Encuentro Científico Internacional ECI2016v

Encuentro Cient铆fico Internacional 2016 de verano Lima, 2 - 4 de Enero del 2016 www.encuentrocientificointernacional.org www. eciperu.net Ingreso libre previa inscripci贸n en este enlace y posterior difusi贸n de este programa ente sus contactos https://docs.google.com/forms/d/12sq15kaCJvPEuPRz-F_Rnj19jBTfYsQyHQcs bFsk/viewform

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Ciencias de la salud Cadena de la Polimerasa Loop mediated isothermal amplification as an alternative to Polymarase Chain Reaction Michelle C. Chirinos-Arias 1, 2,4, Ada P. Mendoza-Farro 1, 3, Ana M. H

1,3

4 Universidad Nacional Agraria La Molina. Av. La Molina S/N, La Molina

Resumen gradiente de temperatura, LAMP como su nombre lo indica, es una costosos como los termocicladores para obtener un alto nivel desplazamiento de hebra (1, 2)

de cebadores que

estructuras de ADN tallo-lazo (stem-loop) con bastantes repeticiones invertidas de ADN extremos designa F1c y B1. Mientras que, otro par de iniciadores que se encuentran fuera de la re F1c, un espaciador TTTT y la secuencia F2 complementaria a F2c. El Backward Inner Primer (BIP) contiene la secuencia B1c (complementaria a B1), un espaciador TTTT y B2. La t amplicones resultantes pueden ser discriminados visualmente y detectados por

lado, LAMP requier

simple vista y el hecho de usar SYBR Green I aumenta el riesgo de observar Tomita et al. (5) ha logr debido a la alta especifi tiempo y con menor costo, LAMP es una excelente alternativa a la PCR. El requiere personal especiali Descriptores (LAMP), PCR. Abstract Loop-mediated isothermal amplification (LAMP) is a fast, simple, cost effective and specific technique. Unlike PCR based on temperature gradient, LAMP as its name implies, is an amplification that occurs at a constant temperature in the range of 60 to 65 or water bath. It is not necessary to use costly equipment such as thermocyclers for a high level of accuracy. Such amplification requires the enzyme Bst polymerase (isolated from Bacillus stearothermophilus) it polymerized and also has a strand displacement activity (1, 2).

This technique uses four pairs of primers that recognize six regions of the target DNA and the amplification products are structures stem-loop DNA with enough inverted repeats sequences of target DNA (3). A pair of primers from 23 to 24 nucleotides on each end of the target region, called F2c and B2, another pair of primers are approximately 40 nucleotides from the ends of the target and designated as F1c and B1.

While, another pair of primers that are outside of the target region are called F3c and B3. The Forward Inner Primer (FIP) contains F1c primer, a spacer TTTT and F2 (a sequence complementary to F2c). The Backward Inner Primer (BIP) contains the B1c sequence (complementary to B1), a spacer TTTT and B2. LAMP technique is fast, lasting from 30 minutes to 1 hour, low cost, the resulting amplicons may be visually discriminated and detected by precipitation using magnesium pyrophosphate or visualizing adding SYBR Green I after reaction (1, 2, 4). It is more sensitive than conventional PCR and real time PCR (3), because the performance of these techniques is affected by inhibitors present in organic samples and PCR is sensitive to contamination. LAMP needs to be improved, it is difficult to identify white precipitates and the fact of using SYBR Green I increases the risk of contamination or false positive with other amplicons (primers dimers) can also fluoresce (3). However, Tomita et al.

(5) has achieved a better result adding calcein, a fluorescence indicator metal and manganese ion which allows to observe if the reaction is a positive color change from orange to green fluorescent. In conclusion, due to the high specificity, high sensitivity and high amplicon yield in less time and with low cost, LAMP is an excellent alternative to PCR. The main drawback of this technique is the design of specific primers, because it requires a specialized researcher with knowledge of bioinformatics and programming, especially if the method will be used for diagnosis.

Keywords: Nucleic acid amplification, Loop-mediated isothermal amplification (LAMP), PCR. El libro de completo se encuentra disponible https://eciperu.files.wordpress.com/2015/12/eci2016vlibroderesumenes1.pdf !

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