Avances biotecnológicos de los últimos 20 años y tendencias a futuro
GeneXpert Systems
1. PAGE | PAGE | 1 Cambio de Cultura PCR en Tiempo Real ® Dra. Yamile Sandoval Pruebas diagnósticas PROMED Lic. Catherine Herrera Aplicaciones de Biología Molecular PROMED
2. PAGE | PAGE | 2 PRESENTAMOS EL LABORATORIO DE DIAGNÓSTICO MOLECULAR MAS PEQUEÑO DEL MUNDO GeneXpert® System
3. PAGE | PAGE | 3 Sistema GeneXpert® 1). Primer instrumento de diagnóstico molecular homologado por la FDA 2). El sistema GeneXpert es el único en combinar una preparación de muestra abordo con PCR en tiempo real 3). Resultados de muestras no procesadas en aproximadamente una hora.
22. PAGE | PAGE | 5 GeneXpert® PCR en tiempo real 1). Extracción automatizada de ADN o ARN de una muestra biológica 2). Cartucho cerrado de un solo uso
23. GeneXpert ® Escalas posibles del sistema PAGE | GX-I GX-IV GX-XVI GeneXpert Infinity Series GeneXpert Module
29. PAGE | Xpert MTB/RIF – Romain Prieur FÁCIL Uso RÁPIDO Respuestas CONFIABLES Resultados Pruebas Moleculares en corto tiempo y con resultados confiables
30. PAGE | PAGE | 4 GeneXpert® PCR en tiempo real Sources: Eveillard et.al., J Hosp Infect (v. 59, 2005); Salgado et.al., SHEA 2003, abstract 28, p. 61; Bhalla et a, Infect Control Hosp Epidemiol (v. 25, 2004) – Special thanks to Becton, Dickinson and Company. Los cultivos de bacteriología solo captan “ la punta del Iceberg” La transmisión se da no solo por pacientes infectados, sino también por pacientes colonizados
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32. PAGE | PAGE | 19 GeneXpert® PCR en tiempo real Xpert® vanA/vanB Xpert® MRSA complete Xpert® MRSA/SA SSTI Xpert® MRSA/SA Nasal Xpert® MRSA/SA BC Xpert® MTB/RIF Xpert® C. Difficile Xpert® GBS Xpert® EV Xpert® Flu A Panel Xpert® HemosIL® FII & FV Xpert® BCR-ABL
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34. PAGE | PAGE | 22 MTB/RIF - Xpert® permite 1- La detección del ADN del complejo MTB en muestras de esputo o sedimento concentrado ya sea de esputo o LBA, BAAR + o BAAR - 2- La detección de la mutación del gen rpoB /región 81bp que esta asociada a la resistencia a la rifampicina en pacientes de alto riesgo 3- La prueba MTB/RIF-Xpert® esta indicada en pacientes con sospecha clínica de TBC y en pacientes que no han recibido tratamiento, pacientes con menos de 7 días de tx. o pacientes que no han recibido tx en los últimos 60 días Cepheid Confidential & Proprietary GeneXpert® PCR en tiempo real
35. PAGE | Sensibilidad de las Pruebas de susceptibilidad Genotípicas Droga Blanco Sensibilidad Rifampicina rpoB 98% INH (katG), inhA 85% PZA pncA Etambutol emB
43. PAGE | BOX 2. Procedures for processing clinical specimens for culture of group B Streptococcus (GBS ---Detection of GBS can be determined directly from broth media using latex agglutination, probes or nucleic acid amplification tests (NAAT) such as PCR . Comprison of culture and 2 real-time polymerase chain reaction assays to detect group B Streptococcus during antepartum screening. Diagn Microbiol Infect Dis 2007;59:17–22. Block T, Munson E, Culver A, Vaughan K, Hryciuk JE.
44. PAGE | Para que una prueba sea considerada clinicamente útil en el período intraparto como método de tamizaje para GBS debe poder ser realizada de manera sencilla al lado de la cama del paciente, ya sea por el personal de la sala de labor o parto, deben obtenerse resulatdos en menos de 30 min y debe poseer una sensibilidad y especificidad >90% Revisión de las Guías CDC, 2010 Preparación : 2 minutos Resultado: 30 minutos Muestra: hisopado vaginal y rectal
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46. PAGE | PAGE | 31 GeneXpert® PCR en tiempo real
53. PAGE | PAGE | 41 Cambio de Paradigma Sistema GeneXpert® GRACIAS
Notas del editor
Billions of amplicon products are produced through PCR, in conventional PCR it is at this end point that PCR products are visualized, they are visualized on . . . . .
The Cepheid GeneXpert Diagnostic System automates and integrates sample purification, nucleic acid amplification, and detection of the target sequence in simple or complex samples using real-time PCR and RT-PCR methodologies. The system requires the use of single-use disposable GeneXpert cartridges that hold the PCR reagents and host the PCR process.
Smart beads are incorporated in to all Cepheid products. Utilizes proprietary dried reagent technology for ambient temp stability
• Utilize families of cartridges to address wide range of applications and specimen either RNA or DNA purification. We have developed assays to identify pathogens such as GBS, MRSA. For EV we purify RNA from CSF and for BCR we purify RNA from blood. Processing chambers—Hold samples, sample purification reagents, primers-probe and enzyme beads, processed sample, and waste solutions. • Valve body—Rotates and allows fluid to move to different cartridge chambers and to the reaction tube. Bacteria from the specimen are captured on a filter, washed to remove inhibitors and are ultrasonically lysed to release nucleic acid. The nucleic acid is mixed with PCR reagents (primer-probe bead and enzyme bead) and moved into the integrated reaction tube. • Reaction tube—Enables rapid thermal cycling, optical excitation, and detection of the target DNA. Advantages: Precision of microfluidics-based reconstitution of dry reagents No wet interface between instrument and cartridge to eliminate carry-over Integrated ultrasonic horn for rapid lysis of bacteria Unique filter capture/concentration technique Total internal control of reagent system – No separate external positive or negative controls required Smart fluidics Encoded software driven motors for valve movement and integral hydraulic drives Advanced micro fluidics technologies to enable complex sample prep processing protocols (many milliliters down to 10 micro liters)
The Cepheid GeneXpert Diagnostic System automates and integrates sample purification, nucleic acid amplification, and detection of the target sequence in simple or complex samples using real-time PCR and RT-PCR methodologies. The system requires the use of single-use disposable GeneXpert cartridges that hold the PCR reagents and host the PCR process. .
Cepheid offers the only platform to deliver an accurate, fast, and easy-to-use molecular solution in a single package. This unique solution is the best way for an institution to fully realize the benefits of molecular testing.