PCR KLINIK.pdf

7/26/2019 PCR KLINIK.pdf

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PENGGUNAAN PCR

UNTUK KLINIKDebbie S. Retnoningrum

Sekolah Farmasi

Institut Teknologi Bandung


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Penggunaan klinik

Mempelajari atau mendeteksi polimorfisme / mutasi

Menegakkan diagnosis penyakit

Memantau terapi obat

Menentukan kesembuhan

Mengetahui terjadinya breakthrough dan relapse


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Polymorphism

A variation in the DNA that is too common to

be due merely to new mutation. A

polymorphism must have a frequency of at

least 1% in the population.

Examples of polymorphisms include the

genes for sickle cell disease, thalassemia and

G6PD deficiency.


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PCR - RFLP

Fragmen DNA yang mengandung mutasi /

polimorfisme diamplifikasi dengan PCR

Produk PCR dipotong dengan enzim restriksiyang dapat membedakan antara alele mutan

dan non-mutan


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Enzim restriksi?

Enzim endonuklease yang mengenali dan

memotong urutan spesifik dan reproducible

pada framen DNA

Urutan yang dikenali merupakan palindrom Palindrom?


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1000 bp

Varian 1

1000 bp

Varian 2

400 bp 600 bp

Varian 1

dan 2

1000 bp

400 bp 600 bp

600 bp

400 bp


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PCR DAN RT-PCR:

KONVENSIONAL DAN REAL TIME

KONVENSIONAL:

AMPLIFIKASI DAN DETEKSI DILAKUKAN SECARA

KONSEKUTIF

REAL-TIME:

AMPLIFIKASI DAN DETEKSI DILAKUKAN SECARA

SIMULTAN


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PCR KONVENSIONAL

COBAS AMPLICOR

HOME BREW PCR


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PEMANTAUAN PCR

Agarose Gel Blotting LightCycler


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Struktur VIRUS HEPATITIS B

YANG MENJADI TARGET ADALAH DNA HBV preCore/Core


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Perbandingan cobas amplicor vs

taQman hBv

Kinerja / metode Cobas Amplicor Taqman

Batas deteksi 60 IU/ml 6 IU / ml

Rentang linearitas 60 3,8 x 104 IU/ml 29 1,1 x 108 IU/ml

Pembacaan End point Real time

Sampel Serum Plasma (serum kurang

sensitif)

Deteksi Kolorimetri Fluorometri

Genotipe A, B, C, D, E, F A, B, C, D, E, F, G

MUTANT PRECORE

Kontrol kontaminasi Ada AdaKontrol mencegah

negatif palsu

Ada Ada

Spesifisitas 100 % 100 %


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StruKtur VIRUS hepatitis C

(RNA)


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PATTERNS OF VIROLOGIC RESPONSE,

BREAKTHROUGH AND RELAPSE


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EVOLVING DEFINITIONS OF TREATMENT RESPONSE

TERMS DESCRIPTIONS

RAPID VIROLOGIC RESPONSE (RVR) UNDETECTABLE HCV RNA AT WEEK 4

EARLY VIROLOGIC RESPONS (EVR) 2 LOG 10 DROP IN HCV RNA FROM BASELINE OR

UNDETECTABLE HCV RNA AT WEEK 12

COMPLETE EVR NO RVR, UNDETECTABLE HCV RNA AT WEEK 12

PARTIAL EVR NO RVR, 2 LOG 10 DROP IN HCV RNA FROM BASELINE AT

WEEK 12 BUT STILL DETECTABLE RNA HCV

NULL RESPONSE < 2 LOG 10 DROP IN HCV RNA FROM BASELINE OR NO

UNDETECTABLE HCV RNA AT WEEK 12

END OF TREATMENT UNDETECTABLE HCV RNA AT END OF TREATMENT

SUSTAINED VIROLOGIC RESPONSE

(SVR)

UNDETECTABLE HCV RNA AT 24 WEEKS AFTER TREATMENT

BREAKTHROUGH DETECTABLE HCV RNA DURING TREATMENT AFTERINITIALLY ACHIEVING UNDETECTABLE HCV RNA

RELAPSE RECURRENCE OF HCV RNA IN PATIENTS WHO ACCHIEVED

AND MAINTAINED UNDETECTABLE RNA THROUGHOUT THE

DURATION OF TREATMENT


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KRAS and the EGFR pathway

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The EGFR signaling pathway is activated inresponse to ligand binding to the cell-surfacereceptors: these ligands include TGF and EGF

The signaling cascade that is activated isinvolved in regulating genes that control cellcycle progression

Cell cycle progression gives rise to tumorsurvival, growth and proliferation as well asmetastasis and angiogenesis

In the early part of the signaling cascade, theprotein KRAS regulates downstream proteinsinvolved in these effects

The KRAS protein plays a central role in tumor development,

regulating downstream proteins that are involved in

proliferation, survival, metastasis and angiogenesis

Th EGFR h d h i


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The EGFR pathway and the importance

of

KRAS status The KRAS gene may benormal (wild-type) ormutated

Wild-type KRAS protein isactive for a short period

when the EGFR is stimulated The effects of the protein are closely controlled

When KRAS is mutated theprotein is permanentlyturned on, even without

being triggered by EGFR-mediated signaling The effects of KRAS that lead to tumor growth and

spread continue unregulated

The KRAS status of a tumor 25


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The KRAS test

A tumor sample is takenand sent to the laboratory the test can use fresh,frozen or paraffin-

embedded tissue

A pathologist confirmsthat the tissue iscancerous, and a sample

of DNA is prepared for theKRAS test

The polymerase chain 26*Direct DNA sequencing can also be used


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TIB MolBiol Assay

DNA Extracted

from FFPET

Each sample added to 2 or 3

different reaction mixtures;

4 controls/standards

required per carousel run

concon

00LowLow

concon concon concon

HigHig

hh

The PCR reaction and

melting curve take around

60 minutes

The analysis is completed

by analyzing the Melting

curve on the instrument


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PCR untuk kuantitasi EBV

PCR KLINIK.pdf

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