Laboratory Diagnosis: Molecular Techniques. Goals Provide an overview of the molecular techniques used in public health laboratories Explain how commonly.

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 SequencesSample 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 ExampleA ribotype image showing two strains of Salmonella Newport (3)

Lane 1: a strain that is

drug-sensitive

Lane 2: a strain that is

drug-resistant

1

2

Differences in the banding pattern indicate that the strains

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

References1. 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.