Recombinase Polymerase Amplification

Nucleic Acid Test to Diagnose Cryptosporidiosis: Lab Assessment in Animal and Patient Specimens

Objetivo:

Desarrollar y evaluar el rendimiento de una técnica de RPA para detectar Trypanosoma Cruzi en muestras de heces.

Métodos:

- Realizar experimentos de laboratorio controlados con “muestras con oocitos de un rango de concentraciones clínicamente relevantes” para optimizar los parámetros del ensayo y evaluar el rendimiento de la prueba. (Controlled laboratory experiments were first performed to optimize assay parameters and evaluate assay performance using samples spiked with oocysts spanning a clinically relevant range of concentrations.)

- Prueba piloto del ensayo con animales infectados y no infectados (control)

- Medir el rendimiento de la prueba RPA con muestras de heces de pacientes con la enfermad de Chagas y pacientes sanos

- Comparar esos resultados con los de la prueba PCR (gold standard)

*Seleccionar target de RPA

*Realizar reacciones de RPA con las instrucciones del fabricante y detectar los productos con gel de electroforesis

Recombinasas se unen a los primers y les permiten juntarse con su secuencia homóloga en la candena de DNA dúplex, desplazando una hebra, a la cual se le unen SSB para evitar que desplacen a los primers (unidos con su cadena complementaria)

RPA necesita de 3 enzimas:

• Recombinasa: Recombinases are genetic recombination enzymes. DNA recombinases are widely used in multicellular organisms to manipulate the structure of genomes, and to control gene expression. These enzymes, derived from bacteria and fungi, catalyze directionally sensitive DNA exchange reactions between short (30–40 nucleotides) target site sequences that are specific to each recombinase. These reactions enable four basic functional modules, excision/insertion, inversion, translocation and cassette exchange, which have been used individually or combined in a wide range of configurations to control gene expression. Recombinases are capable of pairing oligonucleotide primers with homologous sequence in duplex DNA

Types include:

- Cre recombinase

- Hin recombinase

- RecA/RAD51

- Tre recombinase

- FLP recombinase

• SSB: Single stranded binding proteins prevent premature annealing, to protect the single-stranded DNA from being digested by nucleases, and to remove secondary structure from the DNA to allow other enzymes to function effectively upon it.

• Strand-displacing polymerase: The term strand displacement describes the ability to displace downstream DNA encountered during synthesis. No tiene 5´→3´ exonuclease activity

...