infectious diseases
Nucleic Acid–Based Identification Methods for Infectious Disease
Nucleic acid amplification
Nucleic acid amplification techniques (NAAT) take tiny amounts of DNA or RNA, replicate them many times, and thus can detect minute traces of an organism in a specimen, avoiding the need for culture. These techniques are particularly useful for organisms that are difficult to culture or identify using other methods (eg, viruses, obligate intracellular pathogens, fungi, mycobacteria, some other bacteria) or that are present in low numbers. These tests may involve Target amplification (eg, polymerase chain reaction [PCR], reverse transcriptase–PCR [RT-PCR], strand displacement amplification, transcription amplification) Signal amplification (eg, branched DNA assays, hybrid capture) Probe amplification (eg, ligase chain reaction, cleavase-invader, cycling probes) Postamplification analysis (eg, sequencing of the amplified product, microarray analysis, and melting curve analysis, as is done in real-time PCR) Appropriate specimen collection and storage before arrival at the molecular diagnostic laboratory are critical. Because amplification methods are so sensitive, false-positive results from trace contamination of the specimen or equipment can easily occur. Despite high sensitivity, false-negative results sometimes occur even when a patient is symptomatic (eg, in West Nile Virus ). False-negative results can be minimized by the following: Avoiding use of swabs with wooden shafts or cotton tips (the swab that has been validated for the amplification assay must be used) Transporting specimens rapidly Freezing or refrigerating specimens if transport is likely to take>2 hours Freezing is the typical storage method for nucleic acid amplification assays. However, specimens should be refrigerated rather than frozen if labile viruses (eg, varicella-zoster virus, influenza virus, HIV-2) are suspected or if viral cultures are also to be done (frozen specimens may not be usable for standard cultures). 