50 25 gt1a ns3 mutant -1 0 1 2 3 - rsd · 2021. 1. 7. · gt1a ns3 mutant log[compound, nm]) •...
TRANSCRIPT
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www.wuxiapptec.com
Enabling Unbounded Possibilities
Enabling Unbounded Possibilities
• HCV full-length replicon construct templates• HCV chimeras
>100 replicon chimeras constructed and evaluated
• HCV replicon mutants>1000 GT1-6 SDMs of clinical isolates with NS3, NS5A or NS5B gene generated and evaluated
• Stable HCV replicon cell lines>500 GT1-6 replicon cell lines generated and evaluated
• Determination of fitness and drug sensitivity of HCV replicons>1500 GT1-6 replicons evaluated
• High quality services
-1 0 1 2 3
0
25
50
75
100
GT1a wt
GT1a NS3 mutant
Log[compound, nM]
Inh
ibitio
n (%
)
• Full-length and core gene replication-competent shuttle vectorsFull-length chimeras
Core gene chimeras
• Fitness test for HBV clinical constructs>400 constructs evaluated
• Determination of fitness and drug sensitivity of HBV constructs>1000 compounds/constructs evaluated
Detection of subpopulation of a pol inhibitor resistant variant
0 1 2 3 4 5 6 7 8 9 101
10
100
1000
10000replicate 1
replicate 2
replicate 3
Compound
IC5
0 (
nM
)
Cpd A in three experiments
0 1 2 3 4 50
20
40
60
80
100exp 1
exp 2
exp 3
Clinical isolate No.
IC5
0 (
nM
)
High precision and reproducibility
Ratio (M204I+V173L/wt)
-3 -2 -1 0 1 2 3 4-40
-20
0
20
40
60
80
100
1200%
10%
20%
40%
60%
80%
90%
100%
log[GLS4, (nM)]
Inh
ibit
ion
(%
)
Ratio (M204I+V173L/wt)
0 1 2 3 4 5 6-40
-20
0
20
40
60
80
100
1200%10%
20%
40%
60%
80%
90%
100%
log[LAM, (nM)]
Inh
ibit
ion
(%
)
Zhengxian Gu
Executive Director
Mobile: +86 189-3083-0132
Tel: 86-21-5046-3753
Email: [email protected]
Wei Chang
Associate Director
Mobile: +86 137-6186-3280
Tel: 86-21-5046-4003
Email: [email protected]
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Genotyping: Pharmacogenomics,
IL28B genotyping, MET exon 14
skipping, etc.
Microsatellite instability: MSI
Viral genotyping, viral load
determination and viral sequencing:
HBV, HCV, RSV.
Gene expression: Fresh tissue, blood,
etc.
Line probe assay: LiPA
Automated sample preparation, amplification and quantitation
- HCV RNA viral load
- HBV DNA viral load
Amplilink
Software
COBAS®
TaqMan 48 Analyzer
COBAS® AmpliPrep
Instrument
PCR performance
Reagent preparation Sample preparation
Post-PCR process
Lab overview: separate
working areas
1. CYP2D6 genotyping (Taqman SNP, CNV, Sanger
sequencing): 180 samples
2. UGT1A9 genotyping (Taqman SNP, STR): 28 samples
3. Gene expression:>2000 samples per year
4. HCV clinical support
• HCV clonal sequencing: >400 clinical samples
• HCV resistance Sanger sequencing: >700 samples
• HCV deep sequencing (1a, 1b, 2a, 3a, 6a): >1300
samples.
• HCV genotyping: >200 samples
• IL28B genotyping: validated
• HCV RNA: >200 samples
5. HBV clinical support
• INNO-LiPA HBV Multi-DR assay: 340 samples
• Sanger sequencing of an HBV pol-RT gene:
>100 samples
• Sanger sequencing of an HBV Core gene:
validated
• HBV whole genome sequencing: >300
samples
• HBV DNA: validated
6. RSV genotyping: 250 clinical samples
Population sequencing assay of HBV whole genome:
Serum or plasma samples
The average sequence concordance rate
with the reference lab was 99.6%
The average sequence concordance
rate of duplicate samples was 99.9%
The average sequence concordance
rate of independent runs was 99.6%
Accuracy (Consistency) Precision (intra-run) Precision (inter-run)
100% detection rate was obtained
at 5x102 (IU/ml)
Sensitivity (amplification) Specificity
Report of amino acid differences from a reference sequence
Extract of RNA from serum or plasma samples
Amplification of HCV genes by nested-PCR
Gel electrophoresis and DNA purification
Sanger Sequencing
Sequence analysis
Qiasymphony DNA/RNA
extraction QIAxcel DNA/RNA QC
3730xl Genetic Analyzer
COBAS® AmpliPrep
Instrument ABI 7900HT
Assay HCVHBV pol-RT
gene
HBV core
gene
HBV whole
Genome
Accuracy 99.6% 99.85% 99.6% 99.6%
Intra-run
reproduci
bility
99.9% 99.50% 99.6% 99.8%
Inter-run
reproduci
bility
99.6% 99.30% 99.6% 99.9%
Amplificati
on
sensitivity
100%
detection
rate at 5x102
(IU/ml)
100% detection
rate at 5x103
(copies/ml)
100%
detection rate
at 5x102
(copies/ml)
90% detection
rate was
obtained at 2~3
log10(IU/ml)
Minor
species
sensitivity
20% 20% 20% NA
Specificity
validation
No false
positive
No false
positive
No false
positiveNo false positive
Assay HCV Viral Load HBV Viral Load
Accuracy Bias≤3.29% Bias≤2.02%
Precision %CV ≤9% %CV ≤16%
Reportable range
15IU/ml ~1.00E+8IU/ml
20IU/ml ~1.70E+8IU/ml
Carry-Over Not Detected Not Detected
Validation summary of viral population sequencing: Validation summary of HCV RNA/HBV DNA assay:
Viral load (IU/ml) No. of samples Positive rate (%)
2log10 3 100
3log10 7 85.7
4log10 2 100
5log10 6 100
6log10 4 100
7log10 7 100
8log10 1 100
No. of samples 6 4
Expected result Positive Negative
Actual result Positive Negative
Concordance rate 100% 100%
90% detection rate was obtained at 2~3 log10(IU/ml).
No false positive was observed.