Rapid DNA fingerprinting of pathogens by flow cytometry.
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.
Larson
E J
EJ
Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
Hakovirta
J R
JR
Cai
H
H
Jett
J H
JH
Burde
S
S
Keller
R A
RA
Marrone
B L
BL
D016172:DNA Fingerprinting; D004262:DNA Restriction Enzymes / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis* / Q000187:drug effects; D004926:Escherichia coli / Q000145:classification* / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D009930:Organic Chemicals; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000145:classification* / Q000235:genetics
D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D004262:DNA Restriction Enzymes