Evaluation of the NucliSens Basic Kit for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in genital tract specimens using nucleic acid sequence-based amplification of 16S rRNA

Abstract

We evaluated a new RNA amplification and detection kit, the NucliSens Basic Kit (Organon Teknika), for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in genitourinary specimens. The Basic Kit provides an open platform for RNA amplification and detection and contains isolation reagents for nucleic acid extraction, nucleic acid sequence-based amplification (NASBA) reagents (enzymes and buffers), and a generic ruthenium-labeled probe for electrochemiluminescent (ECL) detection of amplified product. Using freshly purified and titrated stocks of C. trachomatis and N. gonorrhoeae and in vitro-generated RNA transcripts for sensitivity determinations, the Basic Kit detected 1 inclusion-forming unit of C. trachomatis, 1 CFU of N. gonorrhoeae, and 100 RNA molecules of 16S rRNA for both bacteria. The clinical performance of the Basic Kit was evaluated by testing a total of 250 specimens for N. gonorrhoeae by culture and NASBA and a total of 96 specimens for C. trachomatis by PCR and NASBA. The Basic Kit detected 139 of 142 N. gonorrhoeae culture-positive specimens and gave a negative result for 73 of 74 culture-negative specimens, for a sensitivity and specificity of 97.9 and 98.7%, respectively. For C. trachomatis, the Basic Kit detected 24 of 24 PCR-positive specimens and gave a negative result for 71 of 72 PCR-negative specimens, for a sensitivity and specificity of 100 and 98.6%, respectively. The Basic Kit also detected specimens containing both N. gonorrhoeae and C. trachomatis, using a multiplex NASBA assay using primers for both bacteria. The NucliSens Basic Kit offers a versatile platform for the development of sensitive RNA detection assays for sexually transmitted diseases.

FIG. 1

Relative positions of NASBA primers and probes specific for N. gonorrhoeae and C. trachomatis. Nucleotide sequences for N. gonorrhoeae and C. trachomatis are from Rossau et al. (21) and Pudjiatmoko et al. (20), respectively. The antisense P1 primers for both N. gonorrhoeae and C. trachomatis (NGP1 and CTP1, respectively) include the T7 promoter sequence at the 5′ end. The sense P2 primers contain sequence complimentary to the ECL universal detector probe.

FIG. 2

Optimal KCl concentration for NASBA amplification of N. gonorrhoeae and C. trachomatis 16S rRNAs. NT, no template; PC, positive control.

FIG. 3

Sensitivity of NASBA and PCR for detecting N. gonorrhoeae. Serial dilutions of N. gonorrhoeae nucleic acid (extracted by the Boom silica method) were amplified by NASBA and PCR. Aliquots of NASBA reaction products were analyzed for a specific product by hybridization with a biotinylated capture probe and a ruthenium-labeled detector probe followed by ECL detection in the NucliSens Reader. PCR products were analyzed by agarose gel electrophoresis. NT, no template.

FIG. 4

Sensitivity of NASBA and PCR for detecting C. trachomatis. Serial dilutions of C. trachomatis nucleic acid were amplified by NASBA and PCR. NASBA products were detected by ECL using the Basic Kit and by Northern blot analysis. PCR products were analyzed by agarose gel electrophoresis. NT, no template.

FIG. 5

Detection of C. trachomatis and N. gonorrhoeae in genital swab specimens by NASBA and ECL detection with the Basic Kit. Twelve specimens—four positive for N. gonorrhoeae (NG1 to NG4), four positive for C. trachomatis (CT1 to CT4), and four positive for both N. gonorrhoeae and C. trachomatis (NG-CT1 through NG-CT4)—and four controls (NT, no template; NGPC, N. gonorrhoeae positive; CTPC, C. trachomatis positive; and NG-CTPC, N. gonorrhoeae and C. trachomatis positive) were amplified and probed separately with a C. trachomatis-specific probe and an N. gonorrhoeae-specific probe. Dotted line represents cutoffs of positivity. y-axis, RLU.