Seminario PCR

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  • The article wants to explain with the title the objective of the investigation. It is based on trying a non conventional PCR method for a faster detection of the MRSA.
  • La  envoltura celular  de las bacterias Gram-positivas comprende la  membrana citoplasmática  y una  pared celular  compuesta por una gruesa capa de  peptidoglucano , que rodea a la anterior. La pared celular se une a la membrana citoplasmática mediante moléculas de ácido lipoteicoico . La capa de  peptidoglicano  confiere una gran resistencia a estas bacterias y es la responsable de retener el tinte durante la tinción de Gram. A diferencia de las  Gram-negativas , las Gram-positivas no presentan una segunda membrana lipídica externa a la pared celular y esta pared es mucho más gruesa. 3
  • Morfológicamente los  Staphylococcus  son cocos grampositivos. Los estafilococos crecen fácilmente sobre casi todos los medios bacteriológicos, en cultivos su crecimiento es mejor en el medio sal manitol y agar sangre, esto puede llegar a dar problemas en el corazón ó hígado, tales como la perdida de un hígado. Es un coco anaerobio facultativo, esto significa que puede crecer tanto en condiciones con oxígeno como carente de éste. Su mayor velocidad de crecimiento es a 5 - 25 °C; pero también se puede ver en activa fisión binaria entre 30 y 27 °C. Además, producen catalasa , lo que los diferencia de los  estreptococos . Tiene importancia médica principalmente el  S. aureus , y en humanos además de éste, el  S. saprophyticus  y el  S. epidermidis .
  • Presencia de B lactamasa, que rompe el anillo b lactámico de los antibióticos con esta estructura.
  • Prokaryotic fission , which is binary fission, is a form of  asexual reproduction  and  cell division  used by all prokaryotes , (bacteria and archaebacteria), and some organelles within  eukaryotic  organisms (e.g., mitochondria ). This process results in the reproduction of a living prokaryotic  cell  (or organelle) by division into two parts that each have the potential to grow to the size of the original cell (or  organelle ). This type of division takes place without the formation of spindles. The single DNA molecule first replicates, then attaches each copy to a different part of the cell membrane. When the cell begins to pull apart, the replicate and original chromosomes  are separated. The consequence of this asexual method of reproduction is that all the cells are genetically identical, i.e. have the same genetic material.
  • S. aureus  infections can spread through contact with pus from an infected wound, skin-to-skin contact with an infected person by producing hyaluronidase  that destroys tissues, and contact with objects such as towels, sheets, clothing, or athletic equipment used by an infected person. Deeply penetrating  S. aureus  infections can be severe. 
  • S. aureus  most commonly colonizes the  anterior nares  (the  nostrils ). The rest of the  respiratory tract , open wounds,  intravenous   catheters , and the urinary tract  are also potential sites for infection. Healthy individuals may carry MRSA asymptomatically for periods ranging from a few weeks to many years. Patients with  compromised   immune systems  are at a significantly greater risk of symptomatic  secondary infection . Open wounds  heridas abiertas.
  • Can produce a wide range of diseases, ranging from skin infections and mucosal relatively benign, such as folliculitis, furunculosis or conjunctivitis, to life-threatening diseases such as cellulitis, deep abscesses, osteomyelitis, meningitis, sepsis, endocarditis or pneumonia. Furthermore, it may also affect the gastrointestinal tract, either by physical presence of Staphylococcus aureus or the ingestion of staphylococcal enterotoxin secreted by the bacteria. Anthrax is a staph skin infection composed of a cluster of boils with spread of infection to the subcutaneous tissue. Deep discharge the lesions are slow to heal and produce scars. Sometimes the term is used to refer to anthrax Anthrax
  • s  oxacillin  (used for clinical antimicrobial susceptibility testing),  flucloxacillin , and dicloxacillin are used medically.
  • Like other beta-lactam antibiotics, meticillin acts by inhibiting the synthesis of bacterial cell walls. It inhibits cross-linkage between the linearpeptidoglycan polymer chains that make up a major component of the cell wall of Gram-positive bacteria. It does this by binding to and competitively inhibiting the transpeptidase enzyme used by bacteria to cross-link the peptide ( D-alanyl-alanine ) used in peptidoglycan synthesis. Meticillin and otherbeta-lactam antibiotics are structural analogs of  D-alanyl-alanine , and the transpeptidase enzymes that bind to them are sometimes called penicillin-binding proteins (PBPs). [4]
  • resistance does make MRSA infection more difficult to treat with standard types of antibiotics and thus more dangerous.
  • Cloning consist of obtaining a clone, understood as a set of genetic elements identical to its precursor. This elements can be molecules, cells, tissues, organs or even complex multicelular organisms. It’s the process of multiplication of DNA or RNA using PCR. PCR is a technique developed by kary mullis in the 1980’s the objective of this techinique is the amplification of genes or a DNA fragment or indirectly through RNA, present in diverse mixtures of different sources. The teqhnique is based on enzimatic in vitro amplification. Geometrical increase in the number of copies of a particular DNA secuence. The amplification starts with low concentrations od DNA and millions of copies can be obtained in a short time.
  • PERFORMANCE: each cycle products are used as templates for the next the accumulation of copies is exponential insted of linear. DURATION: Depends on the specific secuence to be amplified/ SPECIFICITY: Is very high, determined by the used primers secuences and the alignment conditions DETECTION CAPABILITY: allows for detection of a single DNA molecule FIDELITY: Ability to copy secuences with high accuracy without iintroducing mutations
  • Tabla 1. Especies y cepas bacterianas utilizadas en este estudio y los resultados de ensayo de PCR
  • Tabla 2. Las secuencias de los cebadores utilizados para el ensayo de PCR
  • A major drawback of most published PCRs, surprisingly even to date, is that they do not contain an internal amplification control (IAC). An IAC is a nontarget DNA sequence present in the same sample reaction tube which is coamplified simultaneously with the target sequence. In a PCR without an IAC, a negative response (no band or signal) can mean that there was no target sequence present in the reaction. But it could also mean that the reaction was inhibited due to malfunction of the thermal cycler, incorrect PCR mixture, poor polymerase activity, and, not least, the presence of inhibitory substances in the sample matrix. 
  • Seminario PCR

    1. 1. Deisy Tatiana Sánchez Zapata Manuela Rincón Muñoz Facultad de Medicina 2013
    2. 2. Micrografía electrónica de barrido de Staphylococcus aureus resistente a meticilina (MRSA, marrón), rodeada de restos celulares.
    3. 3.  Gram-positive bacteria.  Round (cocci)- grape like clusters.  40 different species • 9 2 subspecies • 1 3 subspecies
    4. 4.  Staphylococci grows on almost every bacteriological Medium.  Facultative anaerobic cocci.  Growth rate 5 - 25 °C.  Catalase. 2 H2O2  2 H2O + O2
    5. 5. Presence of B lactamase.
    6. 6.  Facultative anaerobic Gram-positive coccal bacterium.  S. aureus reproduces asexually by binary fission.  Catalase-positive differentiate
    7. 7.  Responsible for many infections, it can also be a commensal. nosocomial.  It can survive from hours to months dry environmental surfaces.  Infection hyaluronidase destroys tissues.
    8. 8.  Skin and mucous membrane infections benign Folliculitis, furunculosis, conjunctivitis, cellulitis, deep abscesses, osteomyelitis, meningitis, sepsis, endocarditis, pneumonia
    9. 9.  Narrow-spectrum beta-lactam antibiotic of the penicillin class. Previously used to treat infections  gram- positive bacteria.  Replaced by  Oxacillin, flucloxacillin, dicloxacillin.
    10. 10.  Inhibition synthesis of bacterial cell walls.  Inhibits cross-linkage between the linear peptidoglycanpolymer chains that make up a major component of the cell wall of Gram- positive bacteria.  Binds to  transpeptidase used to cross- link in peptidoglycan synthesis.
    11. 11.  Resistance difficult treatment dangerous.
    12. 12. Characteristics • Performance exponential • Duration • Specificity high • Detection capability det. Single DNA molec. • Fidelity clone w/out mutations
    13. 13. Molecular diagnosis for MRSA nasal Rapid detection and screening of MRSA Colonization
    14. 14. No. cepas de referencia 16S rRNA un mec A b luk S fem A Control interno Staphylococcus spp. cepas ( n = 7) 1. S. aureus (ATCC 33591) + + - + + 2. S. aureus (ATCC 43300) + + - + + 3. S. aureus (ATCC 25923) d + - + + + 4. S. aureus (ATCC 49775) + - + + + 5. S. aureus (ATCC 51153) e + - - + + 6. S. epidermidis (ATCC 14990) + - - - + 7. ECN resistentes a la meticilina e + + - - + Las bacterias Gram-positivos ( n = 8) 1. Streptococcus spp. Grupo A (ATCC 19615) e - - - - + 2. Streptococcus spp. Grupo B (ATCC 12401) e - - - - + 3. Streptococcus spp. Grupo G e - - - - + 4. Bacillus subtilis (ATCC 6633) e - - - - + 5. Listeria monocytogenes (ATCC 7644) e - - - - + 6. Enterococcus faecium LMG 16192 c - - - - + 7. Enterococcus faecalis (ATCC 29212) e - - - - + 8. Corynebacterium spp. correo - - - - + Patógenos entéricos gram-negativos ( n = 15) 1. Escherichia coli (EHEC) e - - - - + 2. E. coli (EPEC) e - - - - + 3. E. coli (ETEC) e - - - - + 4. Klebsiella pneumoniae (ATCC 10031) e - - - - + 5. Shigella sonnei (ATCC 25931) e - - - - + 6. Shigella flexneri (ATCC 12022) e - - - - + 7. Shigella boydii (ATCC 9207) e - - - - + 8. Proteus mirabilis (ATCC 29245) e - - - - + 9. Salmonella typhi correo - - - - + 10. Pseudomonas aeruginosa (ATCC 27853) e - - - - + 11. Yersinia enterocolitica (ATCC 23715) e - - - - + 12. Vibrio cholerae (O1 clásica) e - - - - + 13. Citrobacter freundii (ATCC 8090) e - - - - + 14. Gardnerella spp. correo - - - - + 15. Candida albicans (ATCC 10231) e - - - - +
    15. 15. Primers Secuencia del cebador (5'-3 ') Objetivo Gene Gen Banco número de acceso Tamaño del producto 16S ARNr-F GCA AGC ATC GTT CGG AAT T 16S rRNA D83356 597 pb 16S ARNr-R CTT AAT GAT GGC AAC TAA GC mec A-F ACG TCA AGT AGA TGC ATA TAA mec A NC_003923M 293 pb mec A-R CTT TTA AGT TCT GCG ATT GC Luks -F CAG GAG GTA ATG TTG CAT TT luk S AB186917 151 pb Luks -R ATG TCC AGA CAT TAA TTT ACC fem A-F CGA TCC ATA TTT ACC ATA TCA fem A CP000255 450 pb fem A-R ACG ATC CTC TTC GTT TAG TT IC-F AGC GTC TGT CAT GAG A hem M AF227752 759 pb IC-R ATT CTC AGA TAT GTG TGG
    16. 16. Molde de ADN, que van desde 100 ng a 1 pg.
    17. 17. REFERENCE WHAT THEY SAID? AGREE DESAGREE Rossney , Herra and et al. “The Thermostabilized PCR simpler and easier to use than real-time PCR” Klatser , Kuijper and et al. “The thermostabilized PCR has been used similarly for mycobacteria and Salmonella typhimurium” Gillet , Issartel and et al. “The Luks gene partly encodes PVL toxin, which is the main virulence factor responsible for nosocomial “ Zhang K, McClure and et al. “Detection of PVL toxin has been described and has proved useful for identifying strains of MRSA often associated with epidemics”
    18. 18. • The molecular nasal diagnosis to identify MRSA , is important for the epidemiological level especially in nosocomial diseases. • The thermostabilized PCR assay has many advantages: does not require cold chain, the less margin for error, makes a high-performance rapid analysis. The thermostabilized PCR assay is a very useful technique to identify MRSA one important pathogen in nosocomial infections • Molecular methods like PCR facilitate early detection of MRSA patients and helps in infection control and prevention. • Comparing other methods like chromogenic media with molecular methods explain why molecular methods take de lead in the implementation of antibiotics and reduced hospital bed stays.
    19. 19. Gracias….

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