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EPID 525 Lecture 3

EPID 525 Lecture 3. Introduction to Molecular Methods in Clinical Microbiology. Specimen processing Target detection Post-amplification detection “Real-time” detection Quality assurance. Specimen processing. efficient target recovery maintain integrity of nucleic acid target

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EPID 525 Lecture 3

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  1. EPID 525 Lecture 3 Introduction to Molecular Methods in Clinical Microbiology

  2. Specimen processing Target detection Post-amplification detection “Real-time” detection Quality assurance

  3. Specimen processing • efficient target recovery • maintain integrity of nucleic acid target • removal of amplification inhibitors • elimination of components affecting substrates • sterilization of potentially hazardous organisms

  4. Specimen processing (cont.) Nucleic acid extraction (total) - proteinase K digestion - phenol-chloroform extraction - precipitation with salts and ethanol - wash pellet with cold ethanol - resuspend in sterile water

  5. Specimen processing (cont.) Nucleic acid extraction (RNA only) - treat with guanidinium ITC RNA complexes with guanidinium DNA and protein are removed - precipitation with salts and ethanol - wash pellet with cold ethanol - resuspend in sterile RNase-free water RNazol, Tel-Test

  6. Specimen processing (cont.) Heat treatment of specimens - specimen is added to water or buffer - heating or boiling releases NA - aliquot used as template for assay

  7. Specimen processing (cont.) Silica particle methods - take advantage of NA binding properties of silica - add digested specimen to column - wash with buffer - elute with water - can specifically elute RNA by adding a DNase step Qiagen spin columns

  8. Specimen processing (cont.) Tissue: prior homogenation required CSF: “clean” specimen Blood (whole or fractionated) DO NOT use heparin tubes HIV plasma (RNA), CMV whole blood (DNA) Urine : “clean” specimen Sputum: prior digestion and decontamination req. Feces: “dirty” specimen with inhibitors

  9. Automation Specimen processing MagNA Pure (Roche) TECAN (Roche) BioRobot (Qiagen) NucliSens (BioMerieux)

  10. TECAN MagNA Pure

  11. Step A: The sample material, e.g. cells (up to 5x106), blood (up to 1000µl), or 10 mg of homogenized tissue is placed into the wells of the sample cartridge. Step B/C: The binding/lysis buffer and proteinase K is added to the sample. Step D: Magnetic glass particles are added to the lysed sample. The DNA binds to the surface of the particles. Step E: The magnetic glass particles with the adsorbed DNA are recovered from the wells of the processing cartridge by applying a magnet from the outside of the pipette tips. Step F-H: The DNA is washed and eluted from the glass particles whereas the glass particles are retained in the reaction tip and discarded.

  12. Target detection Target-directed probes - Gen-Probe for culture confirmation - Digene Hybrid Capture for HPV Target amplification - PCR for DNA targets - Reverse transcriptase (RT)-PCR for RNA - NASBA, TMA Signal amplification - Bayer Branched DNA (bDNA) assays

  13. Target detection (cont.) Target-directed probes - Synthetic genomic sequences specific for an organism of interest - Hybridize with DNA or RNA targets in specimen (direct specimen or culture isolate) - Probes are labeled with enzymes, antigenic, chemiluminescent moieties, or radioisotopes

  14. Digene Hybrid Capture Gen-Probe Culture ID http://www.vysis.com/Hybridization_12956.asp

  15. Target detection (cont.) Target amplification - Polymerase chain reaction (PCR) of DNA targets - Primer-initiated; requires thermostable polymerase Denaturation of ds DNA 94°C Annealing of primers 50°C or higher Extension of primers 72°C ~1 min. each, repeat for 25 to 40 cycles

  16. http://bric.postech.ac.kr/labinfo/n_protocol/service_view.php?nProtocolId=961http://bric.postech.ac.kr/labinfo/n_protocol/service_view.php?nProtocolId=961

  17. http://bric.postech.ac.kr/labinfo/n_protocol/service_view.php?nProtocolId=961http://bric.postech.ac.kr/labinfo/n_protocol/service_view.php?nProtocolId=961

  18. Target detection (cont.) Target amplification - Reverse transcriptase (RT)-PCR of RNA targets Conversion of RNA to cDNA using RT Primer-initiated; 42°C, 1 hr. Random hexamers oligodT PCR primers cDNA is used as template for PCR

  19. Target detection (cont.) Target amplification - Nucleic acid sequence-based amplification (NASBA) - Transcription-mediated amplification (TMA) Multiple RNA copies of targets generated Use RT and/or T7 RNA polymerase Detect product with labeled probes Isothermal reaction

  20. Target detection (cont.) Signal amplification - Branched DNA (bDNA) assay (Bayer) Target probes Label probes Preamplifiers Substrate Amplifiers HIV-1 Add lysis buffer to disrupt virus and release RNA Hybridize target probes, preamplifiers and amplifiers to microwell and HIV-1 RNA Hybridize label probes to amplifiers, add dioxetane substrate, and measure chemiluminescence

  21. Post-amplification detection Gel electrophoresis Colorimetric microtiter plate Chemiluminescence Real-time fluorescence detection

  22. http://bric.postech.ac.kr/labinfo/n_protocol/service_view.php?nProtocolId=961http://bric.postech.ac.kr/labinfo/n_protocol/service_view.php?nProtocolId=961

  23. Post-amplification detection (cont.) Gel electrophoresis Agarose or polyacrylamide gel with EtBr

  24. Post-amplification detection (cont.) Colorimetric microtiter plate - product captured by probe attached to plate - enzyme-conjugated probes catalyze colorimetric reaction - read on a spectrophotometer Chemiluminescence - conjugated capture probes catalyze chemiluminescent reaction - read on a luminometer

  25. Post-amplification detection (cont.) Real-time fluorescence detection - dual-labeled probe included in reaction “reporter” and “quencher” - intact probe emits low flourescence - Taq cleaves reporter from bound probe - amount of reporter cleaved is proportional to the amount of amplicon produced Other detectors: SYBR Green dyes Molecular beacons

  26. Molecular beacons SYBR green FRET hybridization Sunrise TaqMan hydrolysis Scorpion ChemBioChem 2003, 4, 1120-1128

  27. Melting curve analysis

  28. Real-time quantitation

  29. Automation Amplification and detection COBAS (Roche) CT/NG, HCV, HBV, CMV COBAS TaqMan 48 LightCycler

  30. Quality assurance RNase-free materials and environment - wear gloves - DEPC-treated water Separation of work areas and unidirectional flow - reagent prep - specimen prep - amplification and detection

  31. Quality assurance (cont.) Prevention of amplicon cross-contamination - UNG - barrier tips, topical decontamination - closed systems - blank wells or tubes Processing controls - spiked specimens Amplification controls - internal controls - failure indicates inhibition

  32. Interpretation of results Dead vs. live pathogens - molecular “tests of cure” ex. persistent dead Chlamydia Presence of nucleic acid vs. disease - results taken in context of clinical presentation ex. HSV in CSF Causal vs. casual - difference between “necessary” and “necessary and sufficient” ex. HPV and cervical cancer

  33. Applications of Molecular Methods in Clinical Microbiology

  34. Laboratory detection of microbial pathogens • Culture (“biological amplification”) • Growth • Analysis of macromolecular composition • Analysis of metabolic by-products • Detection of: • Organism’s protein components (antigens) using antibodies • Patient’s immune response (serology) • Specific, characteristic nucleic acid sequences (“enzymatic amplification”)

  35. Why Nucleic Acid Amplification Tests [NAAT] ? • When conventional methods are: • 1. Too slow (e.g., mycobacteria, legionella) • 2. Too insensitive (asymptomatic HIV, viral infection of central nervous system, etc.) • 3. Too cumbersome (e.g., virus isolation) • 4. Not available (unculturable agents: HPV, HCV)

  36. The Promises of NAATs Higher sensitivity and specificity Shorter turn-around-time Overall reduction in patient care costs

  37. Some organisms detected by PCR M. tuberculosis Legionellae B. burgdorferii H. influenzae B.pertussis N. meningitidis T. pallidum H. pylori F. tularensis C. difficile E. coli T. whipelii van, mec HIV HTLV CMV HSV HHV VZV EBV Hepatides HPV Rubella Influenza Rhino Env • Adeno • Rabies • Parvo B19 • Arbo • Yellow Fever • Lassa • JC/BK • Candidae • Cryptococcus • Trypanosoma • Toxoplasma • Naegleria….

  38. Applications Detection of uncultureables or slow growers Organism ID via 16S rRNA sequencing Prognostication by subtype analysis Disease monitoring by quantitation Genotypic approaches to resistance testing Outbreak investigations

  39. Case 1 Patient S.M., 8 month old female Full term delivery, normal weight gain 1 month PTA (Dec, 02): Resp failure w/ presumed pneumonia Sputum cx pos for H. influenzae and P. aeruginosa Responded to medical and abtc management in PICU Discharged home F/U at Pulm. Clinic (Jan, 03): Noted resp. distress, tachypnea, low oxygen sat CXR: changes consistent with viral pneumonia

  40. Current admission Labs: WBC 6.4 K/µl (low normal) ALC ~1 K/µl (low) HCT 30.6 (low) Basic metabolic panel WNL Cultures: Respiratory: P. aeruginosa neg for fungi, AFB, viruses neg for Chlamydia, B. pertussis Stool: neg for viruses Serum: neg for Mycoplasma BAL: PCR+ for Pneumocystis

  41. What is going on?

  42. Immunodeficiency workup Sweat chloride testing CF gene testing CH-50 analysis Ig testing Oxidative burst testing T cell analysis by flow Normal Negative Normal Slight dec. IgG & IgM Normal CD4+ 117 (LLN 967) CD4:CD8 0.3 (LLN 0.8) Patient HIV+ by serology and qual. PCR Mother HIV+ (previously unknown)

  43. Pneumocystis jiroveci (carinii) Previously classified as a protozoa Currently classified as a fungus - based on nucleic acid and biochemical analysis Most healthy children exposed by age 4 Reactivation pneumonia in immunosuppressed - Adults: CD4+ T cells <200/µl - Pediatrics: CD4+ normal - low TMP-SMX prophylaxis reducing incidence

  44. GMS  DFA  Calcofluor

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