NON-NA- ACID BASED METHODS

Non-NA- acid based Methods



Non-NA- acid based Methods

Once an organism has been isolated by culture, it must be identified. Non-nucleic acid-based identification methods use phenotypic (functional or morphologic) characteristics of organisms rather than genetic identification. Characteristics of an organism's growth on culture media, such as colony size, colour, and shape, provide clues to species identification and, combined with Gram stain, direct further testing. Numerous biochemical tests are available, each restricted to organisms of a certain type (eg, aerobic or anaerobic bacteria). Some assess an organism's ability to use different substrates for growth. Others assess presence or activity of key enzymes (eg, coagulase, catalase). Tests are performed sequentially, with previous results determining the next test to be used. The sequences of tests are myriad and differ somewhat among laboratories. (Wolfram 2008: 52-65)

Non-nucleic acid-based identification tests may involve manual methods, automated systems, or chromatographic methods. Some commercially available kits contain a battery of individual tests that may be performed simultaneously using a single specimen and may be useful for a wider range of organisms. Multiple test systems can be highly accurate but may require several days to yield results. Microbial components or products are separated and identified using high-performance liquid chromatography (HPLC) or gas chromatography. Usually, identification is by comparison of an organism's fatty acids to a database. Chromatographic methods can be used to identify aerobic and anaerobic bacteria, mycobacteria, and fungi. Test accuracy depends on the conditions used to culture the specimen and the quality of the database, which may be inaccurate or incomplete. (Nadassy 2009: 1999-2017)

It is widely known that two single-stranded nucleic acids with complementary sequences have the inherent ability to form Watson-Crick duplex structures. The simplicity and sequence-specificity of duplex structure formation, the high chemical stability of a duplex, and the convenience of automated synthesis have made DNA oligonucleotides an ideal choice as probes for the detection of nucleic acids. Recently developed in vitro selection techniques permit creation of DNA and RNA “aptamers” that are capable of binding a wide variety of non nucleic acid targets with high affinity and specificity. Aptamers have considerably broadened the utility of nucleic acids as probes for detection of biological and nonbiological targets. In vitro selection also allows generation of artificial ribozymes (catalytic RNAs) and deoxyribozymes (catalytic DNAs) with desirable functions. Aptamers, ribozymes, and dcoxyribozymes have become increasingly valuable molecular tools in the form of switches and sensors. Unfortunately, binding or catalytic actions by these switches and sensors do not usually lead to an easily detectable signal, and the lack of a facile reporting method could substantially reduce their value. To facilitate the exploitation of nucleic acid switches and sensors for detection-related applications many recent studies have explored fluorescence signalling as a convenient approach for the reporting of binding and catalytic events. This chapter is devoted to the discussion of these efforts. The reporter molecules to be described include molecular beacons, signalling aptamers, and signalling ribozymes and deoxyribozymes. (Keith 2002: 95-115)

The suitability of nucleic acids as specific MREs for the ...