Amplification methods for epidemiologic investigations of infectious diseases

Abstract

DNA restriction fragment length polymorphisms (RFLP) are extremely valuable tools for laboratory-based evaluation of hypotheses generated by epidemiologic investigations of infectious disease outbreaks. Recently, polymerase chain reaction (PCR)-based DNA amplification methods have been used to index differences between suspected etiologic agents isolated from cases and suspected source. These methods are attractive because they require minute amounts of target DNA, and can be completed in a very short time. Two types of PCR-based subtyping methods are available. The PCR-RFLP method involves the amplification of previously characterized or phylogenetically conserved targets followed by restriction endonuclease analysis to evaluate polymorphisms within the amplified sequences. The Random Amplified Polymorphic DNA (RAPD) and Arbitrarily Primed PCR (AP-PCR) methods require no prior knowledge of DNA sequences of test organisms because they rely on random amplification of target DNA by arbitrarily chosen primers. RAPD and AP-PCR do not require any restriction analysis of amplified DNA. The PCR-RFLP methods are organism-dependent and do not always provide adequate discrimination between unrelated isolates. RAPD methods often suffer from poor reproducibility because the amplification is performed by using crude target DNA under nonstringent conditions.