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Vol. 22. Issue 6.
Pages 355-360 (June 2004)
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Vol. 22. Issue 6.
Pages 355-360 (June 2004)
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Aplicaciones de las técnicas de PCR a la epidemiología molecular de las enfermedades infecciosas
PCR techniques for Molecular Epidemiology of Infectious Diseases
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Felipe Fernández-Cuenca1
Corresponding author
felipefc@us.es

Correspondencia: Dr. F. Fernández-Cuenca. Departamento de Microbiología y Epidemiología Infecciosa. Hospital Universitario Virgen Macarena. Apdo. 914. 41009 Sevilla. España.
Departamento de Microbiología y Epidemiología Infecciosa. Hospital Universitario Virgen Macarena. Sevilla. España
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En los últimos años se han desarrollo nuevas técnicas moleculares de tipificación basadas en la reacción en cadena de la polimerasa (PCR) que han supuesto un importante avance en el estudio de las enfermedades infecciosas. La PCR que utiliza cebadores arbitrarios (AP-PCR) y la PCR que utiliza cebadores que hibridan con secuencias de ADN repetidas (rep-PCR) son las técnicas de PCR más utilizadas para tipificar bacterias y hongos. Estas técnicas son sencillas de realizar, rápidas y poseen un elevado poder de discriminación. La AP-PCR es relativamente poco reproducible, por lo que tiene que ser validada o estandarizada en cada laboratorio. La digestión con enzimas de restricción de genes amplificados mediante PCR constituye la base de la PCR-RFLP. Esta técnica es sencilla y muy reproducible, pero suele ser menos discriminativa que la AP-PCR o la rep-PCR. El estudio del polimorfismo de la longitud de fragmentos amplificados (AFLP) se basa en la amplificación mediante PCR de fragmentos de ADN obtenidos por restricción enzimática del ADN cromosómico. Esta técnica de tipificación posee mayor poder de discriminación y reproducibilidad que las anteriores, pero es más laboriosa, más costosa y requiere personal especializado. La mayoría de las técnicas de tipificación basadas en la PCR son menos laboriosas, más rápidas y más fáciles de realizar e interpretar que la electroforesis en campo pulsante (PFGE; técnica de referencia para la mayoría de bacterias y hongos), pero suelen ser, por lo general, menos reproducibles y discriminativas que esta última, dependiendo de la especie estudiada y de la técnica de PCR empleada. En resumen, existen diversas técnicas moleculares basadas en la PCR que son muy útiles como método inicial de tipificación para estudiar la relación clonal entre aislados de una misma especie. La elección de la técnica depende de factores de tipo técnico (rapidez, poca laboriosidad, fácil de interpretar y con un elevado poder de discriminación y reproducibilidad) y económico (bajo coste).

Palabras clave:
Reacción en cadena de la polimerasa
Epidemiología molecular
Enfermedades infecciosas

The development of new PCR-based typing methods in the last years have supposed an important advance in the study of infectious diseases. Arbitrarily primed PCR (AP-PCR) and repetitive element sequence-based PCR (rep-PCR) are the most widely used PCR-based fingerprinting methods for bacteria and fungi. Major advantages of these methods are flexibility, technical simplicity and high discriminatory power. The AP-PCR presents problems of low inter-run and inter-laboratory reproducibility which make necessary the optimization of the protocol and reagents. PCR-RFLP is based in the enzymatic digestion of polymorphic genes amplified by PCR. This method is easy to perform and discriminatory, although less than AP-PCR or rep-PCR. Amplified fragment length polymorphism (AFLP) is a highly reproducible and discriminatory typing method based on the amplification by PCR of restriction fragments obtained from chromosomic DNA. This method is more discriminative and reproducible than AP-PCR, rep-PCR and PCR-RFLP, but it is more time-consuming and expensive, and requires specialised personnel. Most of these PCR-based typing methods are less time-consuming, rapid and easy to perform and of interpretation than pulsed-field gel electrophoresis (PFGE; gold standard method for typing most bacterium and fungi), but they usually are less discriminative and reproducible than PFGE, depending on the species studied and the method of PCR used. In summary, there are several PCR-based methods which are useful as a primary approach to the study of the clonal relationship among microbial isolates. The selection of the method to be used depend on technical (rapid, low time-consuming, easy to perform and to interpretate, reproducible and discriminatory) and economical (low cost) factors.

Key words:
Polymerase chain reaction
Molecular epidemiology
Infectious diseases
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Copyright © 2004. Elsevier España, S.L.. Todos los derechos reservados
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