In the beginning of the 1990’s, an increasing number of outbreaks of foodborne infections was detected and investigated in the United States. Such an increase placed a strain on the resources available at the Central Reference Laboratory at the Foodborne and Diarrheal Diseases Branch, the Centers for Disease Control and Prevention (CDC) for subtyping of outbreak-related isolates. To cope with this situation, PulseNet was developed as a result of the geographic decentralization of molecular surveillance of foodborne infections.in the USA (see PulseNet USA history). This decentralization led to the transfer of subtyping by Pulsed-Field gel electrophoresis (PFGE) from the Central Reference Laboratory at CDC to the local Public Health Laboratories in the states. Subtyping data were analyzed locally and forwarded electronically to CDC for further analysis. Besides shifting some of the subtyping workload away from the CDC, this change benefited foodborne disease surveillance in others ways: isolates were subtyped more rapidly with no delay that would be incurred by transporting them to CDC and the results were simultaneously available for analysis both locally and at CDC. Today, most states perform subtyping for their own isolates, however, the CDC still acts as a national reference laboratory that offers its services to states requesting assistance.
PulseNet plays a vital role in the surveillance and investigation of foodborne illness outbreaks that were previously difficult to detect. By identifying bacterial isolates with indistinguishable subtype through PulseNet, scientists may link cases associated with the same outbreak even if the affected individuals are geographically distant. Outbreaks, along with their sources, can now be identified in a matter of days rather than weeks.
Pulsed-Field Gel Elecrophoresis (PFGE) Fingerprint
PFGE Process showing bacterial suspension, mix with aragose, plug mold, chemical lysis and washing, dna in plugs, restriction enzyme, electrophoresis, documentation capture gel image, data analysis
PFGE is a subtyping method that detects polymorphism in restriction fragments of genomic DNA (macrorestriction). Genomic DNA is restricted by enzymes that recognize short DNA sequences and cut the DNA whenever such a sequence occurs. In this way genomic DNA is cut into a small number (usually 10-20) of restriction fragments in a very specific and reproducible manner.
These DNA fragments are usually too large to separate by conventional agarose gel electrophoresis. In stead, they can be effectively resolved according to their size by a process termed pulsed-field gel electrophoresis (PFGE), developed in the early 80’s.
PFGE facilitates the differential migration of large DNA fragments through agarose gels by constantly changing the direction of the electrical field during electrophoresis. The contour-clamped homogeneous electric field (CHEF) gel electrophoresis method has become the method of choice for resolving DNA macrorestriction fragments of bacterial genomic DNA.
PFGE is a universal method that may be used for subtyping of bacteria with small modifications dependent on the bacterial species investigated. Usually, only the choice of the restriction enzyme and conditions for electrophoresis need to be optimized for each species.
DNA restriction patterns generated by PFGE are stable and reproducible at the intra- and inter-laboratory levels when the method is highly standardized (see section 8 below). Due to its relative simplicity, high discriminatory power and reproducibility, PFGE has become the gold standard for bacterial subtyping.
For PulseNet, the quality and uniformity of the data is ensured by the implementation of a quality assurance and quality control (QA/QC) program. The components of the QA/QC program that allow for the comparison of DNA patterns across all laboratories participating in PulseNet are:
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