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

Ligase Chain Reaction 145
2. The LCx procedure requires that the negative control and the calibrator be run in duplicate
with each carousel of specimen.
3. The negative control and calibrator are activated by the addition of 100 µL of LCx
activation reagent. It is important to make sure correct volume is added or the run may
be invalid. After addition, the contents of the bottles are then recapped and vortexed for
20 s. Each bottle of activated negative control or calibrator is designed to be used up to
48 h if stored at 2–8°C.
4. A positive control that monitors the entire assay procedure including the specimen
processing step should be tested. A known positive urine specimen can be processed in
parallel and tested with unknown specimens. The positive control should give a positive
assay value (S/CO ratio >1.00). Each laboratory should establish a target value and limits
from each control batch using statistical control rules. These target values and limits should
be maintained throughout storage. Alternative choices for a positive control are type strains
of the microorganism targeted in the assay, e.g., N. gonorrhoeae.
3.3.6. LCx Amplification Procedure
1. Turn the LCx Thermal Cycler on for at least 15 min prior to use.
2. Collect all LCx amplification vials containing samples, negative control and calibrator
from Area 1 and transfer to Area 2 for thermal cycling.
3. LCx thermal cycling conditions should be edited to the following amplification parameters:
93°C for 1 s, 59°C for 1 s, 62°C for 1 min, 10 s for 40 cycles.
4. Place the amplification vials into the thermal cycler, and initiate run. After completion
of the thermal cycler run, amplification product may remain at 15–30°C for up to 72 h
prior to LCx detection.
3.3.7. Detection of Amplification Product
1. Refer to the LCx Analyzer Operations Manual for detailed instrument operation procedures.
Before running the LCx Analyzer, check to see that LCx Inactivation Diluent (1) contains
a minimum of 100 mL and the LCx System Diluent (2) contains a minimum of 250 mL.
Remove the LCx Amplification Vials from the LCx Thermal Cycler.
2. Place LCx Reaction Cells into a Carousel; lock the carousel.
3. Pulse-centrifuge the LCx amplification vials in a microcentrifuge for 10–15 s before
placing into the LCx reaction cells.
4. Place the amplification vials into the LCx reaction cells in the following order: negative
controls in positions 1 and 2, calibrators in positions 3 and 4, and specimens in the
remaining positions.
5. Place the carousel into the LCx Analyzer.
6. Lock the amplification vial Retainer.
7. Remove the LCx detection reagent Pack from 2–8°C storage, gently invert it five times,
and open the reagent pack bottles in the numeric order: 1, 2, 3, 4.
8. Look for any film that may have formed over the opening of the reagent bottles. If present
burst the bubble.
9. Place the LCx detection reagent pack into the LCx Analyzer.
10. Press Assay, then sample management to log in samples for the run. Press RUN on the LCx
Analyzer control panel. Final assay results will be printed in approx 60 min.
11. Store the detection reagent pack at 2–8°C in original packaging, decontaminated with 1% v/v
hypochlorite or separate from unopened LCx kits.
12. Remove the Carousel, individually remove the LCx reaction cells, and dispose appropriately.
Rinse the carousel with water after each use.