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Molecular Diagnostic Methods Recombinant DNA Technology Nucleic Acid Test for Infectious Disease • Accurate to ID MO – Specific – DNA, RNA – Sensitive - detect low number • Timely ID MO causing disease • Rapid start proper clinical treatment in patient • Guide Public Health to prevent spread of infection Utility Nucleic Acid Test • ID nonculturable agents: – Human papilloma virus – Hepatitis B virus • ID fastidious, slow-growing agents: – Mycobacterium tuberculosis – Legionella pneumophilia • ID highly infectious agents dangerous to culture: – Francisella tularensis – Brucella melitensis – Coccidioidis immitis Utility Nucleic Acid Test • In situ detection of MO: – Helicobacter pylori (stomach) – Toxoplasma gondii (intracellular protozoan) • MO present low number: – Human immunodeficiency virus (HIV) in antibody negative patient – Cytomegalovirus (CMV) in transplant organ • Differentiate antigenically MO • Detect specific virus genotypes associated with human cancers (Papilloma virus 16, 18) • MO in small volume specimens: – Intra-ocular fluid – Forensic sample Utility Nucleic Acid Test • Antiviral drug susceptibility testing: – Decide therapy to use in HIV drugresistant strain • Molecular epidemiology: – Identify point source for hospital and community-based outbreak – Predict virulence • Non-viable MO: – In very labile, readily inactivated – In immune complexes • Confirm positive lab culture isolate Nucleic Acid Molecular Techniques • Direct probe testing: – Short specific nucleic acid sequence – Better for ID than for detection – Not as sensitive as amplification methods • DNA Amplification methods: – Make more copies of DNA – Improve sensitivity of nucleic acid testing – Amplification of: Target (DNA), Probe, Signal, or combinations DNA Structure: Double Helix • Double strand linear DNA: – 5’ carbon at one end – 3’ OH group at other end • Two strands “opposite” orientation with respect to each other (antiparallel, complementary) • Adenine base-pair with thymine (2 hydrogen bonds) • Guanine base-pair with cytosine (3 hydrogen bonds) DNA Helix: Hydrogen Bonds Hybridization: Direct Probe Testing • Hybridization – to come together through complementary base-pairing • Used in ID • Colony hybridization – MO colony treated to release nucleic acid, denatured to single strands • Add single-stranded fluorescent labeled (ss)DNA (probe) unique to test MO • Hybridization allowed to occur • Unbound probe washed away, bound probe detected by presence of fluorescent label Target DNA Amplification: Polymerase Chain Reaction • Number of copies of target DNA increased by in vitro enzyme synthesis • Use cell DNA polymerase to copy target DNA • DNA polymerase cannot initiate synthesis on its own • Need primer to start enzyme reaction • Primer - single strand piece of DNA (or RNA) complementary to unique region of sequence copied DNA Synthesis • Synthesis occurs only in 5’ to 3’ direction DNA Replication Semiconservative • One ds parent strand copied • Two ds parent:daughter strands Polymerase Chain Reaction (PCR) • Enzyme reaction to amplify DNA: – Two primers - one binds one strand of dsDNA, other binds other strand – Four nucleotide DNA precursors (dNTPs) – Thermostable DNA polymerase – Buffer (ions, salts) for enzyme reaction • Primers (~15-20 bases) unique sequence to DNA being amplified • PCR reaction has three basic steps: – 1) Denaturation – 2) Primer Annealing – 3) Primer Extension PCR Assay: 1) Denaturation • Separate dsDNA into ssDNA • Heating 950 C, 15 seconds to 1 minute • The two ssDNA strands serve as templates for DNA synthesis PCR Assay: 2) Primer Annealing • DNA comes together through complementary base-pairing (hybridization) • Primers base-pair with their complementary sequences on ssDNA template • Primer concentration in excess of DNA template concentration • Excess primer ensures base-pairing with complementary sequence on template DNA instead of ssDNA templates base-pairing back together Primer Annealing • Temperature used should ensure annealing will occur only with DNA sequences that are completely complementary. WHY? • The annealing temperature depends upon length and sequence of primers • The longer the primer and the more Gs and Cs in the sequence, the higher the annealing temperature. WHY? • Annealing temperature range from 45-65ºC • Annealing time usually 15 seconds to 1 minute PCR Assay: 3) Primer Extension • DNA polymerase use dNTPs to synthesize DNA complementary to template DNA • Thermostable Taq DNA polymerase (isolated from Thermus aquaticus) extends annealed primers • Temperature used is 720 C, optimum reaction temperature for Taq DNA pol • The extension time is usually 15 seconds to 1 minute • Why is this high temperature used? • Why is a thermostable polymerase used? PCR Assay • The combination of denaturation, primer annealing, primer extension constitute 1 cycle in a PCR reaction • Exponential increase of DNA with each cycle (2, 4, 8, 16, 32, 64, 128, etc.) • PCR assay uses 25 to 30 cycles to amplify target DNA > 1 million copies • Assay time ~5-6 hr. PCR Product DNA Analysis • DNA agarose gel electrophoresis – ID DNA molecular weight by migration in gel – Estimate size using DNA markers (100 bp ladder; 100-1500 bp) – Assay time ~1-2 hr. • Real-Time PCR – ID DNA during PCR assay in closed tube – Use fluorescent reporter dye released during enzyme reaction – Thermal Cylcler with UV light to excite reporter dye and camera for detection – Assay time ~1 hr. MICR 302 Lab PCR Exercise • First PCR assay will amplify DNA sequence in bacterial 16S ribosomal RNA (rRNA) gene • Use primers for DNA consensus sequence found in all bacteria • Primers not unique to a specific bacteria, but unique to a conserved region of 16S rRNA gene of all bacteria Bacterial 16S rRNA Gene: ID Unknown Bacteria • Amplify portion of 16S rRNA gene of unknown bacteria • DNA of all bacteria are amplified and yield DNA product using the consensus primers • Sequence of the amplified DNA will be determined by Automated DNA Sequencer • Identity of unknown found by searching and matching your result in the National Institutes of Health (NIH) DNA sequence database using “BLAST” program MICR 302 Lab PCR Exercise • Second PCR assay will amplify DNA sequence in Shiga Toxin gene • Use two primer sets unique for the two different Shiga toxin genes of EHEC • Only E. coli that carry one or both of these toxin genes yield DNA product using these primers • Positive DNA product thus identifies bacteria isolate as EHEC strain • For lab diagnosis, only this second type of PCR assay is of practical use for MO ID Advantages of Molecular DNA Test • High sensitivity - theoretically detect single MO • High specificity – one MO species – Detect specific genotypes – Determine drug resistance – Predict virulence • Speed - more rapid than traditional MO culture • Simplicity - assays are automated; Realtime PCR Disadvantages of Molecular DNA Test • Expensive • Very specific, must have good clinical data to support infection by MO before testing initiated • Contaminating DNA give false positive • Miss new MO unless sequencing is done (as for your lab molecular unknown); not practical in clinical lab • Problem with mixed cultures – assay for all MOs possibly causing infection • Too sensitive? Are results clinically relevant? • No isolated MO grown in culture DNA Sequencing • Determine actual DNA sequence of MO • Using computer, based on nucleic acid data: – Identify MO – Predict protein sequence and function • The most commonly used sequencing method is the dideoxy method Dideoxy DNA Sequencing • Use dideoxynucleotide triphosphates (ddNTPs) • H on 3’ carbon of the ribose sugar instead of OH normally found in deoxynucleotide triphosphates (dNTPs) • ddNTP is chain terminator DNA synthesis stops as 3’OH necessary for addition of next nucleotide Dideoxy DNA Sequencing • Template DNA to be sequenced mixed with primer, four normal dNTPs (one radioactively labeled) • Mixture split into four tubes labeled A, C, G,T • Each tube “spiked” with different ddNTP (ddGTP tube 1, ddATP tube 2, ddCTP tube 3, ddTTP tube 4) • Add DNA polymerase, synthesis of new complementary strand • Occasionally ddNTP added instead of normal dNTP, synthesis of strand terminated Dideoxy DNA Sequencing • In ddATP tube, newly synthesized DNA will get ddATP in each place there is T • Thus, set of DNA strands in tube A, each of which ends in A • Similar reaction occurs in other tubes, result in DNA that ends in C, G,T respectively • After DNA synthesis, products of four tubes loaded onto four lanes of polyacrylamide gel • DNA strands separated by size • Sequencing gel able to resolve DNA that differ in size by only one base Dideoxy DNA Sequencing • After electrophoresis to separate by size, DNA strands visualized by exposing gel to X-ray film (one nucleotide was radioactive): – Strands in lane A all end in A – Strands in lane C all end in C – Strands in lane G all end in G – Strands in lane T all end in T • The sequence of the DNA read from the gel by starting at bottom and reading upward Automated DNA Sequencing • All four ddNTP reactions done in single tube • Each ddNTP labeled with a different fluorescent dye • Dye present in each synthesized strand corresponds to ddNTP that terminate DNA synthesis • DNA strands in reaction tube analyzed by gel electrophoresis • The Sequencing reaction depends on quality and purity of the amplified DNA product Automated DNA Sequencing • Flourimeter and computer attach to gel • Detect, record dye attached to DNA strands as run off bottom of gel • DNA sequence identified by order of dyes coming off gel MICR 302 Lab Molecular Unknown • Culture of an isolated Unknown bacteria • Isolate and purify DNA from Unknown bacteria • Use PCR to amplify rRNA gene sequence of Unknown bacteria DNA • Do Automated Sequencing reaction on amplified rRNA DNA product using fluorescent dyes • Submit DNA to CSULA Sequencing Lab • Use NIH DNA databank to search for matching DNA sequence to ID Unknown Class Assignment • Textbook Reading: Chapter 11 Applications of Molecular Diagnostics – Introduction to Nucleic Acid Amplification Procedures – Polymerase Chain reaction – Omit: Other Nucleic Acid Amplification Reactions • Key Terms – only from assigned reading • Omit: Learning Assessment Questions Identifying Fecal Unknown • • • • • • • • • • • Gram Stain, Catalase, Oxidase MO TSI Escherichia: A/A Shigella: K/A Enterobacter: A/A Klebsiella: A/A Salmonella: K/A, H2S Citrobacter: K/A, H2S Proteus: K/A, H2S Yersinia: K/A Aeromonas: K/A LIA K/K K/A K/K K/K K/K, H2S K/A, H2S R/A, H2S K/A CIN K/K CIN, Oxidase(+) Lecture Exam II Tue., Feb. 28, 2012 • Vibrio thru New Molecular Methods • Lecture, Reading, Key Terms, Learning Assessment Questions • Case Study 4, 5, 6 (Pseudomonas, Francisella, Bacillus) • Review, Review, Review! • Repetition is the key to retention • Repetition is the key to retention • Repetition is the key to retention Case Study Reports (Revised Due Date) • • • • March 1 - CS #7 Mycobacterium March 6 – CS #8 Clostridium March 8 – CS #9 Chlamydia March 13 – CS #10 Legionella