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

138 Benjamin, Smith, and Waites
the small size of the amplicon in LCR. The potential for contamination requires strict
adherence to physical separation of setup and detection areas and other containment
methods such as bleach treatment of lab benches, Ultraviolet irradiation of setup areas,
unit premixes for setup, as well as use of aerosol barrier pipet tips. The commercial
LCx instrument injects a binary inactivating agent that is capable of inactivating small
amplicons by a factor of up to 109.
1.6. Inhibitors
LCR is less prone to problems with inhibitors from urogenital specimens than PCR
for detection of Chlamydia (14,20). Freeze-thawing and dilution decreased the falsenegative
rate of PCR (21). Possible inhibitors of LCR were removed by a mildly acid
wash to remove CaHPO4 from concentrated specimens being tested for the presence of
acid fast bacteria (22). Potential effect of inhibitors on amplification results mandates
rigorous quality control for all steps of the procedure as described in the technical
procedures presented here.
1.7. LCR Applications
1.7.1. Noncommercialized Methods
Over the past few years, numerous publications have appeared that describe a
number of potential applications of the LCR procedure, each with its own particular
modifications of the basic procedure, using the varied available ligases, probe designs,
relative positions, and detection methods. Table 1 shows references and lists the
methods used for several diverse applications. A basic LCR procedure that can be
adapted to detect nucleic acid of a variety of etiologic agents as well as eukaryotic
polymorphisms, especially SNPs, is presented in further detail in Subheading 2.
1.7.2. Commercially Available LCR Kits*
As an artifact of the way the companies interested in molecular diagnostics have
carved up the field, mostly driven by the ownership of rights to specific procedures,
in the United States, LCR is primarily used to diagnose the sexually transmitted
diseases caused by C. trachomatis and N. gonorrhoeae. Abbott Diagnostics markets
a commercial LCR kit, the LCx, in which four oligonucleotide probes target and
hybridize with a specific complementary single-stranded nucleotide sequence within
the multicopy cryptic plasmid gene present in all serovars of C. trachomatis that is
exposed during sample preparation in which the heating process causes the release
of single-stranded DNA, leaving a gap of a few nucleotides between the probes (23).
Polymerase then fills the gap with nucleotides in the LCR reaction mixture. Once
the gap is filled, thermostable ligase covalently joins the pair of probes to form an
amplification product that is complementary to the original target sequence that then
serves as an additional target sequence for further rounds of amplification. Amplification
occurs when the LCR reaction mixture and sample are incubated in a thermal
cycler. During thermal cycling, the temperature is raised above the melting point of
*Abbott Diagnostics, which marketed the LCx Uriprobe that was FDA approved in 1994, is to be
discontinued in June 2003 and after that time no commercial kits will use the LCR technology.