Pulsed-field gel electrophoresis
Pulsed-field gel electrophoresis (PFGE) is a sophisticated DNA fingerprinting technique that allows researchers to separate large DNA fragments, typically over thirty kilobase pairs, within an agarose gel. By applying an alternating electric field, this method generates a distinctive pattern of DNA bands, which can vary in size based on genetic differences among organisms. PFGE is particularly useful in analyzing genetic variation, identifying different strains of bacteria, and monitoring outbreaks of infectious diseases. The technique has applications in various fields, including food safety, cancer research, and microbial genetics, where it helps trace the source of infections and assess genetic stability in food products.
In public health, PFGE played a crucial role during foodborne illness outbreaks by linking bacterial strains from patients to contaminated food sources, thereby aiding in rapid response efforts. The Centers for Disease Control and Prevention (CDC) has standardized PFGE protocols, facilitating global consistency in its application. While PFGE remains an important tool in epidemiology, advancements such as Whole Genome Sequencing (WGS) are increasingly being adopted for more detailed genetic analysis. Overall, PFGE continues to be a valuable method for understanding genetic relationships and monitoring public health threats.
Pulsed-field gel electrophoresis
- ALSO KNOWN AS: Deoxyribonucleic acid (DNA) fingerprinting
Definition
Pulsed-field gel electrophoresis (PFGE) is a DNA fingerprinting technique that separates large DNA fragments (of more than thirty kilo-base pairs) in an agarose gel by applying an alternating electric field among pairs of electrodes. The DNA fragments ultimately form the distinctive pattern of bands in a column, or lane, in the gel. Small DNA fragments form bands at the bottom, and large DNA fragments form bands at the top of the gel. Genetic variation among strains causes fragments of differing sizes to be produced when DNA is digested with enzymes called restriction endonucleases. PFGE can thus detect differences in DNA as minor as one base change.
Genetic variations revealed after digestion with restriction endonucleases are called restriction fragment length polymorphisms. The pattern of banding is analyzed visually, then it is digitized and archived to determine the relatedness of biological samples.
Applications
PFGE is used, for example, in quality control of the genetic identity and stability of grapes and hops fermented for wine and beer and in quality control of the microbes used in food production. Cancer researchers use the technique to analyze damage to DNA caused by chemicals and radiation. Genetics research laboratories use the technique to map genetic defects routinely. Outbreaks of infectious diseases can be monitored to determine the similarity of the bacterial strains involved. While genetic matching does not ensure that isolates from two infected persons came from the same source, epidemiologists can detect the rise in prevalence of a bacterial strain when an outbreak does occur. Microbes infecting hospitalized persons can be analyzed to determine if multiple patients are affected by the same strain, raising the possibility the hospital itself is the source of the infection. PFGE is also used for bioterrorism investigations, to map and understand genetic diseases, and in myriad fields, including ecology, biology, veterinary science, and plant pathology.
Public Health
In 1992, an outbreak of food-borne illness occurred in the western United States. Using traditional microbiological techniques, the bacterium Escherichia coli O157:H7 was implicated in the outbreak. The PFGE patterns of isolates from infected persons and hamburger patties from the restaurant where those persons ate were the same. Scientists concluded that the infected persons had acquired the E. coli from that particular restaurant.
The Centers for Disease Control and Prevention (CDC) led the effort to develop a standardized protocol for PFGE for analysis of clinical isolates of E. coli. The materials, methods, and controls are now standardized and can be used to provide reliable results worldwide. The protocol served as a platform for the development of PFGE typing of a host of microbial pathogens.
The CDC then developed PulseNet, a national health and food regulatory agency lab network. The lab networks perform PFGE fingerprinting of foodborne bacteria. In addition to E. coli, Salmonella, Shigella, Listeria, and Campylobacter are fingerprinted, and their patterns are entered into the CDC database. Member labs can access the database in real-time and compare clinical specimens.
Impact
PFGE and PulseNet allow for the far more rapid identification of food-borne disease clusters in as little as a week. The origin of the infection can be identified and isolated, preventing further transmission. PFGE has also been used to trace the source and spread of antibiotic-resistant tuberculosis. Epidemiologists learned that the infected persons had acquired the infection in the hospital. While PFGE remained a vital tool, Whole Genome Sequencing (WGS) began to replace its application in some cases of outbreak investigation and surveillance of foodborne pathogens. This updated technique analyzed entire DNA sequences, was more accurate, and could be used with advanced data analysis tools. PulseNet began transitioning to WGS from PFGE in 2017, and many worldwide public health organizations also began to switch.
Bibliography
Brachman, Philip S., and Elias Abrutyn, editors. Bacterial Infections of Humans: Epidemiology and Control. 4th ed., New York: Springer, 2009.
Coveny, McKenna Madison. "WGS and PFGE DNA Testing: What is the Difference?" Food Poisoning News, 25 July 2023, www.foodpoisoningnews.com/wgs-and-pfge-dna-testing-what-is-the-difference. Accessed 13 Oct. 2024.
"Detecting Outbreaks with Whole Genome Sequencing - Advanced Molecular Detection (AMD)." CDC, 4 Mar. 2024, www.cdc.gov/advanced-molecular-detection/about/detecting-outbreaks.html. Accessed 13 Oct. 2024.
Forbes, Betty A., Daniel F. Sahm, and Alice S. Weissfeld. Bailey and Scott’s Diagnostic Microbiology. 12th ed., St. Louis, Mo.: Mosby/Elsevier, 2007.
"Outbreak Detection - PulseNet." CDC, 18 Mar. 2024, www.cdc.gov/pulsenet/hcp/about/outbreak-detection.html. Accessed 13 Oct. 2024.
Tortora, Gerard J., Berdell R. Funke, and Christine L. Case. Microbiology: An Introduction. 10th ed., San Francisco: Benjamin Cummings, 2010.