Use of Peptide Nucleic Acid Probes
3.5. Quality Assurance
Quality assurance tests are conducted to define the sensitivity of probes in the matrix under consideration and their specificity, as well as to delineate possible matrix limitation and interferences with probe detection.
1. Prepare a twofold dilution of a log culture of S. aureus in PBS to produce a low- to-medium range population density. Measure the optical density of culture as a starting guide (A620 nm values between 0.08 and 0.1 of a log phase culture is approx 100 million cells per milliliter).
2. Introduce the bacteria into sterile sand and sea water to yield numbers like 2, 4, 8, 16, 32 CFUs/gram of sand or 100 mL of water, respectively (CFU is determined by plating on the selective differential agar S110).
3. Prepare samples as described above and correlate PNA detection with CFU deter- mined by plate count.
4. Repeat step 2 with mixed bacterial strains to test probe specificity.
5. Negative controls and matrix interference tests follow the same protocols (9).
4. Notes
1. Custom or published PNA probe sequences can be purchased from www.Applied Biosystems.com. The new PNA probe design software www.appliedbiosystems.
com/support/pnadesigner.cfm is a good guide to test new sequences before manu- facture. Detailed protocols for in-house manufacture of the probes are cumber- some and have been described previously (16). Alternative labels to those described in this chapter are also available. Examples include Cye3, modified Fluo tags, and many more. The type of labeling can be selected at purchase. It also is possible, for example, to purchase biotin-coupled probes and then use coupling kits to link an enzyme label.
2. Contains formamide, which is harmful. Appropriate protective clothing must be worn.
3. These could be custom made: 8 cm deep, 8 ⫻ 20 cm rectangular sieves with parti- tions placed in a compatible trough.
Fig. 2. Staphylococcus aureus detected by peptide nucleic acid probes in sea water (A), wet sand (B), and dry sand (C).
4. The dilutions are plated by means of classical microbiological assay. Calibration is by plate count technique correlated to optical density where pure cultures are concerned. This step does not have to be performed every time samples are ana- lyzed. Previous determinations constitute a reasonable guide to decide on which dilutions and volumes to use for the PNA–CISH. The only exceptions are after a storm event and other sporadic episodes.
5. On the one hand, vortexing for longer than 1 min yields lower culture counts and PNA probe counts because of lysis of bacteria. On the other hand, bacteria remain attached to sand crevices when a sample is vortexed for a shorter time, (N. Esiobu unpublished data).
6. If condensates form on the membrane, discard it because it will interfere with subsequent hybridization and detection. Replication is always recommended.
Acknowledgments
The research described in this article was funded in part by the United States Environmental Protection Agency through grant number R 828830. It has not been subjected to the Agency’s required peer and policy review and therefore does not necessarily reflect the views of the agency and no official endorse- ment should be inferred. The author will also like to thank David Esiobu for technical and computer assistance.
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From: Methods in Molecular Biology, vol. 345: Diagnostic Bacteriology Protocols, Second Edition Edited by: L. O‘Connor © Humana Press Inc., Totowa, NJ