Download - GeneXpert MTB/RIF
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GeneXpert MTB/RIFGeneXpert MTB/RIF
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GeneXpert MTB/RIF
• teste que utiliza a plataforma GeneXpert (Cepheid)
• permite a detecção de resistência a rifampicina (mutações no gene rpoB)
• baseado na técnica de PCR em tempo real
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Reação em Cadeia da Polimerase(PCR)
• Um das principais ferramentas de biologia molecular
• Desenvolvida por Kary Mullis em 1983, resultando no prêmio Nobel em Química
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Importância dos oligonucleotídeos iniciadores (primers) na síntese de DNA
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PCRMelting
94 oC
Tem
pera
tura
100
0
50
T e m p o
5’3’
3’5’
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PCRMelting
94 oC
Tem
pera
tura
100
0
50
T e m p o
3’5’
5’3’
94ºC
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PCRMelting
94 oCAnelamento
50 oC
Extensão72 oC
Tem
pera
tura
100
0
50
T e m p o
3’5’
5’3’5’
5’
Melting94 oC
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PCRMelting
94 oCMelting
94 oCAnelamento
50 oC
Extensão72 oC
Tem
pera
tura
100
0
50
T e m p o
30x
3’5’
5’3’
94ºC
94ºC
5’
5’
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PCRMelting
94 oCMelting
94 oCAnelamento
50 oC
Extensão72 oC
Tem
pera
tura
100
0
50
T e m p o
30x
3’5’
5’3’5’
5’
5’
5’
5’
5’
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PCRMelting
94 oCMelting
94 oCAnelamento
50 oC
Extensão72 oC
Tem
pera
tura
100
0
50
T e m p o
30x
5’
5’
5’
5’
94ºC
94ºC
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PCRMelting
94 oCMelting
94 oCAnelamento
50 oC
Extensão72 oC
Tem
pera
tura
100
0
50
T e m p o
30x
5’
5’
5’
5’
5’
5’
5’
5’
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Fragmentos de tamanho definido
PCRMelting
94 oCMelting
94 oCAnelamento
50 oC
Extensão72 oC
Tem
pera
tura
100
0
50
T e m p o
30x
5’
5’
5’
5’
5’
5’
5’
5’
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Mais Ciclos = Mais DNANúmero de ciclos
0 10 15 20 25 30
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30 ciclos geram aproximadamente 109 moléculas
Amplificação
Permite aumentar a quantidade Permite aumentar a quantidade de uma determinada amostrade uma determinada amostra
Pode aumentar a quantidade Pode aumentar a quantidade de um contaminantede um contaminante
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DNA polimerase de DNA polimerase de E. coliE. coli
DESVANTAGENS: Temperatura ótima baixa e não é termoestável
Klenow (derivada da DNA Pol.I de E.coli)
extensãoextensão desnaturaçãodesnaturaçãoanelamentoanelamento
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Breve Histórico
• O primeiro termociclador foi desenvolvidona Cetus Corp. – Mr Cycle• Projetado para que a DNA polimerasepudesse ser adicionada a cada ciclo
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Yellowstone National Park
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DNA Polimerase Termoestável
• 1969 – T. Brock e H. Freeze relataram a descoberta de uma nova espécie de bactéria – Thermus aquaticus – sobrevive na água a temperatura média de 75ºC
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• Várias enzimas isoladas desse microorganismo incluindo a primeira DNA polimerase termoestável
Taq DNA polimerase (1976)
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Taq DNA polimerase – temperatura ótima de 72ºC e é estável até temperaturas superiores a 94ºC
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Kary Mullis
Prêmio Nobel de Química em 1993
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1. Template (molde) (1–500 ng).2. Primers (0.05–1.0 μM).3. Mg2+ (0.5–5 mM).4. dNTP (20–200 μM cada).5. 1× PCR buffer 6. DNA polimerase (0.5–2.5 U para cada 50 μL de reação).
Componentes de uma reação de PCRComponentes de uma reação de PCR
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PCR “padrão”PCR “padrão”
Permite amplificar uma determinada seqüência de DNA
- Clonagem gênica
- Sonda (northern blot, southern blot, hibridização in situ,...)
- Seqüênciamento (identificação de amostra, análise filogenética,
confirmação de transfecção)
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CURVA DE PCRCURVA DE PCR
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GeneXpertGeneXpert(Cepheid)(Cepheid)
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GeneXpertGeneXpert(Cepheid)(Cepheid)
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GeneXpert MTB/RIFGeneXpert MTB/RIF
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GeneXpert MTB/RIFGeneXpert MTB/RIF
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Avaliação dos módulosAvaliação dos módulos
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Avaliação dos módulosAvaliação dos módulos
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Preparação da amostraPreparação da amostra
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Preparação da amostraPreparação da amostra
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Cartucho GeneXpert MTB/RIFCartucho GeneXpert MTB/RIF
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Cartucho GeneXpert MTB/RIFCartucho GeneXpert MTB/RIF
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Identificação do Cartucho GeneXpert MTB/RIFIdentificação do Cartucho GeneXpert MTB/RIF
Manual Automática
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OPÇÕES DE UTILZAÇÃOOPÇÕES DE UTILZAÇÃO
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Cancelamento de teste(s)Cancelamento de teste(s)
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Resultado – Amostra 1Resultado – Amostra 1
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Resultado – Amostra 1Resultado – Amostra 1
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Resultado – Amostra 2Resultado – Amostra 2
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Resultado – Amostra 2Resultado – Amostra 2
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Diagnóstico Laboratorial da Tuberculose
• Métodos bacteriológicos . Métodos Moleculares
-Baciloscopia -24hs - Fita Hain 3 dias ou mais
(baciloscopia positiva +)
-Cultivo – MGIT – 10 dias
- Genexpert -2hrs
- Sólido – 30 dias
- Teste de Sensibilidade
- MGIT -15 dias
- Método das Proporções 28 a 42 dias
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Diagnóstico Laboratorial da Tuberculose
Vantagens e Desvantagens
-Bacteriológico - Moleculares(Genexpert) - Mais barato - Mais caro (baciloscopia) - TSA Mais demorado (MGIT) - Mais rápido Rif resit - Outras drogas (INH, etc.) - Só para Rif - Bacilos vivos - Bacilos Vivos e “mortos” - Cultivar para TSA confirmatório e outras drogas (quantidade de escarro) - Triagem - Três códons relacionados a resistência a RIF - Não detecta em amostras extra-pulmonares - Não detecata MNT