DOMAIN-DIRECTED POLYMERASE CHAIN-REACTION CAPABLE OF DISTINGUISHING BACTERIAL FROM HOST DNA AT THE SINGLE-CELL LEVEL - CHARACTERIZATION OF A SYSTEMATIC METHOD TO INVESTIGATE PUTATIVE BACTERIAL-INFECTION IN IDIOPATHIC DISEASE

The use of a broad-range PCR to target conserved sequences in the bact erial ribosome has long been recognized as an approach to investigatin g idiopathic human diseases for the presence of bacteria. An important example is rheumatoid arthritis where the hypothesis of a bacterial e tiology remains viable despite failure of previous methods to identify a causative organism. Practical implementation of this strategy, howe ver, was impeded by requirements unique to the study of these diseases . Hence, an adequately characterized method for achieving this has not appeared. We now describe such a method based on the use of a broadly reactive pair of deoxyinosine-containing primers. Detailed characteri zation addressing these requirements provided evidence that DNA from a t least 96% of potential prokaryotic pathogens would be detected. The sensitivity was shown to approximate that of a single organism per rea ction. Importantly, this sensitivity was shown to be maintained among multiple targets and to be unimpaired by a large excess of human DNA. Similar results were obtained with extracts of inflamed human synovial fluid to which as little as 0.01 pg of bacterial DNA or, alternately, a single organism per reaction was added. This method was also shown correctly to detect the causative bacteria in clinically infected syno vial fluids, further documenting its applicability to such specimens. Finally, it was converted to an in situ method by which bacterial sequ ences were histochemically localized to Michaelis-Guttman bodies in ti ssue sections from a patient with malakoplakia, a poorly understood in fectious disease. The broad reactivity and high sensitivity achieved b y this approach translate into a high likelihood of detecting an unkno wn bacterium if present in a clinical specimen. Conversely, if properl y controlled, the failure of this method to detect such organisms can provide evidence supporting rejection of the bacterial etiology hypoth esis, an important aspect unique to this approach. For those bacterial sequences that are detected, the ability to localize them in situ wou ld help define their histologic context and hence facilitate their pat hogenetic interpretation. The present method should therefore be appro priate for use in systematically studying rheumatoid arthritis as well as a number of other important idiopathic disorders where a bacterial etiology is suspect. (C) 1997 Academic Press