Applications in Molecular Diagnostics PDF

HML 3013 APPLICATIONS IN MOLECULAR DIAGNOSTICS New Molecular Based Methods of Diagnosis  Why take a molecular test to diagnose infectious diseases? To ensure accurate and timely diagnosis Important for initiating the...

HML 3013 APPLICATIONS IN MOLECULAR DIAGNOSTICS New Molecular Based Methods of Diagnosis  Why take a molecular test to diagnose infectious diseases? To ensure accurate and timely diagnosis Important for initiating the proper treatment Important for preventing the spread of a contagious disease Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Leading uses for nucleic acid-based tests 1. Antimicrobial resistance detection 2. Nonculturable agents HPV HBV Curr. Issues Mol. Biol. 9: 87–102. 3. Fastidious, slow-growing agents Mycobacterium tuberculosis Legionella pneumophilia 4. Highly infectious agents that are dangerous to culture Francisella tularensis Brucella species Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Leading uses for nucleic acid based tests In situ detection of infectious agents Helicobacter pylori Toxoplasma gondii Agents present in low numbers HIV in antibody negative patients CMV in transplanted organs Organisms present in small volume specimens Intra-ocular fluid Forensic samples Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Leading uses for nucleic acid-based tests Differentiation of antigenically similar agents May be important for detecting specific virus genotypes associated with human cancers (Papilloma viruses) Antiviral drug susceptibility testing May be important in helping to decide anti-viral therapy to use in HIV infections Non-viable organisms Organisms tied up in immune complexes Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Leading uses for nucleic acid-based tests Molecular epidemiology To identify point sources for hospital and community- based outbreaks To predict virulence Culture confirmation Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  What are the different types of nucleic acid molecular techniques that are used? Direct probe testing – better for identification than for detection because it is not as sensitive as amplification methods Amplification methods – used to improve the sensitivity of the nucleic acid testing technique Target amplification Probe amplification Signal amplification Combinations of the above Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Direct probe testing Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Amplification methods Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Human Genetics Phenotype: observed physical and functional traits Genotype: complete set of genes and alleles Alleles: Different versions of homologous genes ex. B and b Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Human genetics How are gametes made? How does chromosome behavior affect inheritance of traits? Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Somatic cells are diploid. is any biological cell forming the body of an organism; that is, in a multicellular organism, any cell other than a gamete, germ cell, gametocyte or undifferentiated stem cell. Gametes are haploid, with only one set of chromosomes Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  a SPERMATOGENESIS b OOGENESIS Spermatogenesis is the process by which haploid spermatozoa develop spermatogonium oogonium from germ cells in the seminiferous tubules of the testis. primary primary Oogenesis, ovogenesis, or spermatocyte oocyte oögenesis is the differentiation of the ovum into a cell competent to meiosis l further develop when fertilized. secondary secondary spermatocyte oocyte Meiosis is a special type of cell polar meiosis ll body division that reduces the chromosome number by half, creating four haploid cells, each genetically distinct from the parent spermatids cell that gave rise to them polar bodies egg (will be degraded) Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  1st law - segregation of alleles Cells contain 2 copies (alleles) of each gene Alleles separate during gamete formation (meiosis) Gametes carry only one copy of each gene Mendel's first law is also known as the law of segregation. The law of segregation states that, 'the alleles of a given locus segregate into separate gametes.' Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Punnett squares show parental gametes and the genotypes of next generation Homozygous: BB and bb Heterozygous: Bb Possible genotypes and their probabilities Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Law of Independent Assortment During gamete formation, genes for different traits separate independently into gametes Why? random alignment of homologues at Meiosis I Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Chromosome behavior accounts for Mendel’s principles Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Tetrad A B a b Crossing over A B a a B b A b Gametes Genes on the same chromosome tend to be inherited together = linked genes Crossing over produces gametes with recombinant chromosomes Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Variations On Mendel’s Principles P GENERATION Red White Incomplete dominance RR rr an offspring’s phenotype is intermediate betweenGametes the phenotypes R r of its parents Pink F1 GENERATION Rr 1/ R 1/ r 2 2 1/ R 1/ R Eggs 2 2 Sperm Red 1/ r 1/ r 2 RR 2 Pink Pink F2 GENERATION Rr rR White rr Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  A recessive allele is a variety of genetic code that does not create a phenotype if a dominant allele is present. In a dominant/recessive relationship between two alleles, the recessive allele's effects are masked by the more dramatic effects of the dominant allele. Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Incomplete dominance in human hypercholesterolemia GENOTYPES: HH Hh hh Homozygous Heterozygous Homozygous for ability to make for inability to make LDL receptors LDL receptors PHENOTYPES: LDL LDL receptor Cell Normal Mild disease Severe disease Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Many genes have more than two alleles in the population Ex. three alleles for ABO blood type in humans IA, IB, i Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Genetic traits in humans can be tracked through family pedigrees The inheritance of many human traits follows Mendel’s principles and the rules of probability Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Family pedigrees are used to determine patterns of inheritance and individual genotypes Dd Dd D_? D_? Joshua Abigail John Hepzibah Lambert Linnell Eddy Daggett D_? dd Dd Abigail Jonathan Elizabeth Lambert Lambert Eddy Dd Dd dd Dd Dd Dd dd Female Male Deaf Hearing Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Inherited Genetic Disorders Normal Normal Most mutations usually involve recessive PARENTS Dd Dd alleles Phenylketonuria, PKU D D Tay-Sachs disease Eggs DD Sperm Cystic fibrosis Normal d d Dd Dd OFFSPRING Normal Normal (carrier) (carrier) dd Deaf Figure 9.9A Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  – Examples: achondroplasia, Huntington’s disease A few are caused by dominant alleles Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Sex-linked disorders affect mostly males Most sex-linked human disorders are due to recessive alleles Ex: hemophilia (blood no clot), red-green color blindness These traits appear mostly in males. Why? If a male receives a single X- linked recessive allele from his mother, he will have the disorder; while a female has to receive the allele from both parents to be affected Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Pedigree Chart: Inheritance Pattern for an X-linked Recessive Disease Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk Figure 19.12  A high incidence of hemophilia has plagued the royal families of Europe Queen Albert Victoria Alice Louis Alexandra Czar Nicholas II of Russia Alexis Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Variations on Mendel’s Principles Codominance (relation between 2 versions of the gene), multiple alleles Pleiotropy (one gene interfere 2 other unrelated phenotypes) Polygenic traits Sex-linked genes Environmental effects Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Accidents during meiosis can alter chromosome number Nondisjunction in meiosis I Abnormal chromosome count is a result of nondisjunction homologous pairs fail to Normal separate meiosis II during meiosis I Gametes n+1 n+1 n–1 n–1 Number of chromosomes Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Or sister chromatids fail to separate during meiosis II Normal meiosis I Nondisjunction in meiosis II Gametes n+1 n–1 n n Number of chromosomes Figure 8.21B Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk An extra chromosome 21 causes Down syndrome The chance of having a Down syndrome child goes up with maternal age Figure 8.20C Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Fetal testing can spot many inherited disorders early in pregnancy Karyotyping and biochemical tests of fetal cells can help people make reproductive decisions Fetal cells can be obtained through amniocentesis Amniotic fluid withdrawn Centrifugation Amniotic fluid Fluid Fetus (14-20 Fetal weeks) cells Biochemical tests Placenta Uterus Cervix Figure 9.10A Cell culture Karyotyping Several weeks later Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Chorionic villus sampling is another procedure that obtains fetal cells for karyotyping Fetus Several hours (10-12 later weeks) Placenta Suction Fetal cells Karyotyping (from chorionic villi) Some Chorionic villi biochemical tests Figure 9.10B Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Examination of the fetus with ultrasound is another helpful technique PGD - Preimplantation Genetic Diagnosis genetic analysis of embryos from in vitro fertilization (IVF) before inserting into womb Figure 9.10C, D Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Review of the Structure of DNA Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  DNA structure In double stranded linear DNA, 1 end of each strand has a free 5’ carbon and phosphate and 1 end has a free 3’ OH group. The two strands are in the opposite orientation with respect to each other (antiparallel). Adenines always basepair with thymines (2 hydrogen bonds) and guanines always basepair with cytosines (3 hydrogen bonds) Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  The Structure of DNA Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  DNA overview DNA deoxyribonucleic acid 4 bases Pyrimidine (C4N2H4) Purine (C5N4H4) A = Adenine T = Thymine C = Cytosine G = Guanine Nucleoside Nucleotide base + sugar (deoxyribose) base + sugar + phosphate O- O- PO -- P4 O OH 5’ CH2 O O 4’ 1’ H H H H sugar 3’ 2’ Numbering of carbons? OH H Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  3’ 5’ Linking nucleotides 3’ Hydrogen bonds 3’ N-H------N N-H------O 3’ 3’ Linking nucleotides: The 3’-OH of one What next? 3’ nucleotide is linked Thymine 3’ to the 5’-phosphate 2nm of the next 3’ nucleotide Adenine 3’ Cytosine 3’ 5’ Guanine 3’ Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  3’ 5’ Base pairing A T 3’ Base pairing (Watson- 3’ C Crick): 3’ A/T (2 hydrogen bonds) G G/C (3 hydrogen bonds) 3’ A T 3’ Always pairing a purine and a pyrimidine yields a 3’ T constant width 3’ A DNA base composition: C A + G = T + C (Chargaff’s rule) 3’ G 5’ 3’ Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  DNA structure Some more facts: 1. Forces stabilizing DNA structure: Watson-Crick-H-bonding and base stacking (planar aromatic bases overlap geometrically and electronically → energy gain) 2. Genomic DNAs are large molecules: Eschericia coli: 4.7 x 106 bp; ~ 1 mm contour length Human: 3.2 x 109 bp; ~ 1 m contour length 3. Some DNA molecules (plasmids) are circular and have no free ends: mtDNA bacterial DNA (only one circular chromosome) Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  DNA structure 5. Percentage of non-coding DNA varies greatly among organisms Organism # Base pairs # Genes Non-coding DNA small virus 4 x 103 3 very little ‘typical’ virus 3 x 105 200 very little bacterium 5 x 106 3000 10 - 20% yeast 1 x 107 6000 > 50% human 3.2 x 109 30,000? 99% amphibians < 80 x 109 ? ? plants < 900 x 109 23,000 - >50,000 > 99% Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  RNA structure RNA 3 major types of RNA ribonucleic acid - messenger RNA (mRNA); template for protein synthesis 4 bases Thymine (DNA) Uracil (RNA) A = Adenine U = Uracil Pyrimidine (C4N2H4) Purine (C5N4H4) - transfer RNA (tRNA); adaptor C = Cytosine molecules that decode the G = Guanine genetic code Nucleoside Nucleotide - ribosomal RNA (rRNA); base + sugar (ribose) base + sugar + phosphate catalyzing the synthesis of - O proteins O- PO -- P4 O OH 5’ CH2 O O 4’ 1’ H H H H sugar 3’ 2’ OH OH Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Amino acids/proteins The “central dogma” of modern biology: DNA → RNA → Protein Getting from DNA to Protein: Two parts 1. Transcription in which a short portion of chromosomal DNA is used to make a RNA molecule small enough to leave the nucleus. 2. Translation in which the RNA code is used to assemble the protein at the ribosome Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  The genetic code - The code problem: 4 nucleotides in RNA, but 20 amino acids in proteins - Bases are read in groups of 3 (= a codon) - The code consists of 64 codons (43 = 64) - All codons are used in protein synthesis: - 20 amino acids - 3 stop codons - AUG (methionine) is the start codon (also used internally) - The code is non-overlapping and punctuation-free - The code is degenerate (but NOT ambiguous): each amino acid is specified by at least one codon - The code is universal (virtually all organisms use the same code) Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  The genetic code Base 2 T C A G Phenylalanine Tyrosine Cysteine T In-class exercise F Y C C Serine T S STOP A 1. Which amino acids are Leucine STOP Tryptophan specified by single codons? L G W methionine and tryptophan Histidine T H C Leucine Proline Arginine C L P R 2. How many amino acids Glutamine A Base 1 Base 3 Q are specified by the first G two nucleotides only? Asparagine Serine T five: proline, threonine, Isoleucine N S I Threonine C valine, alanine, glycine A T Lysine Arginine A Methionine M K R G 3. What is the RNA code for Aspartate T the start codon? B C Valine Alanine Glycine AUG G V A G Glutamate A Z G Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Reading frames - a reading frame is not always easily recognizable - each strand of RNA/DNA has three possible starting points (position one, two, or three): Position 1 CAG AUG AGG UCA GGC AUA gln met arg ser gly ile Position 2 C AGA UGA GGU CAG GCA UA arg trp gly gln ala Position 3 CA GAU GAG GUC AGG CAU A asp glu val arg his Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Reading frames Wildtype CAG AUG AGG UCA GGC AUA GAG Up to 30% of mutations gln met arg ser gly ile glu causing humane disease are due to premature termination Mutant CAG AUG AGU CAG GCA UAG AG of translation (nonsense gln met ser gln ala mutations or frameshift) Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Mutations Mutation: any heritable change in DNA Sources of mutation: Spontaneous mutations: mutations occur for unknown reasons Induced mutations: exposure to substance (mutagen) known to cause mutations, e.g. X-rays, UV light, free radicals Mutations may influence one or several base pairs a) Nucleotide substitutions (point mutation) 1) Transitions (Pu  Pu; Py  Py) 2) Transversions (Pu  Py) Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Mutations b) Insertion or deletion (“indels”) - one to many bases can be involved - frequently associated with repeated sequences (“hot spots”) - lead to frameshift in protein-coding genes, except when N = 3X - also caused by insertion of transposable elements into genes “Weighting” of mutation events plays important role for phylogenetic analyses (model of sequence evolution) In-class exercise How many transition and transversion events are possible? 2 transitions: T  C; A  G 4 transversions: T  A; T  G C  A; C  G Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Mutations Mutations may influence phenotype a) Silent (or synonymous) substitution - nucleotide substitution without amino acid change - no effect on phenotype - mostly third codon position - other possible silent substitutions: changes in non-coding DNA b) Replacement substitution - causes amino acid change - neutral: protein still functions normally - missense: protein loses some functions (e.g. sickle cell anemia: mutation in ß-globin) Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Mutations c) Sense/nonsense substitution - sense: involves a change from a termination codon to one that codes for an amino acid - nonsense: creates premature termination codon Mutation rates = a measure of the frequency of a given mutation per generation - mutation rates are usually given for specific loci (e.g. sickle cell anemia) - the rate of nucleotide substitutions in humans is on the order of 1 per 100,000,000 - range varies from 1 in 10,000 to 1 in 10,000,000,000 - every human has about 30 new mutations involving nucleotide substitutions - mutation rate is about twice as high in male as in female meiosis Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Mutations A single amino acid substitution in a protein causes sickle-cell disease Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Review of protein structure Primary structure Proteins are chains of amino acids joined by peptide bonds Polypeptide chain The structure of two amid acids The N-C-C sequence is repeated throughout the protein, forming the backbone The bonds on each side of the C atom are free to rotate within spatial constrains, the angles of these bonds determine the conformation of the protein backbone The R side chains also play an important structural role Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Review of protein structure Secondary structure: Interactions that occur between the C=O and N-H groups on amino acids Much of the protein core comprises  helices and  sheets, folded into a three-dimensional configuration:  helix  sheet - regular patterns of H bonds are formed between neighboring amino acids - the amino acids have similar angles - the formation of these structures neutralizes the polar groups on each amino acid - the secondary structures are tightly packed in a hydrophobic environment - Each R side group has a limited volume to occupy and a limited number of interactions with other R side groups Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Secondary structure Other Secondary structure elements (no standardized classification) - random coil - loop Super-secondary structure - In addition to secondary structure elements that apply to all proteins (e.g. helix, sheet) there are some simple structural motifs in some proteins - others (e.g. 310 helix, - These super-secondary structures (e.g. transmembrane -hairpin, paperclip) domains, coiled coils, helix-turn-helix, signal peptides) can give important hints about protein function Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Secondary structure Structural classification of proteins (SCOP) Class 1: mainly alpha Class 2: mainly beta Class 3: alpha/beta Class 4: few secondary structures Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Secondary structure Alternative SCOP Class  : only  helices Class  : antiparallel  sheets Class / : mainly  sheets with intervening  helices Class + : mainly Membrane structure: Multidomain: contain segregated  helices with hydrophobic  helices with more than one class antiparallel  sheets membrane bilayers Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Review of protein structure Q: If we have all the Psi and Phi angles in a protein, do we then have enough information to describe the 3-D structure? A: No, because the detailed packing of the amino acid side chains is not revealed from this information. However, the Psi and Phi angles do determine the entire secondary structure of a protein Tertiary structure Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Tertiary structure The tertiary structure describes the organization in three dimensions of all the atoms in the polypeptide The tertiary structure is determined by a combination of different types of bonding (covalent bonds, ionic bonds, h-bonding, hydrophobic interactions, Van der Waal’s forces) between the side chains Many of these bonds are very week and easy to break, but hundreds or thousands working together give the protein structure great stability If a protein consists of only one polypeptide chain, this level then describes the complete structure Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Quaternary structure The quaternary structure defines the conformation assumed by a multimeric protein. The individual polypeptide chains that make up a multimeric protein are often referred to as protein subunits. Subunits are joined by ionic, H and hydrophobic Hemoglobin (4 subunits) Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  Summary protein structure Primary structure: Sequence of amino acids Secondary structure: Interactions that occur between the C=O and N-H groups on amino acids Tertiary structure: Organization in three dimensions of all the atoms in the polypeptide Quaternary structure: Conformation assumed by a multimeric protein The four levels of protein structure are hierarchical: each level of the build process is dependent upon the one below it Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  What are the advantages of using a molecular test? High sensitivity Can theoretically detect the presence of a single organism High specificity Can detect specific genotypes Can determine drug resistance Can predict virulence Speed Quicker than traditional culturing for certain organisms Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  What are the advantages of using a molecular test? Simplicity Some assays are now automated Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk  What are the disadvantages of using a molecular test? Expensive So specific that must have good clinical data to support infection by that organism before testing is initiated. Will miss new organisms unless sequencing is done as we will be doing in the lab for our molecular unknowns (not practical in a clinical setting). May be a problem with mixed cultures – would have to assay for all organisms causing the infection. Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk Thank You  What are the disadvantages of using a molecular test? Too sensitive? Are the results clinically relevant? Aktive Grotesk Aktive Grotesk Aktive Grotesk Aktive Grote sk Aktive Grote sk