Advanced Techniques in Diagnostic Microbiology

22. Molecular Techniques for STDs Diagnosis 359
ranged respectively from 94.5% to 100% and 98.5% to 100% (Vincelette et al.,
1999; Lister et al., 2004).
Ligase Chain Reaction
Use of the ligase chain reaction assay to detect C. trachomatis was commercialized
by Abbott Diagnostics and requires a dedicated thermocylcer and detection module.
Like the PCR, LCR targets the cryptic plasmid of C. trachomatis using four
oligonucleotide primers, a thermophilic ligase for continuous primers ligation, and
Taq polymerase for PCR. The primers incorporate two labels, one for capture and
the other for detection. The ligated product is captured on microparticles and detected
by an alkaline phosphatase–labeled antibody and a fluorescence-producing
substrate. The sensitivity and specificity of LCR assay for C. trachomatis ranged
from 86.4% to 100% and 99.6% to 100%, respectively (Stary et al., 1998; Moncada
et al., 2003). In early 2003, the Abbott LCx for C. trachomatis and N. gonorrhoeae
was globally withdrawn due to continuing batch to batch problems with the performance
of the test.
Q-β Replicase-Amplified Hybridization
Developed on the principles of sandwich hybridization, reversible target capture,
and Q-β replicase amplification, the Q-β replicase-amplified hybridization
(QBRAH) assay (Gene-Trak, Framingham, MA, USA) (An et al., 1995) is a 4-h
test that detects C. trachomatis rRNA or rDNA. Two types of probes are used in
this assay including a test-specific capture probe immobilized to magnetic beads
and a replicatable RNA detector molecule containing a sequence complementary
and adjacent to the capture probe site on the target. After reversible target capture,
the signal is detected by replication of the detector molecule with Q-β replicase
in the presence of propidium iodide. The lower detection limit of QBRAH is said
to be five elementary bodies (EB) (An et al., 1995).
Transcription-Mediated Amplification
AMP-CT and APTIMA Combo 2 are transcription-mediated amplification (TMA)
assays developed by GenProbe. The assay targets the 16S rRNA of C. trachomatis
with hybridization of a DNA primer at its 5 ′ end to a phage RNA polymerase
promoter sequence. After primer extension by reverse transcription, RNA complement
is removed by RNase H. A second primer binds to the end of the DNA
and is extended backwards to form a double-stranded DNA template that is transcribed
by a phage T7 polymerase to give multiple transcripts. The RNA product
is then detected in a luminometer by a hybridization protection assay (HPA) using
an acridinium-labeled DNA probe and an enzyme-labeled anti–DNA-RNA duplex
antibody. TMA has recently been shown to be just as sensitive and specific as PCR
and LCR, with sensitivities ranging from 88.5% to 100% and specificities from
98.7% to 100% in urine samples as well as in both female and male urethral swab
samples (Crotchfelt et al., 1998; Verkooyen et al., 2003).