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

140 Benjamin, Smith, and Waites
2.2. Supplies and Reagents
1. LCx kit (if commercial assay is being used, otherwise individual components as listed
in Table 1 and Steps 2–9).
2. Taq ligase or other type as described in Subheading 1.1. with appropriate buffers from
same supplier.
3. Custom probes with primers chosen according to criteria outlined in Subheadings 1.2.
and 1.3.
4. Proteinase K (final concentration 200 µg/ml).
5. Mineral oil.
6. Specimen or analyte.
7. Specimen Collection and Transport System (Uriprobe) containing 0.5 ml transport buffer.
8. Sterile, preservative-free plastic screw-top containers for collection and transport of
specimen (if using commercial LCx).
9. 100 µL aerosol barrier pipet tips and pipets.
3. Methods
3.1. Procedural Precautions
1. Work in laboratory using DNA amplification methods should always flow in a one-way
direction beginning in the specimen preparation and processing area (Area 1), then moving
to the amplification and detection area (Area 2). Do not bring any materials or equipment
from Area 2 into Area 1.
2. Surface cleaning using a 1% (v/v) sodium hypochlorite solution followed by 70% (v/v)
ethanol should be performed on bench tops and pipets prior to beginning the LCR Assay.
3. Chlorine solutions may pit equipment and metal. Use sufficient amounts or repeated
applications of 70% ethanol until chlorine residue is no longer visible.
3.2. Prototype LCR Technique for Detection of Neisseria gonorrhoeae
The type of materials required for LCR assays varies greatly according to the ligase,
amplification, and detection systems used, as well as the primers required that must be
specific for the desired target. Table 1 summarizes major types of LCR procedures and
the various materials and equipment needed to perform the assays, including primers,
ligases, buffers, templates, test conditions, targets, and detection systems. A more
detailed description of the Neisseria gonorrhoeae detection LCR that was eventually
modified and incorporated into the Abbott LCx system is described in Subheadings
3.2.1.–3.3.9. These methods were originally developed by Birkenmeyer and Armstrong
and described in 1992 (36). Although this procedure was developed specifically for the
detection of N. gonorrhoeae DNA, the basic principle can be adapted for the detection
of other microbial agents, provided specific oligonucleotide primers are designed.
3.2.1. Primer Selection
1. Primers should be designed to be homologous to conserved sequences in N. gonorrhoeae
that have mismatches with N. meningitidis, the species most homologous to N. gonorrhoeae.
Because of the empiric nature of developing LCR, multiple primer sites should be
developed. The site demonstrating the best sensitivity and specificity should be chosen and
this must be validated as described previously in Subheading 1.2. In the example given,
three sites were chosen corresponding to sequences immediately upstream of several of
the opa gene family (Opa-2 and Opa-3) and a site downstream of several of the pil gene
family (Pilin-2) (36).