presentasi h5 n1 short
TRANSCRIPT
Roche Applied Science
Deteksi H5N1 dan Analisis dengan Deteksi H5N1 dan Analisis dengan
Real-Time PCRReal-Time PCR
Roche Applied Science
EtiologiEtiologi
Orthomyxoviridae • Envelop • Glycoprotein/antigen permukaan: •
Hemagglutinin (HA) and neuraminidase (NA)• 15 hemagglutinin and 9 neuraminidase • Ada 3 tipe (A – semua mamalia, B & C hanya ada
pada manusia)• Reservoir unggas liar, terutama unggas air
2
Roche Applied Science
Influenza VirusInfluenza Virus
Roche Applied Science
Roche Applied Science
Avian InfluenzaAvian Influenza
Inkubasi : 3-14 hariInkubasi : 3-14 hari Sangat menular (100%)Sangat menular (100%) LPAI - kematian rendah, sembuh LPAI - kematian rendah, sembuh
dalam 3 minggudalam 3 minggu HPAI HPAI 95% mati dalam 24 jam95% mati dalam 24 jam
5
Roche Applied Science
Individu yang terinfeksi Flu Burung Individu yang terinfeksi Flu Burung (1918 -2004)(1918 -2004)
1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010
1918H1N1
1957H2N2
1968H3N2
1997H1N1
1999H9N2
2003H5N1
2004H5N1
2003H7N7
Roche Applied Science
Diagnosa LaboratoriumDiagnosa Laboratorium Isolasi virusIsolasi virus AGID AGID ELISA ELISA Identifikasi Serologis (HA dan NA) Identifikasi Serologis (HA dan NA) Sera dari Sera dari
ayam yang terinfeksi biasanya antibodinya positif ayam yang terinfeksi biasanya antibodinya positif paling cepat 3-4 hari setelah tampak gejala klinis. paling cepat 3-4 hari setelah tampak gejala klinis.
Tes molekular :Tes molekular :- RT - PCR- RT - PCR- Real time reverse transcriptase polymerase - Real time reverse transcriptase polymerase chain reaction (rT-RT-PCR)chain reaction (rT-RT-PCR)
7
Roche Applied Science
Lev
el V
iru
s
Hari Setelah Infeksi0 7 14 21 28
Directigen
AGID
RT-PCR
Isolasi Virus
Uji Avian Influenza
8
Roche Applied Science
Make copies (extend primers)
Starting DNA Template
5’
5’
3’
3’
5’
5’
3’
3’
Add primers (anneal) 5’3’
3’5’
Forward primer
Reverse primer
Amplifikasi DNA dengan PCR
Separate strands
(denature)
5’
5’3’
3’
Roche Applied Science
In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created
In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created
PCR Copies DNA Exponentially through Multiple Thermal Cycles
Original DNA target region
Thermal cycleThermal cycleThermal cycle
Roche Applied Science
But……………..:– Post amplification processing
– Contamination
– Prolonged testing time– Non-automated
– Expensive to implement/expertise needed– Qualitative (difficult to quantify)
Advantages of PCR
The utilisation of PCR conferred many advantages:– Highly sensitive/specific Applicable to RNA or DNA viruses Rapid (turn around time of 24-48 hours) Can detect multiple viruses Products can be sequenced for epidemiological/resistance
study. Improved patient management and disease surveillance
Roche Applied Science
Real time PCR:
Beda dengan PCR konvensional :Beda dengan PCR konvensional :– Amplicon divisualisasi sebagai kurva amplifikasi .Amplicon divisualisasi sebagai kurva amplifikasi .
» Primer terlabel, probes atau amplicons dengan molekul Primer terlabel, probes atau amplicons dengan molekul fluorogenic.fluorogenic.
• 5 metoda untuk mendeteksi produk amplifikasi:5 metoda untuk mendeteksi produk amplifikasi:
1.1. DNA binding fluorophoresDNA binding fluorophores2.2. Linear oligoprobesLinear oligoprobes3.3. Taqman probesTaqman probes4.4. Molecular beaconsMolecular beacons5.5. Self-fluorescing ampliconSelf-fluorescing amplicon
Roche Applied Science
Needed reagents (Two step PCR)
The Influenza A virus is an RNA virus :
High Pure Viral Nucleic Acid Isolation Kit (manual) orMagNA Pure Compact Instrument (automated)
Transcriptor Reverse Transcriptase
Biological sample extraction kit.
Reverse transcription
Thermal cycler.
LightCycler FastStartPLUS DNA Master Hybridization Probes (PCR master mix)
Amplification enzyme and related reagents
LightCycler 1.5 or 2.0 System
Amplification specific reagents and PCR protocolsLightMix Influenza virus A Matrix Protein + IPC -No. 40-0233-16LightMix Influenza virus A H5 -No. 40-0219-16 LightMix Influenza virus A N1 -No. 40-0230-16LightMix Influenza virus A H5+N1 -No. 40-02xx-16
Roche Applied Science
Work Flow (Two step PCR)
RNA Extraction
Reverse transcription
ResultTarget H5
PCR
ResultTarget N1
ResultTarget
Mat. Prot
40 min.
30 min. (incubation)
20-30 min. (set up)
60 min. (PCR)
RNA
To
tal
Ass
ay
tim
e 18
0 m
in.
Han
ds
on
tim
e 80
min
.
5 min.(data analyses)
Roche Applied Science
H5 detection
Roche Applied Science
N1 detection
Roche Applied Science
Avian Influenza summaryAvian Influenza summary Can screen samples for influenza A (matrix PCR)Can screen samples for influenza A (matrix PCR)
Contains internal control in same reaction to check inhibitionContains internal control in same reaction to check inhibition
Positive matrix samples can then be checked with H5 PCR and N1 PCR in Positive matrix samples can then be checked with H5 PCR and N1 PCR in one reaction (multiplex)one reaction (multiplex)
Reaction capillary
On LightCycler
N1H5
Roche Applied Science