Dịch tễ học phân tử: Các phương pháp xét nghiệm dùng trong điều tra dịch tễ học phân tử

Principles of Molecular Epidemiology Lecture 2: Overview of laboratory methods used in molecular epidemiology investigations Conventional and molecular biology methods National Institute of Infectious Disease January 16, 2017 Epidemiologic investigations Outbreak investigations Surveillance Research Application of laboratory techniques to epidemiologic investigations Diagnosis • • • • • • • Microscopy, morphology Imaging tests (x-ray, MRI, PET, etc) Pathology Biochemical tests Serology Gene probes PCR Strain typing (subtyping) • • • • Biochemical tests Functional assays Serologic tests Genotyping tests Laboratory steps used to perform molecular epidemiology investigations— bacterial infections  Isolation in pure culture and identification  Culture-independent diagnostic tests (CIDT)  Limitations:  quantitatively distinguishing the etiologic agent from other microbes present in a clinical specimen,  performing quantitative drug-susceptibility tests  unequivocally or quantitatively distinguishing pathogenic or drug-resistant variants among mixed bacterial populations  transporting isolates to other laboratories for additional testing (e.g., WHO Collaborating Center-directed quality assurance and standardization tests), and  storing isolates for future use and analyses (e.g., for developing and validating new diagnostic tests, identifying new drug targets, designing new vaccines)  Speciation  Further phenotypic subtyping  Serotyping  Antimicrobial susceptibility testing Laboratory steps used to perform molecular epidemiology investigations—viral infections  Separation from complex biological samples Filtration, nuclease treatment, ultracentrifugation Isolation by cell culture Culture-independent diagnostic tests  Speciation  Further phenotypic subtyping Serotyping Laboratory-based data stratification—core principle in molecular epidemiology Microbial population in a “sterile” niche DK Find Out! Pure culture Microbial population in a non-sterile niche Identification to the species level Separation www.hyserve.com HealthDefine.com www.webmd.boots.com San Jose Functional Medicine Conventional laboratory techniques used to subtype organisms  Growth and morphologic characteristics  Biochemical characteristics  Serologic or immunologic characteristics  Functional or physiologic characteristics  antibiotic susceptibility tests  phage typing  colicin typing  cell culture assays  toxigenicity assays  in vitro survival characteristics Triple sugar iron (TSI) slants (alkaline/acid/gas)  Multilocus enzyme electrophoresis (MLEE) Shigella spp Salmonella typhimurium Salmonella typhi Biochemical tests—automated systems API 20E system (identifies Gram negative bacteria) From de la Maza et al, Color Atlas of Medical Bacteriology, 2004 http://www.biomerieux-diagnostics.com/vitek-2 Microscopy Gram-negative rods Gram-positive cocci Acid fast stain (AFB) Gram negative From de la Maza et al, Color Atlas of Medical Bacteriology, 2004 Gram positive Microscopy—Scanning EM Staphylococcus aureus E coli Leptospira interrogans Fungi Yeast Hyphae Single amplified product having different MW: E coli pathovar detection ETEC (LT) EPEC EIEC ETEC (ST) PCR-based strain-typing methods —cont Repetitive elements ERIC (Enterobacterial Repetitive Intergenic Consensus) sequences REP (Repetitive Extragenic Palindromic) sequences BOX (boxA, boxB, boxC) IS elements (e.g., IS6110 in M tuberculosis) PGRS (polymorphic GC-rich repetitive sequences) DRE-PCR (double repetitive element PCR) ITS-PCR (intergenic transcribed spacer) Multiple locus VNTR analysis (MLVA) PCR assays based on repetitive elements Repetitive element Distribution of ERIC sequences among members of Enterobacteriaceae ERIC PCR of selected E coli isolates (from Amee Manges, UCB) BOX PCR analysis of serotype 14 Pneumococcal isolates from meningitis cases, Bahia, Brazil 1996-98 (Ko et al, Clin Infect Dis;2000;30:191-95) PCR-based method: Multiple loci VNTR analysis (MLVA)  VNTR: variable number tandem repeats  Short, repeated segments found in a specific locus  Each locus has direct tandem repeats of variable number  The number of repeats per locus varies between strains  Amplification of these loci (using primers specific to flanking regions) will produce amplicons of different MW that vary between strains Strain A Strain B 41 loci Strain typing methods based on nucleic acid sequencing Whole genome sequence (NGS) comparison • Chain-termination (Sanger) sequencing (capillary electrophoresis) • New generation sequencing methods Comparison of subsegments of a genome • Multilocus sequence typing (MLST) • Single nucleotide polymorphism (SNP) • Micro-satellites Comparison of extrachromosomal element • SNP analysis of sequences within: • Plasmid • Mitochondria • Kinetoplasts Multilocus sequence typing (MLST) Compares allelic differences among a selected set of gene segments, usually