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

144 Benjamin, Smith, and Waites
3.3.3. Urine Specimen Preparation and Processing
1. Allow urine specimen to completely thaw if frozen. Mix urine in the urine collection cup
by swirling to resuspend any settled material.
2. Using a pipet with aerosol barrier pipet tips, transfer 1 mL of mixed urine into the Urine
Specimen Microfuge Tube from the Urine Specimen Preparation Kit.
3. Centrifuge at >9000g for 15 min in a microcentrifuge.
4. Using a fine-tipped, plastic disposable pipet, gently aspirate all of the urine supernatant. Be
cautious not to contact or dislodge the pellet, which may be translucent. The time between
centrifugation and removal of supernatant must not exceed 15 min.
5. Using a pipettor with aerosol barrier pipet tips, add 1 mL of LCx Urine Specimen
Resuspension Buffer. Close lid of microfuge tube and resuspend the pellet by vortexing
until the pellet is resuspended.
6. Secure tube closure with a cap lock until it clicks into place.
7. Insert specimen tubes in preheated dry bath wells. After the temperature of the heat block
is stabilized at 97°C, heat specimens for 15 min.
8. Remove the specimen from the dry bath and allow to cool at room temperature for 15 min.
Remove cap lock and discard.
9. Pulse-centrifuge the processed urine specimen in a microcentrifuge for a minimum of
10–15 s immediately before inoculating the LCx amplification vials.
10. The amplification reagent level in the LCx amplification vial should measure approx
two-thirds of the conical part of the vial. If necessary, the vial may be pulse centrifuged
in a microcentrifuge for 10–15 s.
11. Using a pipet with aerosol barrier pipet tips, add 100 µL of each processed urine specimen
to the appropriately labeled LCx Amplification Vial, making sure each vial is securely
closed and that only one at a time is open. The vial can now be transferred to Area 2 and
placed immediately in the Thermal Cycler for amplification.
3.3.4. Swab Specimen Preparation
1. Allow specimen to completely thaw, if frozen.
2. Insert specimen tubes in preheated dry bath wells. After the temperature of the heat block
is stabilized at 97°C, heat specimens for 15 min. Failure to reach 97 + 2°C could limit
release of the DNA in the specimen causing false negative results.
3. Remove the specimen from the dry bath and allow to cool at room temperature for
15 min.
4. Unscrew cap and express swab along the side of the tube so that liquid drains back into the
solution at the bottom of the tube. Discard swab and original closure, replacing with a new
swab tube closure that is screwed on until it clicks into place.
5. The amplification reagent level in the LCx amplification vial should measure approx
two-thirds of the conical part of the vial.
6. Using a pipet with aerosol barrier pipet tips, add 100 µL of each processed specimen to
the appropriately labeled LCx Amplification Vial, making sure each vial is securely closed
and that only one at a time is open. The vial can now be transferred to Area 2 and placed
immediately in the Thermal Cycler for amplification.
7. The LCx negative control and calibrator must be prepared in conjunction with specimens
to be tested and run in duplicate with each carousel of clinical specimens.
3.3.5. Quality Control Procedures
1. Negative control and calibrator preparations must take place in the dedicated Specimen
Preparation Area (Area 1).