Library Lecture author Onlineby © ESCMID Introduction to spa typing and MLST Dimitar Nashev Typing methods for S. aureus Library • Phage an d Ban d base d • Labourious: 2-6 days • Analysis is subjective and can give non-typable results • Communication of data is problematicLecture • DNA sequence -based methods author • Standardized protocol:Online 1-2 daysby • Analysis is unambiguous© • Sequence data can easily be transferred and compared ESCMID spa typing Library • Protein A (spa) in S. aureus – Surface protein » Viru lence factor-biLecturen ds to Ig G v ia Fc-bin ding doma in » Reduce phagocytosis » X-region - variable authornumber of 24 bp repeats » Highly polymorphic » DeletionOnline and duplicationby of repeats, and point mutations © ESCMID spa typing - single locus sequence typing Library • Sequencing of the repeat region of spa gene • spontaneous point mutations, loss and/or gain of repeats • spa typing detects: • VitiVariations ithin the number oftf repeaLecturets • Variations in the sequence of individual repeats • 386 repp(p)eats known (April 28. 2010) author • Repeats are assigned an numerical code (in Ridom or Bionummerics software)Onlineby • spa type is deduced from the© sequence and order of specific repeats • Data can be sent to central server - Ridom.spa server ESCMID Library Lecture author Onlineby © ESCMID spa typing – single locus sequence typ ing Library Lecture author Onlineby © ESCMID Library Lecture author Onlineby © ESCMID Multilocus sequence typing (MLST) Library • MLST is based on the principle of Multi Locus Enzyme Elec trop hores is (MLEE), w hic h is base d on differen t electrophoretic mobilities within multiple core metabolic gene • DNA sequencing of 450-500Lecture bp fragments of 7 housekeeping genes • hou sekeeping gene is a geneauthor that is req uired for the maintenance of basalOnline cellularby function • different seqqguences are© assigned as alleles Æcombination of the alleles Æ allelic profile or sequence type (ST) • RltdSTRelated STs can be organi zed ditCl into Clona lCl Comp lexes (CC’s).ESCMID Multilocus sequence typing (MLST) Library • Genes which are sequenced in the MLST of S.aureus – arc (()Carbamate kinase) – aro (Shikimate dehydrogenase)Lecture – ggplp ((yGlycerol kinase) author – gmk (Guanylate kinase) Onlineby – pta (Phosphate ©acetyltransferase) – tpi (Triosephosphate isomerase) – yqi (Acetyle coenzyme A acetyltransferase) ESCMID Library Lecture author Onlineby © ESCMID Steps in DNA sequencing methods • DNA preparation Library • PCR amplification of the staphylococcal protein A (spa) repeat region or the MLST loci • PifiiPurification o fhPCRdf the PCR prodLectureuct - to remove un- incorporated dNTPs and primers • DNA cycle sequencing reactionauthor by • Purification of theOnline Sequencing Reaction - to remove unincorporated dye-labeled© ddNTPs and salts • Sequencing electrophoresis • AnalysisESCMID of the sequence Steps in DNA sequencing 1. Purify PCR 2. Set up sequencing product reaction Library 3. Perform cycle Lecture sequencing author Onlineby © 5. Read order of terminators (DNA 4. Resolve sequence ESCMIDsequence) fragments.