Rapid DNA fingerprinting of pathogens by flow cytometry

Abstract

Background A new method for rapid discrimination among bacterial strains based on DNA fragment sizing by flow cytometry is presented. This revolutionary approach combines the reproducibility and reliability of restriction fragment length polymorphism (RFLP) analysis with the speed and sensitivity of flow cytometry. Methods Bacterial genomic DNA was isolated and digested with a rare‐cutting restriction endonuclease. The resulting fragments were stained stoichiometrically with PicoGreen dye and introduced into an ultrasensitive flow cytometer. A histogram of burst sizes from the restriction fragments (linearly related to fragment length in base pairs) resulted in a DNA fingerprint that was used to distinguish among different bacterial strains. Results Five different strains of gram‐negative Escherichia coli and six different strains of gram‐positive Staphylococcus aureus were distinguished by analyzing their restriction fragments with DNA fragment sizing by flow cytometry. Fragment distribution analyses of extracted DNA were ∼100 times faster and ∼200,000 times more sensitive than pulsed‐field gel electrophoresis (PFGE). When sample preparation time is included, the total DNA fragment analysis time was approximately 8 h by flow cytometry and approximately 24 h by PFGE. Conclusions DNA fragment sizing by flow cytometry is a fast and reliable technique that can be applied to the discrimination among species and strains of human pathogens. Unlike some polymerase chain reaction (PCR)‐based methods, sequence information about the bacterial strains is not required, allowing the detection of unknown, newly emerged, or unanticipated strains. Cytometry 41:203–208, 2000. Published 2000 Wiley‐Liss, Inc.

Statistics of single‐molecule measurements: Applications in flow‐cytometry sizing of DNA fragments

“…Staphylococcus aureus Mu50 (#700699; ATCC, Manassas, VA) DNA was extracted from agarose plugs according to previously described procedures (28). Briefly, overnight cultures were used to prepare 1.5% low-melting agarose (InCert; FMC BioProducts, Rockland, ME) plugs, each containing ϳ10 7 cells.…”

“…Note that the fingerprints shown in Figure 8 have been normalized or offset to facilitate direct comparison. While the data used to produce the histogram in Figure 8b represents only 4% of the entire data set, the prominent, identifying features used from FCM data sets to discriminate bacteria at the species and strain level are all present (28,42). Thus, bacterial discrimination by FCM, previously demonstrated with large data sets, is feasible based on the analysis of as few as 10 genomes.…”

“…Bacterial DNA fingerprinting represents a significantly more difficult task, relative to viral fingerprinting, due to the increased number of fragment species as well as the fact that many fragments are larger and are therefore more fragile. Nevertheless, our group has demonstrated bacterial discrimination at the species and strain level based on FCM results with fragments Յ400 kb (28,42,43).…”

“…The data used to create this histogram was collected over several minutes and corresponds to ϳ600 digested Mu50 genomes. This sample corresponds to approximately 10 3 times less DNA than would be required for PFGE analysis (28,42,43). A conservative estimate of N min for this sample is N ϭ 10 which corresponds to the analysis of only 10 digested bacterial genomes.…”

Performance Assessment of DNA Fragment Sizing by High-Sensitivity Flow Cytometry and Pulsed-Field Gel Electrophoresis

“…For instance, PFGE requires relatively large quantities of DNA (Ն200 ng of DNA or ϳ10 7 bacterial cells [33] or at least 1 ng of DNA per band [42]). Typically, bacterial samples must be cultured to obtain this amount of DNA (culturing can require up to 48 h and some bacteria cannot be cultured).…”