Diagnostic molecular in virusologie
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Diagnostic molecular in virusologie
Diagnosticul molecular in virusologie
AMPLIFICAREA TINTEI–PCR–NASBA (nucleic acid sequenced based amplification)
AMPLIFICAREA SEMNALULUI–B-DNA (branched DNA)
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
PCR Amplifies a Targeted Sequence
Target Sequence
DNA StrandDouble HelixDNA StrandSupercoiled
DNA Strand
Chromosome
PCR – 1- Denaturare
Secventa tinta
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’
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
End of the 1st PCR Cycle - Results in Two Copies of Target Sequence
Target Sequence
Target Sequence
Biotin
Biotin
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
PCR: Master Mix
Mg2+
Mn2+
dNTP
Target DNA
Taq DNAPolymerase
Primers •forward primer •reverse primer
RT- PCR
Reverstranscriere + PCR
Reverse Transcription - Step 1 – Aditia Primer la secventa tinta ARN
Target RNA Sequence
Primer
Biotin
5’
3’
5’
3’
Reverse Transcription - Step 2 – Reverstranscriptaze Catalizeaza extensia Primerului prin incorporarea nucleotidelor
complementare
Target RNA Sequence
PrimerBiotin
5’
3’
5’
3’
rTth DNA Polymerase
End of Reverse Transcription - Step 3 – Rezulta sinteza ADNcomplementar (cDNA)
pe secventa tinta ARN
Target RNA Sequence
cDNA
Biotin
Basic Lab Setup
Unidirectional Unidirectional workflow!workflow!
Safety Gloves, Laboratory Coat Safety Glasses
Biohazard Container
0.5% sodium hypochlorite, ethanol,
Biological Safety Hood
Vortex Mixer
Centrifuge and/or Microcentrifuge (min. RCF 12,500 x g), some tests require a refrigerated, high speed centrifuge (min. RCF 24,000 x g)
Dry-heat Temperature Block
Incubator (37 C ± 2 C)0
•Screw-cap tube racks (to hold Master Mix tubes)
Dedicated Pipettor and Screw-cap Tube Rack, Multichannel Pipettor and Tips
PCR System consumables (tubes, base, tray and retainer).
Analyser Consumables/partsD-cups
A-rings, A-ring Holder
Thermal CyclerPCR System 9600 or System 2400
COBAS AMPLICOR™ Analyser
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)
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
Diagnostic molecular in infectia HIV
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)
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
Precizati tipul de reactie reprezentata mai sus si principiul tehnicii.
In ce viroze aceasta tehnica este de electie?
• MC, 25 ani . Se apreciaza ca se gaseste in fereastra serologica pentru infectia HIV. Ce semnifica aceasta notiune si ce etapa urmeaza in diagnostic.
Nou nascut din mama seropozitiva HIV.
Care este testul de electie folosit pentru diagnosticul infectiei, precizand si etapele sale.
• 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.
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