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A simple assay format was developed for the direct detection of C. trachomatis rRNA utilizing ligation of recombinant MDV-1 probe RNA fragments hybridized to 23S rRNA after capture and release from a solid support. Assay background (equivalent to 10(4) targets) was suppressed by blocking sequences in the 5' MDV reporter probe fragment complementary to the 3' fragment by prehybridization of a DNA oligonucleotide. A pair of reporter fragments bearing a deletion within the region, obtained by a hydrid-selection-amplification protocol, yielded a low level of assay background which was reduced to < 2% with a blocker directed against the remaining pairing sequence. This probe set showed a sensitivity of 10(3) molecules of 23S rRNA (> 95% responding) and could detect a single elementary body (EB) of Chlamydia trachomatis or 1-10 EB added to a clinical matrix of pooled negative human cervical swab samples. The time of first appearance of amplification products by real-time fluorescence detection showed a linear response to log increases in the target level over a 10(5)-fold range, permitting the determination of target level within an order of magnitude. The assay showed approximately 10(9)-fold discrimination over Chlamydia pneumonae (TWAR) rRNA. High levels of cultured C. albicans, E. coli, S. aureus, or N. gonorrhoeae had no detectable effect on assay background or the ability to detect a single elementary body.
J E Stefano, and
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J L Burg, and
W King, and
D J Lane
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D J Lane
October 1972,
Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme,
J E Stefano, and
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J McCarty, and
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J L Burg, and
W King, and
D J Lane
May 1996,
Proceedings of the National Academy of Sciences of the United States of America,
J E Stefano, and
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D J Lane
January 1999,
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D J Lane
December 1981,
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J E Stefano, and
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January 1995,
Journal of clinical microbiology,
J E Stefano, and
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D J Lane
July 1984,
Lancet (London, England),
J E Stefano, and
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W King, and
D J Lane
October 1996,
Proceedings of the National Academy of Sciences of the United States of America,
J E Stefano, and
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Q An, and
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D J Lane
December 2010,
Chemical communications (Cambridge, England),
