Laboratory Diagnosis: Molecular Techniques

1. Laboratory Diagnosis: Molecular Techniques

2. 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

3. 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

4. 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

5. 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)

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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)

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

29. Ribotyping Example A ribotype image showing two strains of Salmonella Newport (3) Differences in the banding pattern indicate that the strains are different. Lane 1: a strain that is drug-sensitive Lane 2: a strain that is drug-resistant 1 2 Similarities in the banding pattern indicate that the species of bacteria is the same (Salmonella Newport).

30. 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.

31. 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

32. 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. 1995;61:3849-3855. • 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. • 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.