Laboratory Diagnosis: Molecular Techniques

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Laboratory Diagnosis Molecular Techniques
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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

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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

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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 partnerA with T and C
  with G
• Together they are known as base-pairs

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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)

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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 cells building machinery

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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

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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

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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 organisms DNA
• PCR can start with very small amounts of DNA and
  can be used with viruses or bacteria

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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

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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 occursDNA in specimen matched
  primer
• No amplificationparticular DNA that primer was
  designed to match was not present

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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

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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

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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

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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
  atbhttp//www.ncbi.nlm.nih.gov/entrez/query.fcgi
   

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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

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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

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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

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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

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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

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PCR GelExample

• 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)

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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

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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

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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

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PFGEExample

• Outbreak of Escherichia coli O157H7 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

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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

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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

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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

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Ribotyping Example
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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.

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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

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References

• 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.
  1995613849-3855.
• Centers for Disease Control and Prevention.
  Multistate outbreak of Escherichia coli O157H7
  infections associated with eating ground beef ---
  United States, June--July 2002. MMWR Morb Mort
  Wkly Rep. 200251637-639. Available at
  http//www.cdc.gov/mmwr/preview/mmwrhtml/mm5129a1.
  htm. Accessed November 30, 2006.
• 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. 20039496-499.
  Available at http//www.cdc.gov/ncidod/EID/vol9no
  4/02-0423.htm. Accessed December 14, 2006.