‫عمل الطالبة‪ :‬حنان عقال الشمري‬
‫إشراف الدكتورة ‪ :‬عفاف شحاتة‬
‫‪1‬‬
Pulsed Field Gel Electrophoresis:
A gel electrophoretic method for the separation of megabase fragments of DNA based on
continuous alteration of the angle at which the electrical field is applied.)9(
Conventional methods of gel electrophoresis are carried out by placing DNA
samples in a solid matrix (agarose or polyacrilamide) and inducing the
molecules to migrate through the gel under a static electric field. When DNA
molecules are under the influence of this electric field, they elongate and align
themselves with the field, migrating toward the anode in a process called
reptation. There are several parameters that affect the migration of DNA
through the gel: concentration and composition of the gel, the buffer, the
temperature, and the voltage gradient of the electric field. In DNA
electrophoresis by the standard method however, DNA molecules larger than
20kb show essentially the same mobility in a static electric field, making
differentiation between these DNA molecules impossible. The first attempts to
resolve these larger fragments included using low percentage agarose gels and
low voltage gradients. Even under these extreme conditions, separation of large
DNA molecules was difficult. In 1984, David Schwartz was able to offer a new
technique. He suggested that periodically changing the orientation of the
electric field would force DNA molecules in the gel to relax upon the removal
of the first field and elongate to allign with the new field. It was his assumption
that this process should be size dependent. Schwartz was finally able to
demonstrate the effectiveness of this technique when he successfully separted
yeast chromosomes that were several hundred kilobases in length)6(
PulseNet is a national network of public health and food regulatory agency laboratories
coordinated by the Centers for Disease Control and Prevention (CDC). The network
consists of: state health departments, local health departments, and federal agencies
(CDC, USDA/FSIS, FDA).
PulseNet participants perform standardized molecular subtyping (or “fingerprinting”) of
foodborne disease-causing bacteria by pulsed-field gel electrophoresis (PFGE). PFGE
can be used to distinguish strains of organisms such as Escherichia coli O157:H7,
Salmonella, Shigella, Listeria, or Campylobacter at the DNA level. DNA “fingerprints,”
or patterns, are submitted electronically to a dynamic database at the CDC. These
databases are available on-demand to participants—this allows for rapid comparison of
the patterns.)2(
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Development of the Technique:
The method of pulsed field gel electrophoresis was first utilized in 1982, and
since then several apparatuses have been developed for separating large
molecules of DNA, all using multiple electric fields. All systems seaparte DNA
molecules within the same size range but differ in the speed of separation and
the resolution. Below are schematic diagrams of the various apparatuses:
Figure 1: Schematic diagrams of published pulsed field gel systems. Nomenclature:
PFGE-pulsed field gradient gel electrophoresis, OFAGE-orthogonal field alternation gel
electrophoresis, TAFE- transverse alternating field electrophoresis, FIGE- field inversion
gel electrophoresis, CHEF- contour clamped homogeneous electric field, crossed field gel
electrophoresis (Waltzer), and ST/RIDE- simultaneous tangential/rectangular inversion
decussate electrophoresis. (Figure 2.1, pg. 8, Pulsed Field Gel Electrophoresis: A
Practical Guide).)6(
In 1993, a large outbreak of foodborne illness caused by the bacterium Escherichia coli
O157:H7 occurred in the western United States. In this outbreak, scientists at CDC
performed DNA "fingerprinting" by pulsed-field gel electrophoresis (PFGE) and
determined that the strain of E. coli O157:H7 found in patients had the same PFGE
pattern as the strain found in hamburger patties served at a large chain of regional fast
food restaurants. Prompt recognition of this outbreak and its cause may have prevented
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an estimated 800 illnesses. As a result, CDC developed standardized PFGE methods and
in collaboration with the Association of Public Health Laboratories (APHL), created
PulseNet so that scientists at public health laboratories throughout the country could
rapidly compare the PFGE patterns of bacteria isolated from ill persons and determine
whether they are similar.)2(
How does PulseNet work:
)
1)PulseNet participants perform DNA "fingerprinting" by pulsed-field gel electrophoresis
(PFGE) on disease-causing bacteria isolated from humans and from suspected food using
standardized equipment and methods.
2) Once these PFGE patterns are generated, they are entered into an electronic database of DNA
fingerprints at the state, local, or federal laboratories. 3a) The patterns are then uploaded to the
national database located at CDC. 3b) All participants who are certified have a direct link to the
national database at CDC. 4) Database managers at CDC perform regular searches, looking for
clusters of patterns that are indistinguishable. The results are reported back to the labs, the
epidemiologists at CDC and if relevant, to the WebBoard, the PulseNet listserv. 5) Laboratorians
perform regular searches on their local databases, looking for clusters of patterns that are
indistinguishable. The results are reported to CDC, the state epidemiologists and if relevant, to the
WebBoard, the PulseNet listserv.)2(
PFGE: The protocol described on this website was designed to account for the
differences in equipment used in the European Union. This method has been used to
examine all 100 strains (83 C. jejuni, 17 C. coli) and profiles produced on two
electrophoresis models (Bio-Rad units DR-III and CHEF Mapper), and two
different agarose grades (standard and high resolution). All duplicate profiles
matched at 100% similarity when subjected to numerical analysis using appropriate
parameters. This database was used as the basis of an interlaboratory trial to test
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the suitability of the system for comparitive purposes between European
laboratories. At the time of the final meeting, two laboratories from the UK and
Sweden each provided two gels that included a number of Campynet strain profiles
for identification via the database established at the Danish Veterinary Laboratory.
Gels from one laboratory were supplied in a non-standard format. One gel from
each lab has for far been analysed, and 30/32 strains correctly assigned to type using
default parameters. The remaining two strains (from the non-standard gel) were
successfully identified using minor changes to the analysis parameters. The nonstandard gels will be rescanned to conform to standard format and reanalysed.
Since this time, data has been supplied from two further laboratories for
comparison. It is intended to complete all interlaboratory comparisons in January
and subsequently assess the potential of a common PFGE typing system for C. jejuni
for use in European laboratories. It was proposed that distinct profiles will be given
SMAP (SMA1 Pfge profile) numbers and strains not digested by this enzyme will be
designated SMAP RD (refractory to digestion). Although duplicate C. coli profiles
were successfully clustered together at 100% similarity, the scheme is not wholly
suited for definitive typing of this species since many of the component fragments
fall outside the frame of reference used for C. jejuni. A paper describing the scheme
will be prepared early in 2002.)3(
How do you prepare the DNA?
Large DNA is very easily sheared and often difficult to pipet due to its high
viscosity. Thus, DNA preparation for PFGE is a bit different from standard
DNA preparation methods. Chromosomal DNA must first be embedded in
agarose plugs and these plugs are treated with enzymes to digest the proteins,
leaving behind the naked DNA. The plugs are then cut to size, treated with
restriction enzymes, loaded into the wells of the gel and sealed into place with
agarose. The link below provides a more detailed description of this procedure
as well as a detailed protocol for running the gel. )6(
Use of the PFGE technique :
 By use of the PFGE technique the number and size of the chromosomal bands
were calculated and the total genome size estimated.
 By use of the PFGE technique it was possible to differentiate between all the
investigated CBS strains and the vast majority of the dairy isolates.
 Further the PFGE technique was proved to have a high discriminative power for
strain typing of D. hansenii.)8(
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ATLANTA (CNN) -- Scientists have developed a genetic fingerprinting process that
helps public officials detect different strains of the potentially deadly E. coli bacteria.
E. coli is a bacteria normally found in all humans, but certain strains, such as 0157:H7,
carry a toxin.
The cutting edge technology is called Pulsed Field Gel Electrophoresis (PFGE), which
relies on genetic fingerprinting to determine if different samples of E. coli are related.
E. coli bacteria from victims of an outbreak are cultured to increase their numbers. The
bacteria's DNA is chemically cut into small pieces, which are separated according to size.
A fluorescent dye illuminates the DNA under ultraviolet light, allowing scientists to
compare different samples.
The new technique proved invaluable to public health officials in Georgia last June,
helping them trace an E. coli outbreak to a popular water theme park in suburban Atlanta.
Using their database of E. coli cultures, scientists separated the water park cases from all
the other E. coli cases the Georgia Department of Health was dealing with.
The PFGE process is only available in a handful states, but is expected to be more widely
adapted in the coming years.
The Centers for Disease Control (CDC) has since established a database of the genetic
fingerprints called Pulsenet, allowing public health officials from across the country to
more easily identify and cope with E. coli outbreaks.
The CDC reports that the dangerous strain of E. coli known as E. coli 0157 sickens up to
20,000 people in the United States each year and kills several hundred. )4(
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What is the Role of PulseNet?
Detect foodborne disease case clusters by pulsed-field gel electrophoresis (PFGE)

Facilitate early identification of common source outbreaks

Assist epidemiologists in investigating outbreaks

Separate outbreak-associated cases from other sporadic cases

Assist in rapidly identifying the source of outbreaks
Act as a rapid and effective means of communication between public health laboratories
What is the PFGE process?
Large DNA is very easily sheared and often difficult to pipet due to its high
viscosity. Thus, DNA preparation for PFGE is a bit different from
standard DNA preparation methods. Chromosomal DNA must first be
embedded in agarose plugs and these plugs are treated with enzymes to
digest the proteins, leaving behind the naked DNA. The plugs are then cut
to size, treated with restriction enzymes, loaded into the wells of the gel
and sealed into place with agarose. The link below provides a more
detailed description of this procedure as well as a detailed protocol for
running the gel.)6(
7
DNA macrorestriction analysis utilizes restriction enzymes that cut genomic DNA
infrequently and thus generates a small number (usually 10-20) of restriction
fragments. These fragments are usually too large to separate by conventional agarose
gel electrophoresis. However, these fragments can be effectively resolved by a
process termed pulsed-field gel electrophoresis (PFGE), developed in 1984 to
separate yeast chromosome-sized DNAs. 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.)2(
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Why is PulseNet important to public health?
PulseNet plays a vital role in surveillance for and the investigation of foodborne illness
outbreaks that were previously difficult to detect. Finding similar patterns through
PulseNet, scientists can determine whether an outbreak is occurring, even if the affected
persons are geographically far apart. Outbreaks and their causes can be identified in a
matter of hours rather than days.
Setting the Parameters:
When running a gel using a PFGE system there are several parameters that
must be considered in order for the proper setup to be established. For example,
the voltage gradient must be altered according to the size of the sample to be
electrophoresed. Larger DNA samples require lower voltage gradients in order
to migrate properly through the gel. When choosing an agarose it is also
important to keep the size of the sample in mind. For separation of molecules
larger than 2.5Mbp a low EEO agarose certified for molecular biology is
suffieient, however, for larger molecules "pulsed field" agaroses are better
because of the reduced run times. Temperature also affects the DNA mobility
within the gel. Raising the temperature increases the mobility and 12-15
degrees Celcius is the most frequently used temperature range. Furthermore, it
must be taken into consideration that DNA will migrate more quicly in buffers
of low ionic strength. Finally, one of the PFGE apparatuses shown above must
be selected (Birren et al., 1993). Seen below are examples of gels run using the
FIGE system (Figure 2) and the RGE system (Figure 3).
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Figure 2. Increased separation of the 20-50 kb range with field inversion gel
electrophoresis (FIGE). Run conditions: 230 V, 7.9 V/cm, 16 hrs., 50 msec. pulse,
forward:reverse pulse ratio = 2.5:1, 1% GTG agarose, 0.5X TBE, 10 C. a) 1 kb ladder,
0.5-12 kb; b) Lambda/Hind III, 0.5-23 kb; and c) High molecular weight markers, 8.348.5 kb (Permission pending for the use of this image).
Increasing both the separation range and the resolution of large DNA requires
smaller reorientation angles, generally 96-140ø, with 120ø most common.
Smaller angles (e.g., 100ø) increase the mobility of the DNA generally without
seriously affecting resolution. The lower limit is approximately 96ø. Below
this, separation is seriously compromised. (HSI Laboratories, Hoefer Scientific
Instruments)
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Figure 3. Rotating gel electrophoresis (RGE) separation Saccharomyces cercevisiae
chromosomes (245-2190 kb). Run conditions: 180 V, 5.1 V/cm, 34 hrs., 120 angle, 60120 sec. pulse ramp, 0.5X TBE, 1.2% GTG agarose, 10 C. Two combs were used on the
same gel to load 32 samples, a maximum of 72 are possible (Permission pending for the
use of this image)6(.
How does subtyping help in epidemiologic investigations:
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
Identifies cases within an outbreak

Distinguishes outbreak cases from concurrent sporadic cases


Reduces misclassification
Detects outbreaks through surveillance

Links apparently sporadic cases

in which the cases are too widely dispersed to detect

Organism too common to notice small increase

Identifies related cases and separates them from unrelated ones

DNA “fingerprinting” methods have greatly increased sensitivity of subtyping
Food consumption and practices have changed during the past 20 years in the United
States. We are observing a shift from the typical point source, or “church supper”
outbreak, which is relatively easy to detect to the more diffuse, widespread outbreaks that
occur over many communities with only a few illnesses in each community.
For example, we have observed the establishment of large food producing facilities that
disseminate products throughout the country. We have seen in a few outbreaks that some
low level contamination of food products can occur, and the products are distributed
among many states. Only a few illnesses occur in each community, and this new style of
outbreak is often difficult to detect. However, new laboratory and statistical tools, such as
PulseNet and the surveillance outbreak detection algorithm (SODA), have had an impact
on our ability to identify and investigate these new types of outbreaks
What makes interlaboratory comparison of DNA patterns
possible?
For PulseNet, the quality and uniformity of the data is ensured by the implementation of a
quality assurance and quality control (QA/QC) program. Here are components of the
QA/QC program that allow for the comparison of DNA patterns across all labs:
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
Standardized protocols

QA/QC Manual

Same molecular size standards

Standardized software used by all participants

Standardized nomenclature of PulseNet patterns

Training workshops (lab & software): most participating labs have attended a
week of combined laboratory and analysis software training

Certification: all individuals who submit data must be certified by stringent
PulseNet standards

Proficiency testing: all certified individuals must participate and pass annual
proficiency testing in order to maintain certification

Annual update meetings: provide a forum for the live exchange of information
What are future applications for Pulse?

Increase the number of PulseNet participants

Achieve real-time subtyping and real-time communication

Reduce the time it takes for isolates to go from the clinical lab to the state/local
public health lab

Reduce the time for pulsed-field gel electrophoresis (PFGE) testing of isolates

Critical for timely detection of clusters

Increase the level of communication between laboratorians and epidemiologists

Timely assignment of PulseNet designations for PFGE patterns

Improve bandmarking among all labs

Strengthen collaborations with the food industry

Future protocols

Vibrio parahaemolyticus/V. cholerae

Yersinia enterocolitica

New subtyping methodology

Multiple-locus variable-number tandem-repeats analysis (MLVA)
)2(
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
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Pulsed-field gel electrophoresis (PFGE) can detect genetic differences between various
strains of Mycobacterium avium subsp. paratuberculosis and therefore is a useful tool for
epidemiological studies of paratuberculosis. In order to compare the epidemiological data
from different laboratories and obtain a global prospective, it is imperative that a
standardised procedure and appropriate nomenclature are used. We have optimised a
standard operating procedure for the PFGE analysis of M.a.paratuberculosis and
suggested a system of nomenclature that could be adopted by laboratories using this
technique for genotyping. We have set up this database as a resource for all interested
parties. We will continue to add our own data to the database and maintain a reference
panel of strains representative of the different PFGE profiles described. We encourage
other laboratories to use this facility and contribute their own data. We will assign
numbers to any new PFGE profiles identified and add the data to the database,
acknowledging the source of data and the authors.
Left-hand gel shows the profiles obtained from non-pigmented isolates and the righthand gel shows the pigmented ovine isolates. Numbers above the lanes refer to the
reference isolates, details of which are given in Table 1. Numbers below the lanes denote
the different PFGE SnaB I reference types. Profiles defined and published by Stevenson
et al. (2002)
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Left-hand gel shows the profiles obtained from non-pigmented isolates and the righthand gel shows the pigmented ovine isolates. Numbers above the lanes refer to the
reference isolates, details of which are given in Table 1. Numbers below the lanes denote
the PFGE Spe I reference types. Profiles defined and published by Stevenson et al. (2002)
)5(
tandardized protocols for foodborne bacterial pathogens were developed in priority
order based on the ability of PFGE to discriminate among strains of the organism and the
epidemiologic utility of the resulting data. Standardized PFGE protocols have been
developed for E. coli O157:H7, Salmonella enterica serotype Typhimurium, L.
monocytogenes, and Shigella species. The S. Typhimurium protocol is applicable to most
other nontyphoidal Salmonella serotypes, including S. Enteritidis. However, neither
PFGE nor other molecular subtyping methods provide acceptable discrimination among
strains of this highly clonal serotype. Standard PFGE protocols for Campylobacter jejuni,
C. coli, and Clostridium perfringens[7] are being developed and validated. Although C.
jejuni and C. coli infections are common, developing a standardized PFGE protocol for
these organisms was not a high priority because they infrequently cause outbreaks. On
the other hand, although outbreaks of C. perfringens infections are seldom widespread,
state and local public health laboratories requested a standardized subtyping protocol to
assist with local outbreak investigations. All PulseNet protocols are 1-day procedures
based on the PFGE protocol developed by the Washington State Public Health
Laboratory in response to the need for more rapid techniques[16]. All new protocols and
modifications of existing protocols are evaluated initially at the developing laboratory,
followed by a second evaluation at CDC, alpha-testing at one or two PulseNet
laboratories, and beta-testing at several PulseNet laboratories before they are adopted as
official PulseNet protocols. Evaluation criteria include reproducibility of patterns,
appropriateness of the strain used as the reference standard, and robustness of the
procedure. Once a protocol is officially adopted, no changes can be made except by a
petition to CDC's PulseNet Task Force, discussion of the proposed changes, and adoption
of the proposal by PulseNet laboratories. The PulseNet Task Force at CDC is composed
of personnel who carry out PulseNet-related activities. The Task Force members develop
and evaluate protocols, provide technical support for participating laboratories, organize
and conduct training workshops, administer the certification program and proficiency
testing program, and maintain the national databases of PFGE patterns for the bacteria
under surveillance in PulseNet.)7(
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Diseases caused by Vibrio species have greatly forged the management practices
associated with aquaculture and public health. Of primary concern is the potential for the
transmission of zoonotic pathogens from aquacultured seafoods to humans. One species,
Vibrio alginolyticus, causes illness in a variety of marine seafood species; and can also
cause septicemia, wound infections and gastroenteritis in humans. A study was initiated
to reveal the types of virulence factors expressed by 23 isolates obtained from various
fish species and sources. The organisms were isolated and identified using standard
methods as well as API20E and MIDI microbial identifications systems. The strains
were characterized further by pulsed field gel electrophoresis (PFGE) and plasmid
analyses. Additional information was collected on surface hydrophobicity of the
organisms; and the expression of hemagglutinins, hemolysins, cytotoxins, enterotoxins,
proteases, and siderophores. PFGE analysis using SwaI revealed 22 different genotypes
among the strains. 91% of the strains possessed plasmids. The cell surfaces of all
strains, except for one, were hydrophilic; and 66% of the strains could agglutinate sheep
erythrocytes (RBCs). Only one isolate was hemolytic for sheep, chicken and rabbit
RBCs. Another single isolate was hemolytic for chicken, rabbit, guinea pig, and calf
RBCs. All other isolates were hemolytic for both chicken and rabbit RBCs. Only one
isolate exhibited caseinolytic activity, while all isolates produced siderophores. Results
using Chinese hamster ovary (CHO) cells in tissue culture showed that some of the
strains produced factors that elongated CHO cells, but only a few isolates produced
factors that caused rounding and/or death of the cells. In summary, these results
demonstrate that the pathogenic V. alginolyticus strains isolated from ill fish are
genetically diverse; that most of the strains express multiple virulence factors, including
hemagglutinins, hemolysin(s), and siderophores; and that the organisms also produce
other factors that are cytotonic and cytotoxic toward CHO cells.)10(
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Pulsed-field gel electrophoresis (PFGE) is a powerful molecular biology technique which
has provided important insights into the epidemiology and population biology of many
pathogens. However, few studies have used PFGE for the molecular epidemiology of
Mycobacterium tuberculosis. A laboratory protocol was developed to determine the
typeability, stability, and reproducibility of PFGE typing of M. tuberculosis. Formal dataanalytical techniques were used to assess the genetic diversity elucidated by PFGE
analyses using four separate restriction enzymes and by IS6110 RFLP analyses, as well as
to assess the concordance among these typing methods. One hundred epidemiologically
characterized clinical isolates of M. tuberculosis were genotyped with four different
PFGE enzymes (AseI, DraI, SpeI, and XbaI), as well as by RFLP analysis with IS6110.
Identical patterns were found among 34 isolates known to be genetically related,
suggesting that the PFGE protocol is robust and reproducible. Among 66 isolates
representing population-sampled cases, heterozygosity and information content
dependency estimates indicate that all five genotyping systems capture quantitatively
similar levels of genetic diversity. Nevertheless, comparisons between PFGE analyses
and IS6110 typing reveals that PFGE provided more discrimination among isolates with
fewer than five copies of IS6110 and less clustering in isolates with five or more copies.
The comparisons confirm the hypothesis that the resolution of IS6110 RFLP genotyping
is dependent upon the number of IS6110 elements in the genome of isolates. The general
concordance among the results obtained with four independent enzymes suggests that
M. tuberculosis is a clonal organism. The availability of a robust genotyping technique
largely independent of repetitive elements has implications for the molecular
epidemiology of M. tuberculosis.)1(
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Reference:
1- Use of Pulsed-Field Gel Electrophoresis for Molecular
Epidemiologic and Population Genetic Studies of
Mycobacterium tuberculosis-- Samir P. Singh, Hugh
Salamon, Carol J. Lahti, Mehran Farid-Moyer, and Peter M.
Small—1999—www.JCM.com
2- What is PulseNet—www.CDM.com
3- CAMPYNET NEWS –2001—www.campynet.htm
4- Genetic fingerprinting technique helps identify
E.coli bacteril—1998-www.cnn.com
5-Bacteriology—www.moredun.com
6-Pulsed Field Gel Electrophoresis
7-Developing Standardized Protocols—
www.medscapetoday.com
8-Titre du document / Document titlewww.CAT.INIST.FN.COM
9-WWW.G&P.COM
10- Characterization of Virulence Factors Expressed by
Vibrio alginolyticusIsolates Obtained from Teleosts and
Elasmobranchs Housed in a Public quarium and
Aquaculture Facility.-- . D. Tall , M. H. Kothary , B. A.
McCardell , S. Zhao , J. Abbott , P. Whittaker , S. K. Curtis
, J. Arnold , .. HACU Intern Team , .IFSAN Intern Team , U.S.
FDA, Laurel, MD 20708 , U.S. FDA, College Park, MD 20740
, Nat. Aquarium, Baltimore, MD 21202.-WWW.fda science.com
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