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
Cr. Steinman et al., 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, Analytical biochemistry, 244(2), 1997, pp. 328-339
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