dg molecular hiv 2013 (2)
DESCRIPTION
medicinaTRANSCRIPT
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Diagnostic molecular in virusologie
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Diagnosticul molecular in virusologie
AMPLIFICAREA TINTEI–PCR–NASBA (nucleic acid sequenced based amplification)
AMPLIFICAREA SEMNALULUI–B-DNA (branched DNA)
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PCRPOLYMERASE
CHAIN REACTION
Metoda utilizata pentru detectia ADN-ului de orice tip.
-se bazeaza pe obtinerea unui numar mare de catene tinta prin repetatea unui nr. n de cicluri, ce contin in principal 3 etape:
- denaturarea
- aditia primerilor la capetele secventei tinta
- extensia primerilor
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PCR Amplifies a Targeted Sequence
Target Sequence
DNA StrandDouble HelixDNA StrandSupercoiled
DNA Strand
Chromosome
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PCR – 1- Denaturare
Secventa tinta
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PCR- 2 – CUPLAREA PRIMERILOR LA CAPETELE SECVENTEI TINTA
Target Sequence
Target Sequence
Primer 1Primer 2
Biotin
Biotin
5’
3’
5’
5’
3’
5’
3’
3’
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PCR -3 – Elongarea primerilor sub actiunea Taq Polimerazei
Secventa tinta
Primer 1
Primer 2
Biotina
Biotina
5’
3’
5’
5’
3’
5’
3’
3’
ADN Polimeraza Taq
Secventa tinta
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End of the 1st PCR Cycle - Results in Two Copies of Target Sequence
Target Sequence
Target Sequence
Biotin
Biotin
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Target Amplification
No. of No. Amplicon Cycles Copies of Target
1 2
2 4
3 8
4 16
5 32
6 64
20 1,048,576
30 1,073,741,822
1 cycle = 2 Amplicon
2 cycle = 4 Amplicon
3 cycle = 8 Amplicon
4 cycle = 16 Amplicon
5 cycle = 32 Amplicon
6 cycle = 64 Amplicon
7 cycle = 128 Amplicon
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PCR: Master Mix
Mg2+
Mn2+
dNTP
Target DNA
Taq DNAPolymerase
Primers •forward primer •reverse primer
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RT- PCR
Reverstranscriere + PCR
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Reverse Transcription - Step 1 – Aditia Primer la secventa tinta ARN
Target RNA Sequence
Primer
Biotin
5’
3’
5’
3’
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Reverse Transcription - Step 2 – Reverstranscriptaze Catalizeaza extensia Primerului prin incorporarea nucleotidelor
complementare
Target RNA Sequence
PrimerBiotin
5’
3’
5’
3’
rTth DNA Polymerase
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End of Reverse Transcription - Step 3 – Rezulta sinteza ADNcomplementar (cDNA)
pe secventa tinta ARN
Target RNA Sequence
cDNA
Biotin
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Basic Lab Setup
Unidirectional Unidirectional workflow!workflow!
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Safety Gloves, Laboratory Coat Safety Glasses
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Biohazard Container
0.5% sodium hypochlorite, ethanol,
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Biological Safety Hood
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Vortex Mixer
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Centrifuge and/or Microcentrifuge (min. RCF 12,500 x g), some tests require a refrigerated, high speed centrifuge (min. RCF 24,000 x g)
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Dry-heat Temperature Block
Incubator (37 C ± 2 C)0
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•Screw-cap tube racks (to hold Master Mix tubes)
Dedicated Pipettor and Screw-cap Tube Rack, Multichannel Pipettor and Tips
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PCR System consumables (tubes, base, tray and retainer).
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Analyser Consumables/partsD-cups
A-rings, A-ring Holder
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Thermal CyclerPCR System 9600 or System 2400
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COBAS AMPLICOR™ Analyser
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DETECTIA AMPLICONULUI
• Electroforeza in gel de agar, colorare cu bromura de ethidiu si comparare cu markeri de GM cunoscuta
• Hibridizare cu sonde moleculare marcate complementare si apoi detectie (+/- spectrofotometrica)
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Detectie
spectrofotometrica Add Stop Solution and Measure Absorbance
CaptureProbe
Microwell Plate
Addition of Substrate
Tetramethylbenzidine(TMB) Substrate
Biotin
BSA
AMPLICON
BSA
AMPLICON
HydrogenPeroxide
Avidin-horseradish peroxidase (Av-HRP conjugate)
Hibridizare
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Diagnostic molecular in infectia HIV
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DIAGNOSTIC SEROLOGICTESTE DE TRIAJ- sensibilitate inaltaELISATESTE RAPIDE
TESTE DE CONFIRMARE- specificitate inaltaWESTERN BLOTRADIOIMUNOPRECIPITAREA (RIPA)IMUNOFLUORESCENTA INDIRECTA
DETECTIA VIRUSULUI SAU A PRODUSELOR VIRALE
IZOLARE VIRALADETECTIA ANTIGENEMIEI p24DETECTIA ACIDULUI NUCLEIC VIRAL(ARN HIV PLASMATIC SAU ADN HIV PROVIRAL LIMFOCITAR)
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Utilizarea metodelor moleculare in infectia HIV
• Fereastra serologica
• Nou-nascuti din mame seropozitive HIV
• Initierea si monitorizarea terapiei antiretrovirale
• iv>5-10.000 corelat cu nr. CD4 si status clinic• iv>30-50.000 indiferent de nr. CD4 si status clinic• Monitorizarea: reducerea iv sub 50 copii ARN
HIV/ml la 24 sapt
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Precizati tipul de reactie reprezentata mai sus si principiul tehnicii.
In ce viroze aceasta tehnica este de electie?
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• MC, 25 ani . Se apreciaza ca se gaseste in fereastra serologica pentru infectia HIV. Ce semnifica aceasta notiune si ce etapa urmeaza in diagnostic.
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Nou nascut din mama seropozitiva HIV.
Care este testul de electie folosit pentru diagnosticul infectiei, precizand si etapele sale.
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• CE TEHNICA ESTE UTILIZATA IN ETAPA DE CONFIRMARE A INFECTIEI HIV? CE CRITERII TREBUIE OBLIGATORIU INDEPLINITE PENTRU A SPUNE CA TESTUL ESTE POZITIV? CUM SE PROCEDEAZA IN CAZUL UNUI TEST INDETERMINAT? MENTIONATI PRINCIPALELE CAI DE TRANSMITERE ALE INFECTIEI HIV.