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. 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.
Stefano
J E
JE
GENE-TRAK, Inc., Framingham, MA 01701, USA.
Genovese
L
L
An
Q
Q
Lu
L
L
McCarty
J
J
Du
Y
Y
Stefano
K
K
Burg
J L
JL
King
W
W
Lane
D J
DJ
D001431:Bacteriological Techniques; D001483:Base Sequence; D002584:Cervix Uteri / Q000382:microbiology; D002692:Chlamydia trachomatis / Q000235:genetics*; D011088:DNA Ligases / Q000235:genetics; D005260:Female; D005453:Fluorescence; D017930:Genes, Reporter; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008969:Molecular Sequence Data; D017354:Point Mutation; D011777:Q beta Replicase / Q000235:genetics*; D015347:RNA Probes / Q000235:genetics*; D012338:RNA, Ribosomal, 23S / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015347:RNA Probes; D012338:RNA, Ribosomal, 23S; D011777:Q beta Replicase; D011088:DNA Ligases