA, representing a 10- to 100-fold increase in sensitivity compared with that obtained by purifying total DNA prior to amplification, Detection of the IS6110 sequence in pleural fluid samples from patients with tuberculous pleu risy by sequence capture-PCR gave positive results in 13 of 17 cases, including 3 of 3 culture-positive samples and 10 of 14 culture-negativ e samples, In contrast, when total DNA was purified from these samples by adsorption to a silica matrix prior to amplification, only the thr ee culture-positive samples were positive by PCR, The sensitivity of d etection of the direct repeat sequence in these samples by sequence ca pture-PCR was similar to that oil IS6110 and, in addition, permitted i mmediate typing of the strains from some patients, We conclude that se quence capture-PCR improves the sensitivity of detection of mycobacter ial DNA in paucibacillary samples. This approach should be useful in d etecting rare target sequences from organisms implicated in other path ologic processes.