RAPID AND SENSITIVE DETECTION OF CHLAMYDIA-TRACHOMATIS USING A LIGATABLE BINARY RNA PROBE AND Q-BETA REPLICASE

A simple assay format was developed for the direct detection of C. tra chomatis rRNA utilizing ligation of recombinant MDV-1 probe RNA fragme nts hybridized to 23S rRNA after capture and release from a solid supp ort. 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 pa ir of reporter fragments bearing a deletion within the region, obtaine d by a hydrid-selection-amplification protocol, yielded a low level of assay background which was reduced to < 2 % with a blocker directed a gainst the remaining pairing sequence. This probe set showed a sensiti vity of 10(3) molecules of 23S rRNA (> 95 % responding) and could dete ct a single elementary body (EB) of Chlamydia trachomatis or 1 - 10 EB added to a clinical matrix of pooled negative human cervical swab sam ples. The time of first appearance of amplification products by real-t ime fluorescence detection showed a linear response to log increases i n the target lever over a 10(5)-fold range, permitting the determinati on of target level within an order of magnitude. The assay showed simi lar to 10(9)-fold discrimination over Chlamydia pneumonae (TWAR) rRNA. High levels of cultured C. albicans, E. coil, S. aureus, or N. gonorr hoeae had no detectable effect on assay background or the ability to d etect a single elementary body. (C) 1997 Academic Press Limited.