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

Ligase Chain Reaction 147
of contamination.
3. When using LCx tests for detection of microorganisms in urogenital specimens, the
presence of excessive mucus or blood in the swab or urine specimen as well as use of
feminine powder sprays can interfere with the assay and cause false negative results, thus
proper specimen quality must be monitored.
4. In addition to the usual quality controls, reproducibility of LCx assays can be monitored
by repeating random samples on a frequent basis.
5. Further quality control testing and recording is detailed for the LCx analyzer and
thermocycler in the respective manuals for the equipment.
6. Failure to reach 97°C before amplification could limit release of the DNA in the specimen
causing false negative results.
7. Test the processed urine specimen immediately, or store for up to 60 d at 2–8°C or –20°C
prior to testing. If the processed urine specimen is stored frozen, it must be completely
thawed prior to addition to the LCx Amplification Vial.
8. The LCx Analyzer first verifies that the assay results of the negative controls and calibrator
are within the specified ranges of the LCx assay parameters by comparing the assay results
of the negative control and calibrator to the values listed in the assay parameters. A run is
valid when the individual and average results are within the values listed for CAL HIGH,
CAL LOW, CAL AVE HIGH, CAL AVE LOW, NEG LOW, NEG HIGH, NEG AVE HIGH,
and NEG AVE LOW parameters in the LCx assay parameters.
9. In the event of an invalid negative control or calibrator assay result, the assay results
printout will identify the out-of-range result, the S/CO ratio of the specimen will not be
calculated and a flag indicating an invalid result will occur in the NOTE column of the
printout. Ensure the LCx Negative Controls and Calibrators are in the correct order on the
MEIA carousel to avoid an invalid run.
10. Environmental quality control screening is also recommended. The laboratory should be
monitored for the presence of amplification product by saturating one of the small-tipped
swabs in transport buffer, then using it to wipe the desired area, including equipment,
and processing according to the LCx procedures. If a positive reaction is detected, the
decontamination steps using 1% sodium hypochlorite followed by 70% ethanol should be
performed. The operation manual should be consulted for decontaminating equipment.
References
1. Barany, F. (1991) Genetic disease detection and DNA amplification using cloned thermostable
ligase. Proc. Natl. Acad. Sci. USA 88, 189–193.
2. Jurinke, C., van den Boom, D., Jacob, A., Tang, K., Worl, R., and Koster, H. (1996)
Analysis of ligase chain reaction products via matrix-assisted laser desorption/ionization
time-of-flight-mass spectrometry. Anal. Biochem. 237, 174–181.
3. Luo, J., Bergstrom, D. E., and Barany, F. (1996) Improving the fidelity of Thermus
thermophilus DNA ligase. Nucleic Acids Res. 24, 3071–3078.
4. Khanna, M., Park, P., Zirvi, M., Cao, W., Picon, A., Day, J., et al. (1999) Multiplex
PCR/LDR for detection of K-ras mutations in primary colon tumors. Oncogene 18, 27–38.
5. Khanna, M., Cao, W., Zirvi, M., Paty, P., and Barany, F. (1999) Ligase detection reaction for
identification of low abundance mutations. Clin. Biochem. 32, 287–290.
6. Zirvi, M., Bergstrom, D. E., Saurage, A. S., Hammer, R. P., and Barany, F. (1999) Improved
fidelity of thermostable ligases for detection of microsatellite repeat sequences using
nucleoside analogs. Nucleic Acids Res. 27, e41.
7. Day, D. J., Speiser, P. W., White, P. C., and Barany, F. (1995) Detection of steroid
21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection
reaction. Genomics 29, 152–162.