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

142 Benjamin, Smith, and Waites
2. The primers for this technique are designed to have a gap of 4 to 5 Gs that would be
filled by adding the single deoxynucleotide dGTP and Taq polymerase that would not add
more nucleotides than are needed to fill the gap. The Pilin-2 locus has 6 bp of nontargeted
DNA added to one end that are complementary to the related primer.
4. The ends of the primers are labeled with fluorescein on one pair of homologous primers (one
5′ one 3′) and biotin on the other nonligating ends of the other two primers. The sequences
of the primers are described in the original publication describing this technique (36).
5. The ligating 5′ ends are phosphorylated to facilitate ligation after the gap is filled.
6. All of the modifications of the oligonucleotides can be ordered from numerous companies
that will custom synthesize oligonucleotides for specific purposes and targets.
3.2.2. Clinical Specimens
1. The type of specimen should be chosen according to the site that the organism is most likely
to be detected in association with disease. For N. gonorrhoeae, urethral or endocervical
swabs are collected and placed into 500 µl of specimen buffer containing 50 mM EPPS
buffer [N-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid)] and 5 mM EDTA
(pH 7.8). The tube can be maintained up to 24 to 48 h at room temperature if necessary
for transport to the laboratory. The swabs are vigorously vortexed prior to removal and the
sample is then frozen at -20°C until DNA extraction.
2. Organism lysis to release DNA is accomplished by adding proteinase K to a final
concentration of 200 µg/ml and incubating for 1 h at 60°C.
3. Boiling for 10 min inactivates the proteinase K, further lyses the bacteria and denatures
the DNA.
4. The cell debris is pelleted by centrifuging for 10 min at 4°C at 13,000g.
5. The supernatants can be stored at –20°C until used.
3.2.3. Amplification and Detection
1. Each 50 µl amplification mixture will contain 4 µl of sample, and a final concentration of:
20 mM Tris-HCl (pH 7.6 @25°C) 25 mM potassium acetate, 10 mM magnesium acetate,
10 mM DTT, and 1 mM NAD and 0.1% Triton X-100. This buffer supplied as 10X the
concentrations needed can be purchased commercially (New England Biolabs, Beverly,
MA) along with the Taq ligase.
2. Each of the four oligonucleotides within a set should be diluted in advance and the
appropriate volumes of each added to obtain the 830 fmol of each of the four in a set
in each reaction tube.
3. This mixture is overlaid with sterile mineral oil and heated for 3 min in a boiling water
bath to ensure complete denaturation. After cooling, 1 unit of Amplitaq DNA polymerase
(Perkin-Elmer Cetus, Norwalk, CT) and 15 units of Thermus aquaticus (Taq) DNA ligase
(New England Biolabs) is added.
4. A positive control consisting of 270 cell equivalents of N. gonorrhoeae DNA in 80 ng/µl
human placental DNA (Sigma Chemicals, Saint Louis, MO) and a negative control must
be included in each run.
5. Gap LCR is performed in a thermocycler. The prototype technique for N. gonorrhoeae
used 27 cycles (Opa-2), 33 cycles (Opa-3) or 31 cycles (Pilin-2). Each cycle consists of a
denaturation step 85°C for 30 s and a hybridization, gap filling, ligation step of 60°C for
Opa-2 and Pilin-2 and 53°C for Opa-3) for 1 min. When developing a new LCR procedure
it is necessary to empirically test different numbers of cycles to determine the optimum
for detection and specificity for each primer pair.
6. Detection is performed on 40 µl of each LCR reaction in the automated Abbott Imx,
which uses anti-fluorescein-coated microparticles to capture the products of the gap LCR.