Bacterial fingerprinting by flow cytometry: Bacterial species discrimination

摘要: Background: A flow cytometric measurement (FCM) technique has been developed to size DNA fragments. Individual fragments of a restriction digest genomic DNA, stained with an intercalating dye, are passed through ultrasensitive cytometer. The measured fluorescence intensity from each fragment is proportional the length. Methods: The isolation bacterial and digestion by enzymes were performed inside agarose plug. Rare cutting employed produce manageable number Electroelution was used move plug into solution containing polyamines protect shear-induced breakage. bisintercalating dye thiazole orange homodimer introduced our A histogram intensities (fingerprint) constructed. Results: Gram-positive Bacillus globigii Gram-negative bacteria Escherichia coli Erwinia herbicola distinguished fingerprint pattern their DNA. sizes determined FCM in good agreement pulsed-field gel electrophoresis (PFGE) analysis. Flow cytometry requires only picogram quantities purified takes less than 10 min for data collection When total sample preparation time included, analysis times PFGE similar (≈3 days). Conclusions: FCM attractive identification species. It more sensitive potentially much faster PFGE. Cytometry 36:324–332, 1999. © 1999 Wiley-Liss, Inc.