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J.pi5

  1. 1. PowerPoint Template<br />JurnalDivisiPenyakitInfeksidanMikrobiologi I<br />Rapid detection of Pseudomonas aeruginosa from positive blood cultures by <br />quantitative PCR<br />Annals of Clinical Microbiology and Antimicrobials 2010, 9:21<br />Vincent Cattoir, Audrey Gilibert, Jeanne-Marie Le Glaunec, Nathalie Launay,<br /> Lilia Bait-Mérabet,Patrick Legrand<br />YUNI SETYOWATININGSIH /ARYATI<br />Jumat, 1 April 2011<br />
  2. 2. 2<br />Pseudomonas <br />aeruginosa<br />6,8 % Bakteremiakarenabakteribatang<br />Gram negatif<br />PENDAHULUAN<br />Patogenoportunisutamainfeksinosokomial, terutamadi unit perawatanintensif<br />Di Amerika patogenterbanyakke 7 darialirandarah, <br />
  3. 3. 3<br />Pseudomonas <br />aeruginosa<br />Lanjutan (PENDAHULUAN)<br />Bakteremiasecaraklinistidakdapatdibedakandariinfeksikarenabakteri Gram negatiflainnya<br />Terapiantibiotika<br />awal yang <br />tidaktepatmengakibatkandampak<br />negatifhebat.<br />
  4. 4. Metodeidentifikasifenotipmembutuhkanwaktu lama dankebanyakanpunyapembatasaninheren<br />Beberapametodeberbasis PCR telahditelitiuntukmengidentifikasiP.aeruginosa, terutamapadasampel yang diambildarisistimrespiratoriuspasien fibrosis kistik.<br />Teknikmolekulertelahditelitisebagaimetode yang cepatdandapatdiandalkanuntukidentifikasipatogenbakterial.<br />Lanjutan (PENDAHULUAN)<br />4<br />
  5. 5. 5<br />Lanjutan (PENDAHULUAN)<br />Target MolekulerqPCR<br />ecfX<br />16 S rRNA<br />algD<br />gyrB<br />Pseudomonas <br />aeruginosa<br />oprI<br />toxA<br />oprL<br />Adahasilpositifpalsu<br />Adahasilnegatifpalsu<br />
  6. 6. Kelemahanmetode PCR  masihharusditeliti target gen yang sesuaikarenatelahdilaporkansebelumnya target gen oprIdanoprLtidak 100% spesifikuntukP.aeruginosa.<br />Metode PCR dari BC positifcepat, akuratsampai level spesiesdilanjutkanpengobatanempiris yang sesuai<br />6<br />Lanjutan (PENDAHULUAN)<br />KelemahanteknikidentifikasiP.aeruginosakonvensionaldari BC positifbutuhwaktu minimal 24 jam <br />
  7. 7. 7<br />
  8. 8. Spesimen<br />1<br />IdentifikasiFenotip<br />2<br />Ekstraksi DNA<br />3<br />PCR kuantitatif<br />4<br />Batas deteksi<br />5<br />METODE<br />8<br />
  9. 9. 9<br />Diambildari 100 hasil BC positifdari 100 pasienrawatinap<br />METODE<br />Waktupenelitian : <br />Agustus 2008 <br />sampaiJuni 2009<br />SPESIMEN <br />DarahbotolBacT/ALERT aerobdananaerob<br />DiinkubasidalamalatBacT/ALERT<br />Pengecatan Gram : Batang Gram Negatif<br />87 aerob +<br />13 anaerob +<br />Konsentrasibakteri KulturKuantitatif<br />Identifikasifenotipdenganmorfologikoloni, tesoksidasedanhasil API 20 E<br />Diinokulasisecaraaerobdananaerob, <br />37oC, 24 - 48 jam pada TSA, Drigalski Agar, Blood Agar, Chocolate Agar<br />Diinokulasi<br />SecaraAnaerob<br />Diinokulasi<br />SecaraAerob<br />
  10. 10. Ekstraksi DNA denganRapid Boiling Procedure<br />10<br />0,5 ml darah<br />Sentrifus 850 g 2’<br />Supernatandiambil<br />1% Triton X-100<br />0,5% Tween 20<br />10 mMTris-HCl<br />1 mM EDTA<br />Inkubasi<br />pada 100oC<br />10 ‘<br />Hasil : Template DNA<br />Waktu : 20 menit<br />Sentrifus 11500 g 5’<br /> Pellet diambil,<br />diresuspen<br />200 µL buferpelisis<br />Supernatandiambil<br />Sentrifus 850 g 2’<br />
  11. 11. 11<br />PCR KUANTITATIF<br />CAMPURAN AMPLIFIKASI :<br />2 µl 10x LightCyclerFastStart DNA Master Hybridization (Roche)<br />2 mM MgCl2<br />0,5 µM masing-masing primer<br />0,2 µM masing-masingprobe<br />5 µl template DNA <br />volume akhir 20 µl. <br />Primeroligonukleotida<br />Amplifikasi<br />Kontrolnegatif: SuspensiTris-EDTA <br />KontrolPositif: Ekstrak DNA P. aeruginosaATCC 27853 <br />Gen ecfX<br />fragmen152 bp<br />Probe berlabelfluoresen<br />Deteksi<br />
  12. 12. 12<br />Tabel 1 PrimeroligonukleotidadanProbehibridisasiLightCycler<br /> yang digunakandalampemeriksaanPCR<br />a [FAM], fluorescein; [LC705], LightCycler™-Red 705; Ph, 3’-phosphate. <br />b Extracytoplasmic function gene(GenBank accession no. DQ996551).<br />
  13. 13. 13<br />Lanjutan (PENDAHULUAN)<br />LOKASI ecfX<br />ecfX-HP2<br />154-172<br />ecfX-F<br />46-62<br />ecfX-HP1<br />135-151<br />ecfX-R<br />179-197<br />
  14. 14. Tahap1<br />Tahap 2<br />Tahap3<br />PCR KUANTITATIF<br />SatuSiklus PCR <br />Denaturasiawalpada<br />suhu 95 °C selama 10 menit<br />SEQUENCING/<br />ELONGATION<br />72 °C, 20”<br />DENATURASI<br />95 °C, 10”<br />ANNEALING<br />50 °C, 10”<br />14<br />
  15. 15. PCR KUANTITATIF<br />15<br />DNA teramplifikasidiukurdengandeteksifluoresensipada705 nm<br />WaktuProsesqPCR : 1,5 jam<br />Padasaat yang bersamaan, dilakukankontrolinhibisi PCR untuksemuasampeldenganmenggunakantabungreaksikedua yang berisi 100 ngekstrak DNA darikontrolpositif.<br />
  16. 16. BATAS DETEKSI<br />Ligasi<br />ecfX<br />Plasmid pRT-ecfX<br />Media berisi 30 µg/mL<br />Kanamycin<br />Escherichia Coli<br />Plasmid DNA diekstraksi,dimurnikan, ditambahelution buffer dandi-sequencing untukmenentukanadanyaecfX<br />16<br />
  17. 17. BATAS DETEKSI<br />Konsentrasi DNA  Spektrofotometer<br />Bahan Plasmid DNA rujukandiencerkan serial<br />  plasmid genome equivalen10 sampai 1010<br />AnalisisKurvaStandar.<br />AnalisiskuantitatifdilakukandenganLightCycler software v 3.5 (Roche)<br />Rasio signal diukurpada 705 nm/ signal pada 530 nm untukmenghitungnilaiCrossing Point<br />17<br />
  18. 18. KurvaamplifikasiqPCRdariplasmid DNA reference material dengan 11 konsentrasi DNA Eksternal(dari 1011sampai 10 copies/ml)<br />18<br />
  19. 19. KurvakalibrasiqPCRmenggunakanpengenceran serial dariplasmid DNA reference material<br />19<br />
  20. 20. HASIL<br />20<br />33 strain P. aeruginosa<br />53 strain Enterobacteriaceae<br />100 BC<br />9 strain Stenotrophomonasmaltophilia<br />2 spesies gram negatif lain<br />3 BC adalahpolimikrobial<br />Dijumpaiinhibisi PCR padadua (2 %) <br />preparasi DNA<br />
  21. 21. Tabel 2 HasiltesqPCRecfX Pseudomonas aeruginosadari 98 botol BC positif<br />KulturdarahPolimikrobial . <br />Didapatkan P. aeruginosasebanyak 20 CFU/ml dan K. pneumoniae 108 CFU/ml darikulturkuantitatif<br />21<br />
  22. 22. Lanjutan HASIL<br />22<br />qPCRternyatatidakdapatmendeteksiadanyaP.aeruginosadalamkulturPolimikrobial<br />PenelitimelakukantesqPCRlangsungdariisolatP.aeruginosa ecfXberhasildiamplifikasi<br />KulturkuantitatifditemukanbahwakonsentrasiP.aeruginosadi BC inisangatrendah (20 CFU/ml).<br />Batas deteksi PCR Kuantitatifpenelitianiniadalah<br />102 CFU/ml. <br />
  23. 23. Pada<br />Dari 98 BC yang dapat ditafsirkan (98 %), assay qPCR menunjukkan sensitivitas 97%, spesifisitas 100 %, PPV 98,5 % dan NPV 100%.<br />23<br />
  24. 24. Lanjutan HASIL<br />Sensitivitas 97%<br />Spesifisitas 100 %<br />98 BC<br />NPV 98.5 %<br />24<br />PPV 100 %<br />
  25. 25. DISKUSI<br />DenganteknikFISH (disetujuioleh FDA) yang secaralangsungdigunakanpada BC positif, metodeqPCRterbarumenunjukkanperforma yang serupanamunlebihcepat (1,5 jam vs 2,5 jam) <br />Inhibitor teknikqPCRyang potensialdarispesimen BC positifadalahcharcoal dan hemoglobin<br />25<br />
  26. 26. Lanjutan DISKUSI<br />Informasiakuratdantepatwaktu yang diberikanolehassayqPCRiniakanmembantudoktermengidentifikasibakteremiaP. aeruginosadanmemberiterapilebihcepat 18-24 jam.<br />SulituntukmemprediksikepekaanantibiotikapadaP. aeruginosadenganteknikmolekuler.<br />P. aeruginosaMultidrug resistance  terapiantibiotikadikombinasi<br />26<br />
  27. 27. 27<br />PenelitimengembangkanteknikqPCR<br /> identifikasicepat (< 1,5 jam) dengansensitivitasdanspesifisitastinggiuntukidentifikasiPseudomonas aeruginosa<br />dari BC positif. <br />Penelitianlanjutanmengevaluasi<br />dampakklinisdarimetodeqPCRbaruinidibandingkandenganmetode<br />konvensional.<br />2<br />1<br />KESIMPULAN<br />
  28. 28. www.themegallery.com<br />Thank You !<br />www.themegallery.com<br />
  29. 29. KurvaamplifikasiqPCRdariplasmid DNA reference material dengan 11 konsentrasi DNA Eksternal(dari 1011sampai 10 copies/ml)<br />29<br />Treshold<br />CT<br />CT<br />CT<br />
  30. 30. KurvakalibrasiqPCRmenggunakanpengenceran serial dariplasmid DNA reference material<br />30<br />
  31. 31. Inhibitor PCR <br />Inhibitor PCR: Darah, jaringan, fabrics, soil<br />Sumber lain : material danreagen yang kontakdengansampelselamaproses PCR<br />misal : KelebihanKCl, NaCl, Ion detergen (Sodium Deoxycholate), Isopropanolol, Phenol<br />Cara mengatasi :<br /> - inhibitor PCR daridarahdanjaringantidakbisadihindari<br /> - inhibitor PCR misal saliva dengan media transfer swab pemurnian DNA dengancaraekstraksi  memurnikanTemplate DNA<br />31<br />
  32. 32. 32<br />
  33. 33. PCR CONTAMINATION<br />Contamination is defined as the unwanted presence of DNA (or RNA) amplicons<br />At present, no experimental means<br /> exist which can directly detect the ultra-trace amounts of this template (e.g.,<br />picogram amounts or less). <br />The best way to monitor is to use reagent-only blanks <br />The presence of contamination is then signalled by the appearance of the test amplicon in the reagent-only blank tubes.<br />33<br />
  34. 34. Triton X-100<br /> Triton X-100 (C14H22O(C2H4O)n) is a nonionicsurfactant which has a hydrophilicpolyethylene oxide group (on average it has 9.5 ethylene oxide units) and a hydrocarbonlipophilic or hydrophobic group. <br />34<br />
  35. 35. Tween 20<br />Polysorbate 20 (a common commercial brand name is Tween 20) is a polysorbatesurfactant whose stability and relative non-toxicity allows it to be used as a detergent and emulsifier in a number of domestic, scientific, and pharmacological applications. <br />It is a polyoxyethylene derivative of sorbitanmonolaurate, and is distinguished from the other members in the polysorbate range by the length of the polyoxyethylene chain and the fatty acid ester moiety. <br />35<br />
  36. 36. Tris-HCl<br />TE buffer is a commonly used buffer solution in molecular biology, especially in procedures involving DNA or RNA. <br />"TE" is derived from its components: Tris, a common pH buffer, and EDTA, a molecule that chelatescations like Mg2+. <br />The purpose of TE buffer is to solubilize DNA or RNA, while protecting it from degradation.<br />36<br />
  37. 37. TBE<br />TBE or Tris/Borate/EDTA, is a buffer solution containing a mixture of Tris base, boric acid and EDTA.<br />In molecular biology, TBE and TAE buffers are often used in procedures involving nucleic acids, the most common being electrophoresis. <br />Tris-acid solutions are effective buffers for slightly basic conditions, which keep DNA deprotonated and soluble in water. <br />EDTA is a chelator of divalent cations, particularly of magnesium (Mg2+). As these ions are necessary co-factors for many enzymes, including contaminant nucleases, the role of the EDTA is to protect the nucleic acids against enzymatic degradation. <br />37<br />
  38. 38. KANAMYCIN<br />Kanamycin is used in molecular biology as a selective agent most commonly to isolate bacteria (e.g., E. coli) which have taken up genes (e.g., of plasmids) coupled to a gene coding for kanamycin resistance (primarily Neomycin phosphotransferase II [NPT II/Neo]). <br />Bacteria that have been transformed with a plasmid containing the kanamycin resistance gene are plated on kanamycin (50-100ug/ml) containing agar plates or are grown in media containing kanamycin (50-100ug/ml). <br />Only the bacteria that have successfully taken up the kanamycin resistance gene become resistant and will grow under these conditions. <br />As a powder kanamycin is white to off-white and is soluble in water (50mg/ml).<br />38<br />
  39. 39. Dampaknegatif<br />Pasientidaksembuh<br />Waktuopnamelebih lama<br />Memicuresistensiantibiotika lain<br />39<br />
  40. 40. Kelemahan PCR<br />Tidakbisamenunjukkanbakteriinihidupataumati. <br />KulturtetapdipercayasebagaiGold Standard  karenabakteritumbuhdanhidup<br />Tetapidarisampeldarahbiasanyahanya 20-30% yang positif  menunjukkansulitnyamencapaiGold Standard  butuhmetodealternatifuntukidentifikasi.<br />40<br />
  41. 41. Antimicrobial Resistance in P. aeruginosa<br />Intrinsic resistance to most antibiotics is attributed to:<br />Efflux pumps: Chromosomally-encoded genes (e.g. mexAB-oprM, mexXY, etc) and <br />Low permeability of the bacterial cellular envelope<br />Acquired resistance with development of multi-drug resistant strains by:<br />Mutations in chromosomally-encoded genes, or <br />Horizontal gene transfer of antibiotic resistance determinants <br />41<br />
  42. 42. Metodefenotip<br />MetodekonvensionaluntukidentifikasiPseudomonas aeruginosabutuhwaktu lama<br />Tesgula-gula :<br />Tesoksidasepositif<br />Mamputumbuhpada 42oC<br />TSI slant  tidakadareaksi<br />MH agar : Warnahijaukebiruan, merahataucoklat<br />Produksinitrat, Gas +<br />42<br />
  43. 43. Metodefenotip<br />LanjutanTesGula-gula :<br />Arginindehidrogenase +<br />Gelatin Liquified : variabel<br />Lysine decarboxylase –<br />Urea Hydrolysis : variabel<br />Mengoksidaseglukosa & Xylosa (Lactose -, mannitolvariabel)<br />43<br />
  44. 44. Pseudomonas aeruginosa<br />Scientific Classification :<br />Kingdom : Bacteria<br />Phylum : Proteobacteria<br />Class : Gamma Proteobacteria<br />Order : Pseudomonadales<br />Family : Pseudomonadaceae<br />Genus : Pseudomonas<br />Species : Pseudomonas aeruginosa<br />44<br />
  45. 45. Synonims<br />Bacterium aeruginosumSchroeter 1872<br />Bacterium aeruginosum Cohn 1872<br />Micrococcus pyocyaneusZopf 1884<br />Bacillus aeruginosus (Schroeter 1872) Trevisan 1885<br />Bacillus pyocyaneus (Zopf 1884) Flügge 1886<br />Pseudomonas pyocyanea (Zopf 1884) Migula 1895<br />Bacterium pyocyaneum (Zopf 1884) Lehmann and Neumann 1896<br />Pseudomonas polycolor Clara 1930<br />Pseudomonas vendrellinomennudum 1938<br />45<br />
  46. 46. <ul><li>Although classified as an aerobic organism, P. aeruginosa is considered by many as a facultative anaerobe, as it is well adapted to proliferate in conditions of partial or total oxygen depletion.
  47. 47. This organism can achieve anaerobic growth with nitrate as a terminal electron acceptor, and, in its absence, it is also able to ferment arginine by substrate-level phosphorylation.</li></ul>46<br />
  48. 48. 47<br />
  49. 49. Biofilms and treatment resistance<br />Biofilms of Pseudomonas aeruginosa can cause chronic opportunistic infections. <br />They often cannot be treated effectively with traditional antibiotic therapy. <br />Biofilms seem to protect these bacteria from adverse environmental factors. <br />Pseudomonas aeruginosa can cause nosocomial infections and is considered a model organism for the study of antibiotic-resistant bacteria. <br />48<br />
  50. 50. DIAGNOSIS<br />First, a Gram stain is performed, which should show Gram-negative rods with no particular arrangement.<br />Then, if the specimen is pure, the organism is grown on MacConkey agar plate to produce colorless colonies (as it does not ferment lactose)<br />Cetrimide agarP. aeruginosa may express the exopigmentpyocyanin, which is blue-green in color, and the colonies will appear flat, large, and oval. It also has a characteristic fruity smell. <br />P. aeruginosa is catalase+, oxidase+, nitrase+, and lipase+. When grown on TSI medium, it has a K/K profile, meaning that the medium will not change color. <br />Finally, serology could help, which is based on H and Oantigens.<br />49<br />
  51. 51. TREATMENT<br />aminoglycosides (gentamicin, amikacin, tobramycin)<br />quinolones (ciprofloxacin, levofloxacin, and moxifloxacin)<br />cephalosporins (ceftazidime, cefepime, cefoperazone, cefpirome, but notcefuroxime, ceftriaxone, cefotaxime)<br />antipseudomonalpenicillins: ureidopenicillins and carboxypenicillins (piperacillin, ticarcillin: P. aeruginosa is intrinsically resistant to all other penicillins)<br />carbapenems (meropenem, imipenem, doripenem, but notertapenem)<br />polymyxins (polymyxin B and colistin)[31]<br />monobactams (aztreonam)<br />In the rare occasions where infection is superficial and limited (for example, ear infections or nail infections), topicalgentamicin or colistin may be used.<br />50<br />
  52. 52. 51<br />
  53. 53. PENGGUNAAN INKUBATOR SESUAI KEBUTUHAN LINGKUNGAN UNTUK PERTUMBUHAN BAKTERI<br />52<br />
  54. 54. TOPLES LILIN<br />Komponen<br />Toplesterbuatdarikaca<br />Tutuptoples<br />Lilinputih yang kering<br />53<br />
  55. 55. ANAEROBIC JAR DENGAN GAS PACK<br />KomponenAlat:<br />Toplesterbuatdarimika yang dilengkapikaretmelingkardiatasnya<br />Tutuptoplesdengan 3 lubangsaluranuntuk gas CO2, O2 dan N2<br />Penjepitantaratutupdantoples yang diperkuatsekrupdiatasnya<br />Gas pack<br />Indikatoranaerob<br />Katalis<br />54<br />
  56. 56. INKUBATOR CO2<br />55<br />
  57. 57. The basics<br />Aerobic, opportunistic pathogen<br />Gram-negative bacillus<br />Flagella<br />56<br />
  58. 58. Pathogenesis<br />Virulence Factors (structural components)<br />“Alginate”<br />Adherence proteins and “pili”<br />Lipopolysaccharide (LPS)<br />Procyanin<br />57<br />
  59. 59. Structure<br />58<br />
  60. 60. Structural Components<br />Adherence to host cells mediated by pili and nonpilusadhesins. <br />LPS (lipopolysaccharide) inhibiting antibiotic killing and suppress neutrophil and lymphocyte activity<br />Alginate – mucoidexopolysaccharide that forms a shiny biofilm protecting from antibodies, complement, phagocytosis, and antibiotics<br />Procyanin – impairs ciliary function, mediates tissue damage through production of oxgen radicals<br />59<br />
  61. 61. Pathogenesis<br />Virulence Factors (toxins and enzymes):<br />Exotoxin A<br />Exoenzyme S<br />Endotoxins<br />Phospholipase C<br />Elastase and Alkaline Protease<br />60<br />
  62. 62. Exotoxin A<br />Similar in structure to Diptheria toxin <br />Inhibits protein synthesis by ADP-ribosylating EF-2 (G-protein)<br />Causes Dermatonecrosis in burn wounds, corneal damage in ocular infections, and tissue damage in chronic pulmonary infections.<br />Also this toxin is immunosuppressive<br />61<br />
  63. 63. Exoenzyme S & T<br />ADP-ribosylates G-proteins including p21 RAS interfering with host cell growth<br />62<br />
  64. 64. Phospholipase C<br />Heat labile hemolysin<br />Breaks down lipids and lecithin causing tissue destruction<br />Stimulates inflammatory response<br />63<br />
  65. 65. Elastase and Alkaline Protease<br />Destruction of elastin-containing tissues (blood vessels, lung tissue, skin), collagen, immunoglobulins, and complement factors<br />Can produce hemorrhagic lesions (ecthyma gangrenosum) associated with disseminated infection<br />Inactivation of interferon and TNF-Alpha<br />64<br />
  66. 66. Epidemiology<br />Ubiquitous in moist environmental sites in the hospital as well as nature<br />No seasonal incidence<br />Can transiently colonize the respiratory and GI tract of hospitalized patients<br />Minimal nutritional requirements and can tolerate broad temperature spectrum<br />65<br />
  67. 67. Clinical Disease<br />Pulmonary Infections<br />Burn Wound Infections and other skin and soft tissue infections (life threatening)<br />UTI’s (especially catheterized)<br />External Otitis (malignant OE, swimmer’s ear)<br />Eye Infections and corneal ulceration via contaminated contact lens cleaning fluids<br />Pseudomonal Endocarditis<br />66<br />
  68. 68. Pulmonary Infections<br />Can range from asymptomatic colonization to severe necrotizing bronchopneumonia<br />Colonization is seen in patients with cystic fibrosis, chronic lung disease, and neutropenia<br />Mucoid strains are commonly isolated from chronic pulmonary patients and are more difficult to eradicate<br />Predisposing conditions include previous therapy with broad spectrum abx (disrupts normal protective bacteria population and use of respiratory therapy equipment (can introduce the organism to lower airways)<br />Mortality rate can be as high as 70% for invasive bronchopneumonia<br />67<br />
  69. 69. Ecthyma Gangrenosum<br />Ecthyma gangrenosum is a well recognized cutaneous manifestation of severe, invasive infection by Pseudomonas aeruginosa that is usually seen in immunocompromised, burn patients, and other critically ill patients <br />68<br />
  70. 70. Antibiotic Resistance<br />Inherently resistant to many abx<br />Can mutate to more resistant strains during therapy<br />Penetration of abx highly dependent on outer membrane pores which can be altered<br />Production of B-lactamases<br />Combination of active abx generally required for successful therapy<br />69<br />
  71. 71. Drigalski Agar<br />Drigalski agar is a selective isolation and differentiation medium used to identify Enterobacteriaceae and other Gram-negative bacteria.<br />Microorganisms that ferment lactose form yellow or yellowish-green colonies; the others produce blue, green, or bluish-green colonies.<br />The presence of sodium deoxycholate and crystal violet inhibits the growth of Gram-positive.<br />70<br />
  72. 72. Chocolate Agar<br />A non-selective, enriched growth medium. <br />A variant of the blood agar plate. <br />Contains red blood cells, which have been lysed by heating very slowly to 56 °C. <br />Chocolate agar is used for growing fastidious (fussy) respiratory bacteria, such as Haemophilusinfluenzae. These bacteria need growth factors, like NAD and hematin, which are inside red blood cells; thus, a prerequisite to growth is lysis of the red blood cells. <br />71<br />
  73. 73. Trypticase Soy Agar<br />The medium contains enzymatic digests of casein and soybean meal which provides amino acids and other nitrogenous substances.<br />Dextrose is the energy source. Sodium chloride maintains the osmotic equilibrium, while dipotassium phosphate acts as buffer to maintain pH.<br />The medium may be supplemented with blood to facilitate the growth of more fastidious bacteria or antimicrobial agents to permit the selection of various microbial groups from pure flora.<br />72<br />
  74. 74. Microbiology<br />Family Pseudomonadaceae<br />Aerobic, non-spore forming Gram negative straight or slightly curved rod (1 to 3 um in length), polar flagella<br />Non-fermenters<br />Catalase and oxidase positive<br />Morphologic characteristics on lab media:<br />Production of pigments:<br />Soluble blue-coloured phenazine pigment called pyocyanin)<br />Some strains produce red or black colonies due to pigments termed pyorubin and pyomelanin, respectively<br />P. aerugnosa produces pyoverdin (diffusible yellow-green to yellow-brown pigment) which, when produced with pyocyanin gives rise to green-blue colonies on solid media<br />Term ‘aeruginosa” stems from green-blue hue <br />73<br />
  75. 75. Microbiology<br />Term Pseudo = “false”; monas = “single unit”<br />Term ‘aeruginosa” stems from green-blue hue<br />Pseudomonas are classified as strict aerobes but some exceptions:<br />May use nitrate<br />Biofilm formation<br />74<br />
  76. 76. Microbiology<br />Pseudomonads classified into five rRNA homology groups:<br />Pseudomonas (sensu stricto)<br />Burkholderia species<br />Comamonas, Acidovorax, and Hydrogenophaga genera<br />Brevundimonas species<br />Stenotrophomonas and Xanthomonas genera<br />Genus Pseudomonas contains over 160 species but only 12 are clinically relevant<br />75<br />
  77. 77. Microbiology<br />P. aeruginosa is the type species and may have highly varied morphology<br />Typical colonies may appear to spread over the plate, lie flat with a metallic sheen and frequently produce a gelatinous or “slimy” appearance<br />Most strains produce characteristic ‘grapelike’ or ‘corn taco-like’ odor<br />76<br />
  78. 78. P. aeruginosa on blood agar<br />77<br />
  79. 79. Non-Lactose Fermenter<br />Lactose Fermenter<br />MacConkey Agar<br />78<br />
  80. 80. P. aeruginosa<br />Mucoid P. aeruginosa<br />MacConkey Agar<br />79<br />
  81. 81. Epidemiology and Transmission<br />Natural habitat:<br />Temperature between 4 to 36oC (can survive up to 42oC)<br />Found throughout nature in moist environment (hydrophilic) (e.g. sink drains, vegetables, river water, antiseptic solutions, mineral water, etc.)<br />P. aeruginosa rarely colonizes healthy humans<br />Normal skin does not support P. aeruginosa colonization (unlike burned skin)<br />Acquisition is from the environment, but occasionally can be from patient-to-patient spread<br />80<br />
  82. 82. Range of clinical infections caused by P. aeruginosa<br />P. aeruginosa is an opportunistic infection:<br />Individuals with normal host defenses are not at risk for serious infection with P. aeruginosa<br />Those at risk for serious infections include:<br />Profoundly depressed circulating neutrophil count (e.g. cancer chemotherapy)<br />Thermal burns<br />Patients on mechanical ventilation<br />Cystic fibrosis patients<br />81<br />
  83. 83. Range of clinical infections caused by P. aeruginosa<br />Immunocompetent Host:<br />Most common cause of osteochondritis of dorsum of foot following puncture wounds (running shoes)<br />Hot tub folliculitis<br />Swimmer’s ear<br />Conjunctivitis in contact lens users (poor hygiene or if lenses are worn for extended periods)<br />Other Hosts:<br />Malignant otitis externa in diabetics<br />Meningitis post trauma or surgery<br />Sepsis and meningitis in newborns<br />Endocarditis or osteomyelitis in IVDUs<br />Community-acquired pneumonia in pts with bronchiectasis<br />UTI in patients with urinary tract abnormalitis<br />82<br />
  84. 84. PLASMID<br />A plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. <br />They are double stranded and, in many cases, circular. <br />Plasmids usually occur naturally in bacteria.<br />83<br />
  85. 85. PLASMID<br />Plasmids can be considered to be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer.<br />Plasmids are considered transferable genetic elements, or "replicons", capable of autonomous replication within a suitable host.<br />plasmids are "naked" DNA and do not encode genes necessary to encase the genetic material for transfer to a new host, though some classes of plasmids encode the sex pilus necessary for their own transfer.<br />84<br />
  86. 86. TesOksidase<br />Digunakanuntukmembantuidentifikasispesies yang memproduksienzimoksidase<br />Prinsip<br />Sepotongkertassaringdibasahidenganbeberapatetesreagenoksidase. Jikaorganismememproduksioksidase, phenylenediaminepadareagenakanteroksidasimenjadiwarna deep purple.<br />Reagen yang dibutuhkan:<br />ReagenOksidase yang berisilarutan 10 g/l tetramethyl-p- phenylenediaminedihydrochloride<br />Ctt: reageninimudahteroksidasi. Jikareagentelahberwarnabiru, jangangunakan.<br />85<br />
  87. 87. Metode<br /><ul><li>Letakkankertassaringpadapetri disk yang bersih, tambahkan 2-3 ttsreagenoksidase
  88. 88. Denganbatangkaca, pindahkankoloniorganismetesdanoleskanpadakertassaring
  89. 89. Lihatperubahanwarnamenjadi blue-purple dalambeberapadetik</li></ul>Hasil<br />Warna blue purple (dalam 10 detik) positifmemproduksienzimoksidase<br />Warnabukan blue purple (dlam 10 detik) negatif<br />Ctt:abaikanwarna blue purple yang terjadisetelah 10 detik<br />Kontrolpositif: Pseudomonas aeruginosa<br />Kontrolnegatif: Eschericia Coli<br />86<br />
  90. 90. 87<br />
  91. 91. Pseudomonas aeruginosa<br />Cultural features<br /> blood agar: large, flat, haemolitic colonies. Strict aerob. Most strain produce pyocyanin (yellow green in medium)<br />McConkey:non lactose fermenting colonies with yellow green in medium<br /> KIA: Pink-red slope and butt, gas (-), H2S (-)<br />identifikasi: gram negatif, oksidasepositif, citrate positif<br />MIU: Motility positif, indolnegatif, tesureasetergantung strain <br />88<br />
  92. 92. Citrate utilization test<br />Untukmembantuidentifikasienterobactericiaea. Tesiniberdasarkankemampuanorganismemenggunakansitratsebagaisumberkarbondan ammonia sebagaisumber nitrogen<br />Prinsip:<br />Organismedikulturpada media yang mengandung sodium citrate, garam ammonium, danindikatorbromothymol blue. Pertumbuhanpada media ditandaiolehkekeruhandanperubahanwarnaindikatordarihijauterangmenjadibiru, olehkarenareaksialkali,mengikutipemakaiancitrat<br />89<br />
  93. 93. Bahan yang dibutuhkan:<br /> Media Koser’s citrate (Simon citrate dapatdigunakantapilebihmahal)<br />Dengansterile straight wire, inokulasiorganismedi media kultur broth ke 3-4 ml media Koser’s Citrate<br /> (harushati-hati agar tidakmengkontaminasi media denganpartikelkarbon, sepertikawat yang terbakar<br />Inkubasipada 35-37oC, hingga 4 hari, periksatiaphariadanyapertumbuhan<br />90<br />
  94. 94. Hasil<br /><ul><li>Kekeruhandanwarnabiru, hasiltespositif,citrateterpakai
  95. 95. Takadapertumbuhan, tesnegatif, citrate tidakterpakai
  96. 96. Kontrolpositif : Klebsiellapneumoniae
  97. 97. Kontrolnegatif : Eschericia coli</li></ul>91<br />
  98. 98. CITRATE UTILIZATION TEST<br />92<br />Inkubasi<br />Positifnegatif<br />
  99. 99. 93<br />
  100. 100. Urease Test <br />Tesureasepentinguntukmembedakanenterobacteriaceaeberdasarkankemampuanmemproduksiurease.<br />Prinsip<br />Organismetesdikulturpada media yang mengandung urea danindikator phenol warnamerah. Jika strain memproduksiurease, enziminiakanmemecah urea menghasilkan ammonia dankarbondioksida. Denganmenghasilkan ammonia, media menjadi alkali yang ditunjukkanperubahanwarnamenjadimerahmuda<br />94<br />
  101. 101. Tesurease<br />Bahan yang dibutuhkan<br /> Media Motility Indole urea (MIU)<br />Metode<br />menggunakankawatsterillurus, inokulasi media pada media MIU<br />Tempatkanindole paper strip dilehertabung MIU diatas media. Tutuptabungdaninkubasipada 35-37oC semalam<br />Periksaproduksiureasedenganmelihatwarnamerahmudapada media<br />95<br />
  102. 102. Hasil<br /> Media merahmuda, positifmenghasilkanurease<br /> Media tidakmerahmudanegatifmenghasilkanurease<br />Kontrol<br />kontrolpositifurease : Proteus vulgaris<br />kontrolnegatifurease : Eschericia Coli<br />Motilitas<br />ditunjukkandenganadanyapenyebarankekeruhandarigaristusukan<br />96<br />
  103. 103. 97<br />
  104. 104. TesIndol<br />Tesproduksiindolpentinguntukidentifikasienterobacteriaceae. Kebanyakan strain dariE.Colimemecahasam amino tryptophan denganmenghasilkanindole.<br />Organismetesdikulturpada media yang mengandung tryptophan. ProduksiindoldideteksiolehreagenKovac’satau Ehrlich yang berisi 4 (p)-dimethylaminobenzaldehyde. Bahaninibereaksidenganindolsehinggamemproduksiwarnamerah<br />98<br />
  105. 105. Bahan<br /> media MIU<br />Kovac’s reagent strips<br />Hasil<br /> strip kemerahan, positifmenghasilkanindol<br />bukanwarnamerah, negatifmenghasilkanindol<br />Jikabelumjelas, tambahkan 1 ml reagent Kovacdanlihatadanyawarnamerahdalam 10 menit<br />99<br />
  106. 106. 100<br />
  107. 107. gyrB<br />DNA gyrase subunit B<br />Location : sitoplasma<br />Genomic location : 4275 - 6695 (+) <br />Function : DNA replication, recombination and repair<br />101<br />
  108. 108. gyrB<br />102<br />Dari gyrB dicari primer misalnya 20 bpdengan<br />urutansbb :<br />Diambildari www.pseudomonas.com<br />
  109. 109. Exotoxin A<br />103<br />
  110. 110. algD<br />104<br />Gene name : algD<br />Genomic Location : 3962825 - 3964135 (+) <br />Function : Cell wall / LPS / capsule Adaptation, Protection Secreted Factors (toxins, enzymes, alginate)<br />
  111. 111. oprI<br />Gene Name :oprI<br />Location : 3206915 – 3207166<br />PseudoCAP Function Class : Membrane Protein<br />Product name : Outer membrane lipoprotein OprI precursor<br />105<br />
  112. 112. oprL<br />Gene Name :oprL<br />Location : 1057400 - 1057906<br />PseudoCAP Function Class :Membrane proteins,Transport of small molecules<br />Product name : Peptidoglycan associated lipoprotein OprL precursor<br />106<br />
  113. 113. toxA<br />Gene Name : toxA<br />Location : 1242500 - 1240584<br />PseudoCAP Function Class : Secreted Factors (toxins, enzymes, alginate)<br />Product name : Exotoxin A precursor<br />107<br />
  114. 114. PCR<br />PCR is a technique used to amplify segments of DNA in vitro<br />PCR can generate millions of copies of DNA fragments<br />108<br />
  115. 115. 109<br />
  116. 116. BacT/ALERT<br />Fungsinya sama dengan BACTEC sebagai media pemupuk kuman.<br />Macam Media :<br />110<br />FAN ANAEROB<br />PEDIATRIC FAN <br />FAN AEROB<br /> SA SN FA FN PF MP Lytic<br />
  117. 117. Media BacT/ALERT<br />111<br />
  118. 118. Prinsip dan Cara Kerja BacT/ALERT<br />Menggunakan sensor kolorimetri dan sinar refleksi untuk memonitor CO2 yang larut pada botol media.<br />CO2 dihasilkan oleh mikroorganisme  berdifusi melewati membran dan melepas ion H+perubahan pH ditangkap oleh sensor warna pada dasar botol BacT/ALERT perubahan warna dari abu-abu menjadi kuning.<br />112<br />
  119. 119. 113<br />Perubahan warna botol<br />Prinsip kerja kolorimetri<br />
  120. 120. 114<br />
  121. 121. 115<br />
  122. 122. API 20 E<br />API 20 E is an identification system for Enterobacteriaceae and other non-fastidious Gram-negative rods which uses 23 standardized and miniaturized biochemical tests and a database. <br />The API 20 E strip consists of 20 microtubes containing dehydrated substrates.<br />These tests are inoculated with a bacterial suspension which reconstitutes the media. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents.<br />Incubate at 35-37°C for 18-24 hours.<br />116<br />
  123. 123. Preparation of the strip<br />Prepare an incubation box (tray and lid) and distribute about 5 ml of distilled water or demineralized water [or any water without additives or chemicals which may release gases (e.g., Cl2, CO2, etc.)] into the honeycombed wells of the tray to create a humid atmosphere.<br />Record the strain reference on the elongated flap of the tray.<br />Remove the strip from its packaging.<br />Place the strip in the tray.<br />Perform the oxidase test on a colony identical to the colony which will be tested.<br />Refer to the Oxidase Test Kit Package Insert.<br />This reaction should be recorded on the result sheet as it constitutes the 21st test.<br />117<br />
  124. 124. Preparation of the inoculum<br />Open an ampule of NaCl 0.85 % Medium (5 ml) or an ampule of Suspension Medium (5ml) as indicated in the paragraph "Warnings and Precautions" or use any tube containing 5 ml of sterile NaCl 0.85% Medium, pH 5.5 - 7.0, or sterile distilled water, without additives.<br />With the aid of a pipette, remove a single well-isolated colony from an isolation plate.<br />Carefully emulsify to achieve a homogeneous bacterial suspension.<br />118<br />
  125. 125. Inoculation of the strip<br />With the same pipette, fill both tube and cupule of test CIT , VP and GEL with the bacterial suspension.<br />Fill only the tubes (and not the cupules) of the other tests.<br />Create anaerobiosis in the tests _A_D_H_, _L_D_C_, _O_D_C_, _H_2_S_ and U__R_E_ by overlaying with mineral oil.<br />Close the incubation box.<br />Incubate at 35-37°C for 18-24 hours.<br />119<br />
  126. 126. PCR(Polymerase Chain Reaction)<br />
  127. 127. Pendahuluan<br /><ul><li>PCR ditemukanolehKaryMulispadatahun 1985.
  128. 128. PCR  suatutekniksintesisdanamplifikasifragmen DN A secara in vitro
  129. 129. Proses PCR miripdenganprosesreplikasi DNA in vivo
  130. 130. PCR membutuhkan template untaiganda yang mengandung DNA target yang akandiamplifikasi, enzim DNA polimerase, nukleotidatrifosfat, dansepasang primer oligonukleotida.</li></ul>121<br />
  131. 131. PRIMER<br />Primer oligonukleotidadisintesismenggunakanalatDNA Synthesizer<br />Untukmerancangurutan primer, perludiketahuiurutannukleotidapadaawaldanakhir DNA target.<br />Padakondisitertentu, kedua primer menempelpadauntai DNA komplemennya yang terletakpadaawaldanakhir DNA target. <br />122<br />
  132. 132. Lanjutan (PRIMER)<br />Primer haruspunyasuhuTm(Melting Temperature) yang samadengansuhuprosesanneling menghindarimishybridizedan agar tidakmenempelkesegmen DNA lain<br />Kedua primer menempelpadaawaldanakhir DNA target  berfungsimenyediakangugushidroksilbebaspadakarbon 3’.<br />SetelahituEnzim DNA Polimerasemengkatalisisprosespemanjangankedua primer tersebutdenganmenambahkannukleotida yang sesuai<br />123<br />
  133. 133. Primer dipilihdaridaerah yang khususuntukmenghindarimishybridizationdari sequence yang sama. <br />Method BLASTseringdigunakanuntukmencari primer disemua area tersebut. <br />Basic Local Alignment Search Tool<br />124<br />
  134. 134. Komponen PCR<br />125<br />DNA Template<br />Enzim DNA Polymerase<br />Primer<br />dNTP (deoxynucleosidetriphosphate)<br />Buffer<br />Ion Logam<br />
  135. 135. 16 S rRNA<br />126<br />16S ribosomal RNA (or 16SrRNA) is a component of the 30S subunit of prokaryoticribosomes. It is 1,542 nucleotides in length.<br />Multiple sequences of 16S rRNA can exist within a single bacterium.[2]<br />In addition to highly conserved primer binding sites, 16S rRNA gene sequences contain hypervariable regions that can provide species-specific signature sequences useful for bacterial identification. As a result, 16S rRNA gene sequencing has become prevalent in medical microbiology as a rapid, accurate alternative to phenotypic methods of bacterial identification[9].<br />
  136. 136. 16 S rRNA<br />127<br />It has several functions:<br />Like the large (23S) ribosomal RNA, it has a structural role, acting as a scaffold defining the positions of the ribosomal protein<br />The 3' end contains the anti-Shine-Dalgarno sequence, which binds upstream to the AUG start codon on the mRNA<br />Interacts with 23S, aiding in the binding of the two ribosomal subunits (50S+30S)<br />Stabilizes correct codon-anticodon pairing in the A site, via a hydrogen bond formation between the N1 atom of Adenine (see image of Purine chemical structure) residues 1492 and 1493 and the 2'OH group of the mRNA backbone<br />
  137. 137. 128<br />16 S rRNA<br />
  138. 138. Enzim DNA Polimerase<br />129<br />Enzim DNA Polimerasebersifattermostabil, tahansampaisuhu 95oC<br />BakteriThermusAquaticus(enzimdisebutTaqPolymerase)<br />Meningkatkanspesifisitas PCR karenasintesis DNA dilakukanpadasuhu 72oC <br />
  139. 139. dNTP (deoxynucleosidetriphosphate)<br />130<br />Untuksintesis DNA :<br />Deoksiadenosintrifosfat (dATP)<br />Deoksitidintrifosfat (dCTP)<br />Deoksiguadintrifosfat (dGTP)<br />Deoksitimidintrifosfat (dTTP)<br />dNTPinimenempelpadagugus 3’ hidroksilbebas primer membentukrantaiuntaibarudenganrantai DNA templat<br />
  140. 140. Buffer dan Magnesium klorida<br />131<br />Buffer yang biasanyaterdiriatasbahan-bahankimiauntukmengkondisikanreaksi agar berjalan optimum danmenstabilkanenzim DNA polymerase.<br />Ion Magnesium membentukkomplekslarutantaradNTP.<br />Ion Magnesium menstimulasiaktivitaspolimerase, meningkatkan Tm DNA untaigandadaninteraksidengan template<br />
  141. 141. Primer<br />132<br />Panjangoligonukleotida :15 – 30 bp<br />Temperatur melting ™ : 55 – 65oC<br />Komposisinukleotida<br />Lokasipada DNA target<br />Panjang target yang diamplifikasi<br />
  142. 142. Pradenaturasi<br />133<br />Dilakukanselama 1-10 menitdiawalreaksiuntukmemastikankesempurnaandenaturasidanmengaktifasi DNA Polymerase (jenis hot-start alias baruaktifkalaudipanaskanterlebihdahulu).<br />
  143. 143. DNA<br />134<br />DNA adalahasamnukleat yang mengandungmaterigenetikdanberfungsiuntukmengaturperkembanganbiologisseluruhbentukkehidupansecaraseluler. <br />DNA terdapatpadanukleus, mitokondriadankloroplas. <br />Perbedaandiantaraketiganyaadalah: DNA nukleusberbentuk linear danberasosiasisangateratdengan protein histon, sedangkan DNA mitokondriadankloroplasberbentuksirkulardantidakberasosiasidengan protein histon. <br />Struktur DNA prokariotberbedadenganstruktur DNA eukariot. DNA prokariottidakmemiliki protein histondanberbentuksirkular, sedangkan DNA eukariotberbentuk linear danmemiliki protein histon (Klug & Cummings 1994: 315--316; Raven & Johnson 2002: 94).<br />
  144. 144. Bentuk DNA<br />135<br />ContohGambar Double helix<br />
  145. 145. PenyusunUtama DNA<br />136<br />Sesuaidengannamanya, DNA, Deoxyribose Nucleic Acid. Penyusunutama<br /> DNA adalahgula ribose yang kehilangansatu atom oksigen (deoksiribose).<br />Gambar 2. Perbedaan Ribose danDeoksiribose<br />Perhatikangambardiatas, padadeoksiribose, satu atom oksigen<br />padasalahsatu atom C ribose hilang.<br />
  146. 146. Nukleotida<br />137<br />Tiap pita/rantai double helix terbuatdari unit-unit berulang yang disebutnukleotida. Satunukleotidaterdiridaritigagugusfungsi; satugula ribose, triphosphate, dansatubasa nitrogen.<br />
  147. 147. 138<br />Satuhal yang perludiingatadalahposisitriphosphatedanbasa nitrogen yang terikatpadaribosa. Gugustriphosphatterikatpada atom C no 5′ dariribosa (Lihatgambardiatas). Gugustriphosphateinihanyadimilikiolehnukleotidabebas. Sedangkannukleotida yang terikatpadarantai DNA kehilanganduadarigugus phosphate ini, sehinggahanyasatu phosphate yang masihtertinggal.<br />Ketikanukleotidabergabungmenjadi DNA, nukleotida-nukleotidatersebutdihubungkanolehikatanphosphodiester. Ikatankovalen yang terjadiantaragugus phosphate padasatunukleotida, dengangugus OH padanukleotidalainnya. Sehinggasetiaprantai DNA akanmempunyai ‘backbone’ phosphate-ribosa-phosphate-ribosa-phosphate. Dan seterusnya..<br />
  148. 148. 139<br />GambarStruktur DNA Sederhana<br />
  149. 149. Basa Nitrogen Pada DNA<br />140<br />Padastruktur DNA, gularibosadangugus phosphate yang terikatadalahsama. Yang berbedahanyalahpadabasa nitrogen. Jadisebetulnyaperbedaandisebabkanolehvariasisusunandaribasa-basa nitrogen yang terdapatpadarantai DNA. Adaempatmacambasa nitrogen. Adenin, Cytosine, Guainne, dan Thymine.<br />
  150. 150. 141<br />Ketikabasa-basa nitrogen tersebutterikatdalamnukleotida, makapenamaan-pun berubah. Ingatkembalipenjelasandiawaltentangnukelotida. Nukleotidaterdiridarigugustriphosphatedansatubasa nitrogen yang terikatpadasatumolekul ribose. Nah.. basa-basa nitrogen iniapabilaterikatpada ribose membentuknukleotidamakapenamaannya-pun berubah.<br />Adeninmenjadi 2′deoxyadenosinetriphosphate, cytosinmenjadi 2′deoxycytidinetriphosphate, guainnemenjadi 2′deoxyguanosinetriphosphate, dan Thymine menjadi 2′deoxythymidinetriphosphate. Disingkatmenjadi A, C, G, dan T.<br />
  151. 151. 142<br />Perhatikanbahwaadaduapasangbasa yang mirip. A dan G sama-samamempunyaiduacincinkarbon-nitrogen, disebutgolonganpurine. Sedangkan C dan T hanyamempunyaisatucincinkarbon-nitrogen, masukgolonganpirimidin.<br />
  152. 152. PenyebabBentuk DNA Double Helix<br />143<br />GambarIkatanHidrogenAntaraBasa-Basa Nitrogen<br />
  153. 153. 144<br />Interaksiikatanhidrogenantaramasing-masingbasa nitrogen menyebabkanbentukdariduarantai DNA menjadisedemikianrupa, bentukinidisebut double helix. <br />Interaksispesifikiniterjadiantarabasa A dengan T, dan C dengan G. <br />Sehinggajika double helix dibayangkansebagaisebuahtangga spiral, makaikatanbasa-basainisebagaianaktangga-nya. <br />Lebardari ‘anaktangga’ adalahsama, karenapasanganbasaselaluterdiridarisatuprimidindansatupurin.<br />
  154. 154. 145<br />GambarStruktur Double HeliLengkap<br />
  155. 155. StrukturAsam amino<br />146<br />
  156. 156. Transkripsi<br />147<br />Prosespengkopian DNA menjadi RNA inidinamakantranskripsi.<br />
  157. 157. Translasi<br />148<br />mRNA hasiltranskripsikemudiandikeluarkanmenujusitoplasmasehinggabisadiproseslebihlanjutolehsuatuorganelsel yang bernamaribosom. Ribosomakanmembacaurutanbasa RNA danmenterjemahkannya (translate ) menjadiurutanasam amino tertentusesuaidenganresep yang dibawa mRNA. Di sinilahasam-asam amino itudirakitsesuaiurutan yang diresepkan gen (DNA) dankemudianmelipatmembentukstrukturtigadimensi yang fungsional.<br />
  158. 158. Central Dogma of Molecular Biology<br />149<br />
  159. 159. Replikasi DNA<br />150<br />Prosesreplikasipertama kali dimulaiketika enzyme Helicasememutusikatankimia yang paling lemahdiantaraduarantaipolinukleotida. Untaian DNA diputustepatditengahmemisahkanpasangan-pasanganbasa. Rantaipolinukleotida yang barudipisahkanmenjadirantaitunggalakanmenjadirantaidasar (template) untukmembentukduauntairantai DNA baru<br />Prosesterbentuknyaikatanbasa-basainidibantuoleh enzyme yang disebut enzyme DNA Polymerase III. Enzyme inihanyabekerjadariujung 5’ keujung 3’ darirantai DNA. <br />
  160. 160. MengujiSpesifisitas Primer<br />151<br />Primer yang telahkitadesaintersebutharusdiujispesifisitasnya agar kitayakinbahwa:<br />Primer tersebuthanyaakanmengamplifikasidaerah yang kitamau<br />Tidakmenempelpadadaerah lain digenomorganisme target (dalamcontohini TMV memilikimaterigenetikberupa RNA)<br />Tidakmenempelpada DNA organisme lain yang mungkintercampurbersama DNA TMV ketikaisolasi DNA<br />Pengujianspesifisitasdapatdilakukandenganbantuan BLAST di NCBI. Jenis BLAST yang digunakanadalahblastn (nucleotide blast). <br />
  161. 161. MengujiSpesifisitas Primer<br />152<br />Cara mudahuntukmengetahui primer kitaspesifikatautidakadalahdengancaramelihathasilpencarian BLAST. Jika primer kitamemilikikesamaandengan DNA organisme lain ataubahkansama, berarti primer kitatidakspesifik. Inibisabahayakarena primer menempelkemana-mana, akibatnyaakanmunculbanyakproduk PCR yang tidakkitainginkan.<br />
  162. 162. Hasil BLAST masihbisaditoleransijika<br />153<br />Sekuen DNA tersebutberasaldariorganisme lain yang secarapraktiknyatidakmungkinadabersama-samadenganorganisme target (dalamcontohini virus TMV) ketikakitamengambilsampeluntukekstraksi RNA. MisalnyaOryza sativa alias tanamanpadi, atauFeliscatus alias kucing, yang sepertinyatidakmungkinadabersamatanamantembakau.<br />Hanyasatudarisepasang primer tersebut yang didugatidakspesifik. Jikamemangsulitmenemukanalternatif lain yang lebihbaik, makakondisiinimasihbisaditerima, tapijumlah primer yang ditambahkanketika PCR nantinyaharusdioptimasi.<br />Bolehjadikedua primer didugatidakspesifik, tapiposisipenempelantidakspesifikmerekaitupada gen lain terpisahsangatjauhsehinggasecarateoritidakakanmungkinmenghasilkanproduk PCR, misalnyajikajaraknyalebihdari 10000 bp.<br />Sekuen primer tersebuttidakbenar-benar match 100%, terutamajika 5 basaterakhirnyatidak match.<br />
  163. 163. Reverse Transcriptase PCR<br />154<br />RT PCR alias Reverse Transcriptase PCR adalahteknik yang digunakanuntukmembuatcDNA (complementary DNA) dengan RNA sebagai template-nya. <br />Prosesiniadalahkebalikandaritranskripsi DNA menjadi RNA yang umumterjadipadamakhlukhidup, sehinggadinamakan reverse transcription (transkripsiterbalik). <br />Di alamprosesinihanyaterjadipada virus-virus tertentuketikamenyusupkanmaterigenetiknya yang berupa RNA kedalamgenom ‘korbannya’.<br />
  164. 164. PCR vsqPCR<br />155<br />
  165. 165. 156<br />
  166. 166. 157<br />

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