Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean...
Transcript of Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean...
![Page 1: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/1.jpg)
Fiocruz Paraná Pesquisa, Desenvolvimento e Produção
![Page 2: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/2.jpg)
Gelified real-time PCR assay for Malária Diagnosis
December/2011
Responsável: Cheysa Biondo
Data: 08/12/2011
![Page 3: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/3.jpg)
![Page 4: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/4.jpg)
Comparação de desempenho das Mastermix: Applied, NAT e H1N1
0 1 2 3 4 5 620
25
30
35
40
Mastermix Applied
Mastermix NAT
Mastermix H1N1
log UI/mL
Qu
anti
ficat
ion
Cyc
le
![Page 5: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/5.jpg)
![Page 6: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/6.jpg)
P. falciparumGelified + Liquid
Gel mean Ct ���� 24,6Liq mean Ct ���� 24,7
After 24 hours 4°C
P. vivaxGelified + Liquid
Gel mean Ct ���� 26,8Liq mean Ct ���� 27,3
![Page 7: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/7.jpg)
Company and Technology Introduction to:
24th February 2013
Kieran Curran Ph.DCEO
![Page 8: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/8.jpg)
Mission:
To develop and commercialize better automated solutions for life sciences.
GenCell’s Composite Liquid Cell Tech
✓ Widely applicable
✓ Automation “friendly” microfluidics
✓ No plastic consumables
Revolutionary Technology for Automated Biology
Conventional Automation Tech
✓ Widely applicable
✗ Miniaturization a challenge
✗ Consumable plasticware
![Page 9: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/9.jpg)
Technology Introduction
Natural Biological Cell Microfluidic Capsules (GenCell CLC’s)
![Page 10: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/10.jpg)
GenCell Genotyping: Process Flow
1) Create “Empty”
CLC2) Dispense
Assay3) Dispense
Sample
4) Thermally
Process5) Optically Interrogate
CONFIDENTIAL [Shared under NDA]
Data Acquisition
No Plasticware = Lower running costs for HTP
Option to recover 100% of
product
6) Aspirate to
waste
![Page 11: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/11.jpg)
GenCell NGS Library PreparationHigh Throughput: 960 libraries
per day
20 minutes of user interaction
Absolutely no plastic consumables – a
paradigm shift in automation
Work with us to achieve up to
500 fold* savings in reagent
costs
Barcoded library pooling
capability on-board
Prep with as little as 1ng in
500nL
![Page 12: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/12.jpg)
![Page 13: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/13.jpg)
Gencell Purification Instrument followed by amplification in CLC on Gencell qPCR platform and on ABiStepOne. Magmax kit.
10uL 10uL 10uL 10uL
104
(x4)105
(x2)VLP concentration -
Gencell Purification platform - MagMax protocol
50uL input sample
Eluted viral
RNA:
7uL of each eluted RNA added to 10.5uL MMx
17.5uL
5uL
CLC5uL
CLC5uL
CLC
10uL elution performed x 2 for
each sample
Confidential
![Page 14: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/14.jpg)
Confidential
Viral CopiesSpin Column
IBMPStandard
Magnetic PlateGencell
20,000 ✔ ✔ ✔
2,000 ✔ ✔ ✔
200 ✔ ✗ ✔
20 ✔ ✗ ✔
7 ✔ ✗✔
Review
Viral RNA Purification and amplification on commercial instrument:
![Page 15: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/15.jpg)
Thursday 11/4/13 – Post NTC’s run on Gencell qPCR platform.
12 x 5uL CLCs containing No Template Blanks (NTB’s) performed on Gencell
Purification Platform post run on Wednesday 10/4/13
Confidential
Conclusion: No detectable carry over contamination
![Page 16: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/16.jpg)
![Page 17: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/17.jpg)
![Page 18: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/18.jpg)
POC Real Time qPCR (detecção com quantificação 1 amostra /hora)
![Page 19: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/19.jpg)
Procedimento Experimental
Extração de DNA107 epimastigotas
FONTE Portal Fiocruz
Diluições em PBS: 107 – 10-1
Fator diluição 1:10
106 105 104 103
102 101 100 10-1
![Page 20: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/20.jpg)
Procedimento Experimental
DENATURAÇÃOANELAMENTOEXTENÇÃO
Plástico Silício
Comparação de 2 tipos de equipamentos de PCR em Tempo Real
1 hora e 30 minutos 30 minutos
40.000 DÓLARES 1.000 DÓLARES
VOLUME DOS REAGENTES E AMOSTRA
![Page 21: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/21.jpg)
Resultados e Discussão3 experimentos distintos
ABI7500
Eficiência: 98,2%
Q3
Eficiência: 97,4%
ABI7500 Q3
![Page 22: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/22.jpg)
![Page 23: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/23.jpg)
![Page 24: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/24.jpg)
Desenvolvimento de Testes para Diagnóstico Molecular de patógenos
Associados a Sepse.
![Page 25: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/25.jpg)
![Page 26: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/26.jpg)
![Page 27: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/27.jpg)
Principais microorganismos causadores de sepse:
Enterobactérias(bacilos Gram-negativos)
Cocos Gram-
positivos
Fungos
![Page 28: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/28.jpg)
• Seleção das sequências para identificação dosmicroorganismos:
Genes rRNA Fungos
ITS
Genes rRNA Bactérias
![Page 29: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/29.jpg)
16S rRNA gene sequencing can profile the bacterial communities in a greater detail
than can metagenomics. In addition low abundance species are best identified
through 16S rRNA gene sequencing
![Page 30: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/30.jpg)
![Page 31: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/31.jpg)
![Page 32: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/32.jpg)
Pathogen DNA-free Taq Polymerase (SepseTaq)
SepseTaq IBM
P
← Taq
Western Blot: anti-Taq
ST
aq
ST
aq
![Page 33: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/33.jpg)
![Page 34: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/34.jpg)
![Page 35: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/35.jpg)
![Page 36: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/36.jpg)
Software E@syCheck
Sistema de controle de
Temperatura do Chip(TCS)
Leitor óptico(OCR)
Biochip LC03
Extrator e
purificador de
DNA
Lavador e
Secador do
Biochip
![Page 37: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/37.jpg)
![Page 38: Fiocruz Paraná Pesquisa, Desenvolvimento e Produção · P. falciparum Gelified + Liquid Gel mean Ct 24,6 Liq mean Ct 24,7 After 24 hours 4°C P. vivax Gelified + Liquid Gel mean](https://reader031.fdocument.pub/reader031/viewer/2022022708/5be7053509d3f26f698bf0b6/html5/thumbnails/38.jpg)
Marco Aurelio KriegerInstituto Carlos Chagas FiocruzRua Prof. Algacyr Munhoz Ma der 3775 Curitiba-Paraná, Brasil. Tel. + 55 41 33163230 Fax + 55 41 33163267 Email: [email protected]
[email protected]@pesquisador.cnpq.br