Laboratory Diagnosis:

Molecular Techniques
Goals
 Provide an overview of the molecular
techniques used in public health
laboratories
 Explain how commonly used molecular
techniques such as PCR, PFGE, and
ribotyping are used in outbreak
investigations
What is DNA?
 DNA stands for deoxyribonucleic acid
 DNA is a twisty, ladderlike molecule termed a
‘double helix’
 DNA is the genetic material present in
bacteria, plants, and animals and provides the
code used to build the molecules that make
up a living being
 Some viruses also have DNA while others use
RNA as their genetic material
DNA Structure
 DNA is made up of 4 molecular units called
bases. The bases are:
 Adenine (A)
 Thymine (T)
 Cytosine (C)
 Guanine (G)
 Each base is linked with a partner—A with T
and C with G
 Together they are known as base-pairs
DNA Structure
 Bases are arranged in an exact order
called a sequence
 Example: AATTCGCG or CATAGCGTA
 A particular sequence is like a recipe for
the protein that will be created by that
particular piece of DNA
 DNA can also code for RNA but in RNA
T (thymine) is replaced by U (uracil)
DNA Replication
 To replicate DNA or create proteins, the two
sides of the DNA ladder separate from each
other and new bases pair up with the existing
sequence
 In living cells RNA serves as the copy
messenger to DNA
 From the DNA template a cell makes a copy of
RNA
 RNA then circulates around the cell carrying the
code to all parts of the cell’s building machinery
Why is DNA Useful in
Epidemiology?
 DNA sequences can be used to identify
an organism causing a disease outbreak
 Certain DNA sequences are unique to each
organism
 Samples can be tested for the presence of
DNA from different organisms
DNA Testing
 DNA sequences can vary between different
strains of the same organism
 Comparing variation in certain sequences can
help distinguish one strain from another
 For example, if Norovirus is identified in two
cases of gastrointestinal illness, they may (or
may not) be part of the same outbreak
 DNA testing can help determine whether the same
strain is present in both cases and therefore
whether the cases are related
Polymerase Chain Reaction
(PCR)
 Using molecular techniques such as PCR to
examine DNA sequences can help to identify
what strain of a pathogen is present in a
specimen
 PCR is a technique that makes multiple copies
of a piece of DNA or RNA in a process called
amplification
 Amplification makes it easier to detect the
tiny strands of an organism’s DNA
 PCR can start with very small amounts of
DNA and can be used with viruses or bacteria
Steps in PCR
 PCR starts with a sample of DNA from a
clinical specimen suspected to contain a
pathogen
 A primer is added to the sample
 A primer is a very short sequence of DNA which
will seek out and bind to a specific sequence of the
target DNA
 Primers can be designed to be very specific or
more general
 Example – a primer could be made to “match”
echovirus 30 or to match any echovirus
Steps in PCR (continued)
 After the primer other materials added to the mixture
include:
 A polymerase enzyme that will “read” a sequence of DNA
and create copies
 “Building blocks” of DNA bases to use as raw materials to
make copies
 The polymerase enzyme will make copies only of the
DNA that matches the primer
 Results:
 Amplification occurs—DNA in specimen matched primer
 No amplification—particular DNA that primer was designed
to match was not present
PCR Example
 If you believe Salmonella is causing an
outbreak of diarrheal illness you would
amplify a gene that is unique to Salmonella
 After the PCR reaction you would use the
genes amplified by PCR to confirm the
organism is Salmonella
 Note: It is important to ensure that proper
collection, shipment and storage of your
sample have taken place
Sequencing DNA
 If you are still unsure what the infecting
organism might be after PCR you
probably ran a non-specific PCR reaction
and amplified whatever genetic material
was present
 The next step would be to sequence the
DNA with the genetic material obtained
from amplification
Sequencing DNA
 You can determine the specific order of
the bases in the DNA strand(s) that you
amplified
 This particular sequence can then be
compared with known sequences of an
organism or strain
DNA Sequences
Sample Comparison of the DNA sequences of a nucleoprotein gene
in infections of two patients with different strains of rabies

A. Gene sequence AY138566; rabies virus isolate 1360, India

B. Gene sequence AY138567; rabies virus isolate 945, Kenya
 
 Line 1a gaaaaagaac ttcaagaata tgagacggca
 Line 1b gagaaagaac ttcaagaata cgagacggct

 Line 2a gaattgacaa agactgacgt agcgctggca

 Line 2b gaactgacaa agactgacgt ggcattggca

 Line 3a gatgatggaa ctgtcaattc ggatgacgag

 Line 3b gatgatggaa ctgtcaactc tgacgatgag 

 Full sequence available from query at:bhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi  

DNA Sequences
 The DNA sequence amplified may be that of a
known gene from a specific organism
 Example: laboratory suspects Salmonella and
runs the experiment to amplify the DNA of a
Salmonella gene
 Gene will be amplified if Salmonella is infecting
organism
 Gene will not amplify if Salmonella is not the
infecting organism
PCR Gels
 After PCR amplification the laboratory
technician will run the PCR product on a
special gel that helps visual the DNA
 With a known gene, you know how big the
sequence is
 When sample DNA is seen on a gel, it can be
determined whether the gene is present and
whether it has the correct length segment
and is the expected organism
PCR Gels & DNA Fingerprinting
 The pattern of DNA as it appears on a gel is
called the DNA fingerprint
 DNA fingerprinting is done when a specific
organism is suspected in order to determine
which strain of the organism is present
 Example --Tuberculosis (TB) has very specific
symptoms
 DNA fingerprinting could help determine whether
different TB cases are infected with the same
strain due to an outbreak or common
exposure
How Do Gels Work?
 PCR product is placed in a lane at one end of the gel
 A small electric field is applied which causes the DNA
to migrate from one end of the gel to the other
 The distance traveled by DNA depends on the
sequence and the length of the piece(s) of DNA
 DNA bases have natural electrical charges that determine
speed and direction
 Different sized pieces of DNA move faster/slower
 After a defined time period the electric field is turned
off, freezing the DNA “race” so that the DNA pattern
can be examined
How Do Gels Work?
 Special techniques are used to look at
the clusters of DNA which appear as
solid bands in the gel
 Different organisms have different DNA
patterns
 If samples taken from different patients
have the same DNA pattern, these people
were infected with the same organism
PCR Gel—Example
 Picture of a PCR gel for
diagnosing Cryptosporidium
parvum from a fecal sample
 Each dark band represents
many strands of DNA that are
the same length.
 The lane marked “S” is a DNA
ladder; each band shows DNA
strands with a specific number
of base pairs that can be used
to measure the length of DNA
amplified in the PCR reaction.
 In this case, the 435 base pair
band from C. parvum is a
positive identification. (1)
Pulsed Field Gel
Electrophoresis (PFGE)
 DNA can also be detected by pulsed field gel
electrophoresis (PFGE) which is used for the
analysis of large DNA fragments
 PFGE requires less processing and sample
preparation of the DNA
 To perform PFGE special enzymes can be used to
cut the DNA into a few long pieces
 Instead of applying an electric field so that DNA
fragments race straight to the end, after the
electrical field is applied the direction is changed
several times
PFGE
 PFGE is like a race with only large, slow-
moving runners
 At the start they are so slow and large they
appear only as a mass of runners
 The finish line gets moved to different places and
the “runners” re-orient each time
 Switching directions separates the runners
(the DNA pieces) into two different planes
and separates out the DNA more distinctly
PFGE
 PFGE is used to identify bacteria but not
viruses
 DNA used for PFGE analyses can be
extracted from a microorganism in
culture, a clinical specimen or an
environmental specimen
 Like regular gels, PFGE can be used to
identify an organism or to distinguish
between strains of the same organism
PFGE—Example
 Outbreak of Escherichia coli O157:H7
infections among Colorado residents in June
2002. (2)
 Case definition required that E. coli be cultured
from the patient AND that all cultures exhibit the
same PFGE pattern
 Example of how molecular techniques were used
to fine-tune a case definition
 PFGE patterns are often used this way to link
cases in an outbreak
 PFGE can not be used to fingerprint every bacterial
organism but can be used with a wide variety
of pathogens
Ribotyping
 Ribotyping is another molecular diagnostic
technique.
 Name derives from the ribosome which is part of
the cellular machinery that creates proteins
 Ribotyping can be used to identify bacteria only,
not viruses
 Ribosomes are found only in cells
 Viruses have no cellular structure but are molecules with
genetic material and protein only
Ribosomes & RNA
 A ribosome is composed of RNA that is folded up in a
particular way
 This is referred to as “rRNA” for ribosomal RNA
 DNA codes for RNA and since a wide variety of living
cells create proteins, the DNA genes that code for
rRNA have a lot in common, even across different
species
 Some parts of the (DNA) genes that code for rRNA are
highly variable from one species to the next or between
strains of bacteria
 These variable regions can therefore be used to identify a
particular strain of bacteria
Ribotyping
 How are the variable regions of rRNA
determined?
 DNA-cutter enzymes are used to divide the RNA
only when a specific sequence occurs
 If a strain of bacteria has that sequence in its rRNA,
the rRNA strand will be cut at that location
 The rRNA is then run on a gel so that the number
and size of the pieces can be seen
 rRNA that has been cut in the expected locations
will appear different from rRNA that was not cut
Ribotyping Example
A ribotype image showing two strains of
Salmonella Newport (3)

1 Differenc
Lane 1: a es in the
strain that is banding
drug-sensitive pattern
indicate
Lane 2: a 2 that the
strain that is strains
drug-resistant are
different.

Similarities in the banding pattern indicate that

the species of bacteria is the same (Salmonella
Newport).
Ribotyping
 Advantages of ribotyping as an identification
method:
 Ribotyping is a fully automated procedure
 Procedure involves less labor and is standardized
 Disadvantages of ribotyping:
 Expensive because of the equipment used, therefore
usually only performed in reference laboratories
 Ribotyping is most commonly used for typing
strains of Staphylococcus aureus, but it can also be
used for typing other species of Staphylococcus and
for E. coli.
Summary
 This has been an overview of molecular
techniques, i.e., laboratory analyses that
use DNA or RNA.
 A future issue of FOCUS will provide
further information on the use of these
techniques in an outbreak setting and
provide examples from real
investigations
References
1. Johnson DW, Pieniazek NJ, Griffin DW, Misener L, Rose JB.
Development of a PCR protocol for sensitive detection of
Cryptosporidium oocysts in water samples. Appl Environ Microbiol.
1995;61:3849-3855.
2. Centers for Disease Control and Prevention. Multistate outbreak of
Escherichia coli O157:H7 infections associated with eating ground
beef --- United States, June--July 2002. MMWR Morb Mort Wkly
Rep. 2002;51:637-639. Available at:
http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5129a1.htm .
Accessed November 30, 2006.
3. Fontana J, Stout A, Bolstorff B, Timperi R. Automated ribotyping
and pulsed field electrophoresis for rapid identification of
multidrug-resistant Salmonellas Serotype Newport. Emerg Infect
Dis [serial online]. 2003;9:496-499. Available at:
http://www.cdc.gov/ncidod/EID/vol9no4/02-0423.htm . Accessed
December 14, 2006.