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.