Despite major progress in their treatment and prevention, bacterial infections remain a significant cause of morbidity and mortality worldwide. In responding to a disease outbreak, rapid and accurate identification of the bacterial species involved is of paramount importance. Strain level discrimination is desirable to allow selection of treatment modalities, and in the case of a deliberate release, for identification of the source. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis was used to perform species and strain identification of subgroup I Bacilli, Yersinia, Staphylococci and Escherichia coli. By careful selection of AFLP primers, it was possible to obtain reproducible and sensitive identification to strain level, even within the highly monomorphic species Bacillus anthracis. SE-AFLP fragments can be analyzed using standard gel electrophoresis, and can be easily scored by visual inspection, due to the low complexity of the fingerprint obtained by this method. These features make SE-AFLP suitable for use in either field or laboratory applications.

DNA fragment size distribution analysis is a ubiquitous measurement in molecular biology and is typically done using gel electrophoresis. Applications exist in public health, genomics, medical diagnostics, and forensics. We have fully demonstrated a flow cytometric approach for rapidly and accurately sizing DNA fragments.(1-5) Our work has been focused on bacterial species and strain identification. In our approach, a DNA sample of whole bacterial genome DNA is digested into a characteristic set of DNA fragments using a rare-cutting restriction endonuclease. The fragment set is stained with a fluorescent intercalating dye that binds stoichiometrically to the DNA such that the amount of dye incorporated is directly proportional to the fragment size [number of base pairs (bp)]. The large increase (~1000X) in the fluorescence quantum yield of the intercalating dye upon binding to the DNA makes it unnecessary remove unbound dye from the solution before analysis. The stained fragments are diluted to ~10−14 molar and introduced into an ultrasensitive flow cytometer developed in our laboratory. Fragments pass individually through the laser illuminated detection region of the flow cytometer, each fragment producing a fluorescence burst as it transits the laser beam. Fluorescence bursts from individual fragments are detected and recorded. A histogram of the individual burst sizes is generated that displays the distribution of fragment sizes in the sample (i.e. a DNA fingerprint). For large DNA fragments, our approach is more sensitive (femtograms vs. micrograms), faster (analysis time of five minutes vs. tens of hours), and more accurate (size uncertainty 2% vs. 10%) than pulsed-field gel electrophoresis, commonly used for these analyses.

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 approximately 100 times faster and approximately 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.

As we have discussed previously (1), analysis and isolation of rare cell subpopulations have been of interest to researchers and clinicians in many areas of biology and medicine including: a) detection of somatic cell mutations in mutagenized cells (2), b) detection of human fetal cells in maternal blood for prenatal diagnosis of birth defects (3), c) detection of CALLA+ cells (4), d) detection of minimal residual diseases (5,6), e) detection of stem cells (7), and f) detection of rare HIV-infected cells in peripheral blood (8). Unfortunately, conventional flow cytometer/cell sorters operating at rates below 10,000 cells/sec require many hours to analyze and/or isolate cell subpopulations of low frequencies (e.g. 10-4 – 10-7) making them impractical to use for routine analysis and sorting of such cell subpopulations. One simple method for processing cells at higher speeds on conventional flow cytometers has been described (9). This method triggers the data acquisition or sort signal on a rare fluorescence signal.

A macro function was developed to run in conjunction with the popular image analysis package NIH Image, to allow simultaneous determination of mapping positions of one or two separate probes with respect to cytogenetic bands by dual color fluorescence in situ hybridization (FISH) and DAPI banding, and by determination of their fractional distance from pter (FLpter). In order to allow maximal flexibility, a user-defined line along the chromosome is used for measurements. Algorithms were developed to detect the ends of the chromosome and the cytogenetic bands. Results of the analysis are presented in graphical form, comprising a display of the DAPI intensity along the chromosome, the positions of the probe(s), the locations of bands as determined by analysis of the second derivative of the DAPI intensity profile, and a standard ideogram of the chromosome for comparison. The approach was validated and compared to visual assignment of probes to DAPI bands using the cosmid clone PYGM, which has been previously mapped to chromosome 11q13, and has been used as a landmark for mapping for other probes.