John M. S. Bartlett.pdf - Bio-Nica.info

Ligase Chain Reaction 139
the hybridized amplification product causing it to dissociate from the original target
sequence. Lowering the temperature allows more of the oligonucleotide probes to
hybridize to the targets now available and to be ligated. The temperature continues to
be cycled until sufficient numbers of target amplification product have accumulated.
Ligated product is captured by antibody immobilized onto the surface of microparticles
using a ligand attached to the end of one primer and then detected by an enzymeconjugated
antibody directed at a second reporter molecule at the distal end of the
other primer. Only ligated product with both haptens covalently attached will generate
a chemiluminescent signal. The amplification product accumulates exponentially and
is detected by chemiluminescence on the automated Abbott LCx or IMx Analyzers.
Specimens that can be used include cervical swabs and urethral swabs as well as urine
from either men or women (24–26). The LCx kit also has the advantage of being
multiplexed for the detection of N. gonorrhoeae in the same genitourinary specimen.
The LCx target in N. gonorrhoeae is a 48 base pair DNA sequence in the multicopy Opa
gene that is conserved in all strains studied to date and is specific to N. gonorrhoeae.
There are mixed results on the sensitivity of LCx compared to other methods of
amplification for detection of urogenital infections. One report found PCR more
sensitive than LCx. (27), whereas a second study found LCx more sensitive (28). Stary
et al. compared the sensitivity and specificity of LCx and the Transcription Mediated
Amplification (TMA) assay (GenProbe, Inc., San Diego, CA) using several different
urogenital specimens. They found comparable results with endocervical and vulval
swab samples, male urethral swabs and urine, but found a lower sensitivity with the
TMA method for female first void urine (29). Carroll et al. found the overall sensitivity
of LCx was better than GenProbe PACE 2 for chlamydia with very good specificity
for both (30).
Abbott Diagnostics also makes a Mycobacterium (M.) tuberculosis LCx kit that is
approved for diagnostic use in some European countries but not in the United States
at present. This product is a Gap LCR assay with a few nucleotides that must be filled
before ligation. It targets the protein antigen b genes of M. tuberculosis. This LCx
kit has been evaluated in comparison to culture and/or other amplification methods
such as the Roche PCR and the TMA with generally favorable results. Overall, most
evaluations have found the LCx to perform better for detection of M. tuberculosis in
smear-positive as opposed to smear-negative respiratory specimens (31–34). However,
one investigation (35) found similar high sensitivity and specificity values for the LCx,
exceeding 90% for smear-positive as well as smear-negative specimens, suggesting
that the LCx assay has potential utility as a screening test for the rapid diagnosis of
tuberculosis in high-risk patients.
2. Materials
2.1. Equipment
1. Microcentrifuge capable of speeds of ≥9000g.
2. Vortex mixer.
3. 20°C freezer for sample storage if not processed immediately.
4. LCx Analyzer or other detection system with appropriately labeled reagents specific for
the system employed.
5. LCx Thermocycler.
6. LCx dry bath capable of heating from 60° to 100°C.