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BASIC MOLECULAR TECHNIQUES IN INFECTIOUS DISEASES

BASIC MOLECULAR TECHNIQUES IN INFECTIOUS DISEASES. Clinical laboratory. 1)bacteriology & mycology 2)parasitology & protozoalogy 3)virology 4)hematology 5)biochemistry&hormon&metabolism 6)immunology&serology 7)cytology&histo-pathology & genetics.

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BASIC MOLECULAR TECHNIQUES IN INFECTIOUS DISEASES

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  1. BASIC MOLECULAR TECHNIQUES IN INFECTIOUS DISEASES Dr.Sarookhani

  2. Clinical laboratory • 1)bacteriology & mycology • 2)parasitology & protozoalogy • 3)virology • 4)hematology • 5)biochemistry&hormon&metabolism • 6)immunology&serology • 7)cytology&histo-pathology & genetics Dr.Sarookhani

  3. Types of laboratory methods(for infectious diseases) • Direct methods • look for/detect the agent • Indirect methods • detect host response to the agent Dr.Sarookhani

  4. Ag Ab Reactions • PRIMARY • IF٬ RIA ٬ ELISA ,CLIA • SECONDARY • Percipitation • Agglutination • Fulccolation Dr.Sarookhani

  5. Direct methods(Bacteriology&mycology& Parasitology&Virology) • Macroscopic evaluation • Staining • Direct microscopy • Electron microscopy • Rapid tests • Molecular methods • Propagate the agent (culture&sensitivity) No propagation required Dr.Sarookhani

  6. MOLECULAR TECHNIQUES ADVANTAGES • High speed • high analytical sensitivity • high clinical sensitivity • conceptually simple • highly specific • Amenable to full automation Dr.Sarookhani

  7. BASIC CATEGORIES OF ANALYSIS USED TO CHARACTERIZE DNA&RNA • 1)electrophoretic seperations(total,RE,PFGE) • 2)hybridization assays • 3)amplification techniques (NAAT) • 4)restriction fragment length polymorphism(RFLP) • 5)sequencing Dr.Sarookhani

  8. 1)whole blood& PBMC 2)serum 3)body fluids (urine, semen, CSF,ameniotic fluid,...) 4)biopsies 5) placenta & CVS 6)blastomer cells of embryo 7)others(hair,stool,smears,..) LABORATORY SPECIMENS FOR MOLECULAR TECHNIQUES Dr.Sarookhani

  9. ELECTROPHORETIC SEPERATION OF NUCLEIC ACIDS Dr.Sarookhani

  10. RFLP CONCEPT Dr.Sarookhani

  11. HYBRIDIZATION ASSAY FORMATS 1)liquid or solution phase hybridization 2)solid support hybridization a)DOT/blot(&inverse DOT/blot)hybridization b)southern&northern blot hybridization c)in situ hybridization(tissue,cells,chromosomes ) d)NA chip technology Dr.Sarookhani

  12. HYBRIDIZATION CONCEPT Dr.Sarookhani

  13. SOUTHERN&NORTHERN BLOT HYBRIDIZATION Dr.Sarookhani

  14. FLOURESCENT IN SITU HYBRIDIZATION (FISH) • Whole cells or tissue section affixed to glass slides. • Clinical applications in formalin-fixed paraffin embedded tissues. tissue Dr.Sarookhani

  15. NA CHIP TECHNOLOGY Dr.Sarookhani

  16. MICRO ARRAY TECHNOLOGY Dr.Sarookhani

  17. Application of microarray for pathogen detection Dr.Sarookhani

  18. DNA SEQUENCING Dr.Sarookhani

  19. ‏‏‏‏‏‏‏Nucleic Acid Amplification ‏‏‏ ‏Technologies (NAAT) • 1)TARGET AMPLIFICATION METHODS a)PCR & modifications b)NASBA c)TMA d)SDA • 2)PRIMER(PROB) AMPLIFICATION METHODS a)LCR b)Q-beta replicase c)cleavase / invader technology • 3)SIGNAL AMPLIFICATION METHODS a)b DNA & b)HCA Dr.Sarookhani

  20. principles Dr.Sarookhani

  21. Dr.Sarookhani

  22. PCR-based modification techniques • RT-PCR • nested PCR • hot start PCR • PCR-LiPA • PCR-SSP • PCR-ARMS • PCR-RFLP • multiplex PCR • PCR-SSCP • RACE-PCR • Real time PCR Dr.Sarookhani

  23. Locus C Locus A Locus B A B C small large Schematic of Multiplex PCRIn multiplex PCR more than one target sequence can be amplified by including more than one pair of primers in the reaction. ( Amplifying various genes simultaneously) Dr.Sarookhani

  24. Multiplex PCR Dr.Sarookhani

  25. In the field of infectious diseases the technique has been shown to be a valuable method for identification of: • viruses • bacteria • fungi • parasites • All Dr.Sarookhani

  26. REAL TIME PCR Dr.Sarookhani

  27. Dr.Sarookhani

  28. APPLICATIONS OF MOLECULAR TECHNIQUES IN MEDICAL MICROBIOLOGY Dr.Sarookhani

  29. Microbiology Laboratory • Clinical Microbiology comprises essentially 5 sections. • Aerobic and anerobic bacteriology • Mycology • Mycobacteriology (also called Acid-fast Bacteriology, AFB) • Parasitology • Virology Dr.Sarookhani

  30. IMPACT OF MOLECULAR METHODS ON CLINICAL BACTERIOLOGY • 1)DIAGNOSIS & PATHOGEN IDENTIFICATION a)for slow growing or difficult-to- culture organisms b)further examination & identification of agar-grown pure cultures c)simultaneous isolation of pathogens from specimens • 2)THERAPY • 3)EPIDEMIOLOGY & CONTROL MEASURES Dr.Sarookhani

  31. MOLECULAR METHODS IN CLINICAL BACTERIOLOGY LAB. • PCR & other amplification techniques • nucleic acid hybridization techniques • use of RE,s • DNA sequencing analysis • gene chip technology Dr.Sarookhani

  32. MOLECULAR METHODS FOR IDENTIFICATION OF BACTERIA(1) Dr.Sarookhani

  33. MOLECULAR METHODS FOR IDENTIFICATION OF BACTERIA(2) Dr.Sarookhani

  34. MOLECULAR METHODS FOR IDENTIFICATION OF BACTERIA(3) Dr.Sarookhani

  35. PCR of M.tuberculosis Dr.Sarookhani

  36. Molecular detection of mycoplasma Dr.Sarookhani

  37. PCR DETECTION OF BRUCELLA Dr.Sarookhani

  38. PCR-based detection of H.pylori (cag A gene) Dr.Sarookhani

  39. PCR-based detection of T.pallidum Dr.Sarookhani

  40. PCR-based detection of Mycobacterium lepre in skin biopsy Dr.Sarookhani

  41. PCR-based detection of Yersinia entrocolitica (chromosomal ail gene) Dr.Sarookhani

  42. APPLICATIONS OF MOLECULAR EPIDEMIOLOGY • Detection of identity of strains • detection of genotypes • detection of emergence & spread of strains of an organism with unusual resistance patterns or pathogenicity • determining the efficiency of infection control procedures • identification of source in outbreaks Dr.Sarookhani

  43. APPLICATION OF MOLECULAR METHODS IN VIROLOGY • Hepatitis viruses(HBV,HCV,HDV):PCR&RT-PCR • herpesviridae(CMV,HSV,EBV,VZV,...):PCR • HTLV1 & HIV1,2, : nested RT-PCR • ENTOVIRUSES :RT-PCR • PARVOVIRUS B19 : HB & PCR • HPV : FISH • mumps,adenovridae,LCM,measles : PCR&RT-PCR • rubella : RT-PCR Dr.Sarookhani

  44. QUANTITATIVE AMPLIFICATION RESULTS MAY USEFUL FOR: • Viral load • prognosis • monitoring response to therapy Dr.Sarookhani

  45. HCV RNA ( RT-PCR) Dr.Sarookhani

  46. QUANTITATIVE AMPLIFICATION FOR HIV DETECTION • branched DNA assay Dr.Sarookhani

  47. Laboratory Diagnosis of Influenza Dr.Sarookhani

  48. Specimen Types • Upper respiratory tract • Nasal or naso-pharyngeal (NP) swabs • Throat swabs • NP aspirates or washes • Lower respiratory tract • Tracheal aspirates • Bronchoalveolar lavages • Store at 2-8°C < 72 hours or freeze at < -70°C. • Transport with cool-pack Dr.Sarookhani

  49. Possible contamination due to the throat- wash sampling method Dr.Sarookhani

  50. Dr.Sarookhani

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