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BASIC MOLECULAR TECHNIQUES IN INFECTIOUS DISEASES Dr.Sarookhani 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 Types of laboratory methods (for infectious diseases) • Direct methods – look for/detect the agent • Indirect methods – detect host response to the agent Dr.Sarookhani Ag Ab Reactions • PRIMARY • IF٬ RIA ٬ ELISA ,CLIA • SECONDARY • Percipitation • Agglutination • Fulccolation Dr.Sarookhani Direct methods (Bacteriology&mycology& Parasitology&Virology) 1. 2. 3. 4. 5. 6. 7. Macroscopic evaluation Staining Direct microscopy No propagation required Electron microscopy Rapid tests Molecular methods Propagate the agent (culture&sensitivity) Dr.Sarookhani MOLECULAR TECHNIQUES ADVANTAGES • • • • • • High speed high analytical sensitivity high clinical sensitivity conceptually simple highly specific Amenable to full automation Dr.Sarookhani 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 LABORATORY SPECIMENS FOR MOLECULAR TECHNIQUES • 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,..) Dr.Sarookhani ELECTROPHORETIC SEPERATION OF NUCLEIC ACIDS Dr.Sarookhani RFLP CONCEPT Dr.Sarookhani 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 HYBRIDIZATION CONCEPT Dr.Sarookhani SOUTHERN&NORTHERN BLOT HYBRIDIZATION Dr.Sarookhani 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 NA CHIP TECHNOLOGY Dr.Sarookhani MICRO ARRAY TECHNOLOGY Dr.Sarookhani Application of microarray for pathogen detection Dr.Sarookhani DNA SEQUENCING Dr.Sarookhani 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 principles Dr.Sarookhani Dr.Sarookhani 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 Schematic of Multiplex PCR In 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) Locus A Locus C Locus B A B C small large Dr.Sarookhani Multiplex PCR Dr.Sarookhani 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 REAL TIME PCR Dr.Sarookhani Dr.Sarookhani APPLICATIONS OF MOLECULAR TECHNIQUES IN MEDICAL MICROBIOLOGY Dr.Sarookhani Microbiology Laboratory • Clinical Microbiology comprises essentially 5 sections. • Aerobic and anerobic bacteriology • Mycology • Mycobacteriology (also called Acid-fast Bacteriology, AFB) • Parasitology • Virology Dr.Sarookhani 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 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 MOLECULAR METHODS FOR IDENTIFICATION OF BACTERIA(1) Streptococci group Group A streptococci Pneumococci N.gonorrhea N.gonorrhea N.gonorrhea N.meningitidis Entrococci Staphylococci Staphylococci 16S rRNA Emm gene Lyt A gene rRna CPPB gene Pillin gene rrn gene rRNA HSP60 Nuc&femA RFLP Hybridizat&PCR DNAprobe&PCR DNA probe PCR LCR PCR Probe Colony DOT blot PCR Dr.Sarookhani MOLECULAR METHODS FOR IDENTIFICATION OF BACTERIA(2) H.influenza PBP& rRNA Probe H.influenza BexA&ompP6 PCR Legionella rRNA Probe Legionella mip PCR H.ducreyi 16 S rRNA Probe H.ducreyi groEL PCR Mycoplasma&Ureaplasma MP & MCC Probe Mycoplasma&Ureaplasma Tuf&urease PCR Dr.Sarookhani MOLECULAR METHODS FOR IDENTIFICATION OF BACTERIA(3) H.pylori Ure A,C&rRNA Probe&PCR Y.entrocolytica ail Cholera toxin Elt&ctx&hlyA PCR C.diphteria toxA Probe M.tuberculosis IS probes M.tuberculosis IS6110 PCR&RFLP &LCR Dr.Sarookhani PCR PCR of M.tuberculosis Dr.Sarookhani Molecular detection of mycoplasma Dr.Sarookhani PCR DETECTION OF BRUCELLA Dr.Sarookhani PCR-based detection of H.pylori (cag A gene) Dr.Sarookhani PCR-based detection of T.pallidum Dr.Sarookhani PCR-based detection of Mycobacterium lepre in skin biopsy Dr.Sarookhani PCR-based detection of Yersinia entrocolitica (chromosomal ail gene) Dr.Sarookhani 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 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 QUANTITATIVE AMPLIFICATION RESULTS MAY USEFUL FOR: • Viral load • prognosis • monitoring response to therapy Dr.Sarookhani HCV RNA ( RT-PCR) Dr.Sarookhani QUANTITATIVE AMPLIFICATION FOR HIV DETECTION • • branched DNA assay Dr.Sarookhani Laboratory Diagnosis of Influenza Comparison of Test Methods for Influenza Test Method Time to Results Comments Serology >2 wks Retrospective, requires paired sera Culture 1-10 days Still gold standard(?), requires expertise, provides virus for studies Molecular (RT-PCR) 2-4 hrs Becoming gold standard(?), requires expertise & expensive equipment 2-4 hrs Requires reading expertise & IF microscope 15-30 min Widely available, requires little expertise Antigen Detection (IF) Antigen Detection (Rapid EIA-like) Dr.Sarookhani 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 Possible contamination due to the throat- wash sampling method Dr.Sarookhani Dr.Sarookhani Dr.Sarookhani MOLECULAR DETECTION OF PARASITES &PROTOZOA Hydatidosis (protoscolex) r-DNA ITS 1 PCR-RFLP • Plasmodium sp. A.culcifacies Mt-DNA toxoplasmosis B1 E.histolytica & (dispar var.) r RNA leishmaniosis Dr.Sarookhani Nested PCR-RFLP PCR-RFLP DOT/BLOT& PCR Hybridization & PCR PCR Nested PCR for Leishmania diagnosis Dr.Sarookhani PCR-BASED DETECTION OF TOXOPLASMA GONDII Dr.Sarookhani MOLECULAR DETECTION OF FUNGI • • • • • • • T.verrocosum :HSP 70 :PCR Candida sp. :PCR Cryptococcus neoformans:nested PCR(CSF) Histoplasma capsulatum: probe Coccidioides immitis :probe Blastomyces dermatidis :probe Acanthamoeba :PCRDr.Sarookhani Detection of Cryptococcus neoformans by nested PCR Dr.Sarookhani MOLECULAR METHODS FOR CO-IDENTIFICATION OF MULTIPLE AGENTS Dr.Sarookhani Infectious agent Combination (All agents) Pathogens targeted Clinical manifestation(s) and/or specimen HSV, Genital ulcer disease HPVs, HSV, and Genital swabs H. ducreyi, and T. pallidum C. trachomatis Adenovirus, HSV, and Keratoconjunctivitis C. trachomatis EV, influenza viruses A and Acute respiratory tract B, RSV, PIV types 1 and 3, infection adenovirus, M. pneumoniae, and C. pneumoniae Dr.Sarookhani Application of multiplex PCR for diagnosis of viral infections (viruses in CNS) Clinical manifestation(s) •Meningitis, encephalitis, and/or meningoencephalitis Specimen(s) CSF six herpesviruses Viruses and/or other agent(s) targeted HSV-1, HSV-2, and CMV HSV and VZV; EBV and HHV-6 HSV-1, HSV-2, VZV, CMV, HHV-6, and EBV HSV-1, HSV-2, VZV, CMV, HHV-6, EBV, and Ent.V that may infect the CNS CMV, EBV, HHV-6, HHV-7, and HHV-8 Dr.Sarookhani 16 S rDNA Dr.Sarookhani Advantages of Molecular techniques in Infectious Diseases • • • • • • • increased sensitivity and specificity of identification faster report turnaround time Confirmation of culture Identification of organisms that are non-viable or cannot be cultured Identification of fastidious, slow growing organisms Identification of organisms that are dangerous to culture Identification of organisms in small numbers or in small volume specimens Dr.Sarookhani