Nucleic acids detection methods

The greatest advances in the detection of chlamydial infection involved the development of various nucleic acid detection methods. Similar to EIA, the DNA hybridization probe test is easy to perform, and a large number of samples can be processed at the same time. Overall, the sensitivity and specificity of the DNA probe tests are greater than with EIA. " " ... 

This article is designed to provide readers who are unfamiliar with chemiluminescence with a sufficient theoretical basis to understand the phenomenon and to extend that basis with a survey of its evolution in terms of chemistry, analytical applications, and recent innovations both in high sensitivity clinical assays and in nucleic acid detection methods. 

Collis, M. P. Methods for detecting nucleic acids detection method using multiple pairs of donor flurophores and quencher molecules in the same probe. Eur. Pat. Appl. EP 1586662, 2005 Chem. Abstr. 2005, 143, 400770. 

Serological techniques can detect target bacteria rapidly in mixtures, but their accuracy depends on the specificity of the antibody used. The use of monoclonal instead of polyclonal antibodies may increase specificity.49,52,58 However, because the same epitope can be present in more than one species, a monoclonal antibody against one species may cross-react with other bacteria.50 For this reason serological methods are not always successful for detection of bacteria in environmental samples and nucleic acid-based methods are now commonly used. 

Nucleic acid hybridization methods use oligonucleotide DNA probes with sequences complementary to a portion of the nucleic acid of the target bacterium38,60 and designed to hybridize with immobilized DNA or RNA on a membrane. After any unbound probe has been washed off, the hybridized probe can be detected.64 66... 

Mansfield, E. S. Worley, J. M. McKenzie, S. E. Surrey, S. Rappaport, E. Fortina, P. Nucleic acid detection using non-radioactive labelling methods. Mol. Cell. Probes 1995, 5(3), 145-156. 

Dellunde, S., et al. 2002. A fast and sensitive nucleic acid extraction method for the detection of Cryptosporidium by PCR in environmental water samples. Water Supply 2 95. 

Nucleic acid probe methods which rely on the specificity of nucleic acid hybridization reaction, combined with the powerful methods of nucleic acid amplification, are expected to spawn many new diagnostic tests which may be useful in the detection and treatment of infectious diseases, cancer, and inherited disorders. The impact of development and applications of diagnostic tests based on nucleic acid probes and on Mab technology wiU be felt increasingly as deeper understanding of diseases at a molecular level is gained. 

Instead of specific amplification of one target to improve sensitivity, methods that amplify all genomic DNA or mRNAs are useful when the target is in short supply. For example, multiple-displacement amplification uses exonuclease-resistant random hexamers and a highly pro-cessive polymerase to amplify DNA nonspecificaily. Initial DNA denaturation is not necessary and the reaction proceeds isothermally. Similarly, messenger RNA can be generi-caUy amplified with a poly(T) primer modified with an RNA polymerase promoter. After reverse transcription, second-strand DNA synthesis, and transcription, antisense RNA is produced. Both whole genome and antisense RNA amplification are also useful as nucleic acid purification methods before amplification or detection. 

DNA PGR assay (neither of which has been cleared by the FDA). GMV PGR LDTs that use standard and real-time PGR methods are also widely used in clinical laboratories. These LDTs use various specimen types, nucleic acid extraction methods, target genes, calibrators, and detection methods. As a result, viral load values obtained with the different assays may not always agree. This makes it very difficult to compare results among clinical studies that use these assays and to establish concentrations of GMV DNA that correlate with clinical disease. 

Nucleic acid amplification methods are now considered a standard laboratory tool. They have had a tremendous impact on the diagnosis and treatment of infectious diseases. These highly sensitive methods have the capability to detect and quantitate minute amounts of target nucleic acid in a rapid manner. The polymerase chain reaction (PCR)... 

Mothershed EA, Whitney AM. Nucleic acid-based methods for the detection of bacterial pathogens Present and future considerations for the clinical laboratory. Clin Chim Acta. 2006 363 206-220. 

Sidransky, D. 1997. Nucleic Acid-Based Methods for the Detection of Cancer. Science, November 7 1054-1058. 

PIO. Pollard-Knight, D. V., Rapid and sensitive luminescent methods for nucleic acid detection. In Bioluminescence and Chemiluminescence Current Status (P. E. Stanley and L. J. Kricka, eds.), pp. 83-90. Wiley, Chichester, 1991. 

Wilson, S. G., Chan, S., Deroo, M., Vera-Garcia, M, Johnson, A., Lane, D., and Halbert, D. N. (1990) Development of a colorimetric second generation nucleic acid hybridization method for the detection of Salmonella in foods and a comparison with conventional culture procedure J. Food Prot 55,1394-1398... 

Nucleic-acid based methods A number of detection and identification methods have been developed that are based on specific nucleotide sequences in RNA or DNA and several are noted in Table 2. Synopses of other related methods are presented in Table 3. 

It is beyond the scope of this chapter to discuss all available technologies, just one example of each of three detection principles will be mentioned here, viz., a growth based method, a non-growth based method and a nucleic acid determination method. For an encyclopaedic overview the reader is referred to the literature [71]. 

There are a variety of methods to detect the DNA content of food, which can be used to unequivocally identify the nature of the product [4]. Among the various systems for nucleic acid detection, electrochemical DNA analysis can involve direct detection based on a guanine signal (label-free) [5] or an electrocatalytic mechanism (label-based). Quantum dots (QDs) [6,7], metal nanoparticles (NPs) [8,9], enzymes [10,11], and metal complexes [12, 13] can be employed as labels. This chapter focuses on electrochemical biosensing systems based on DNA hybridization events, which offer novel routes for food safety and security applications. Particularly, it describes in detail different approaches reported in the latest years on the immobilization of oligonucleotides on electrochemical transducers for sensing of various compounds with interest in food industry. In addition, some interesting applications in other fields that can easily be extended to that of food are also included. 

Nucleic Acid—Based Methods Specific DNA probes have been developed to detect segments of genetic material coding for known enzymes. A gene probe developed to detect the hydrogenase enzyme which occurs broadly in SRB from the genus Desulfovibrio has been tested on samples from an oilfield waterflood plagued with iron-sulfide related corrosion problems. The enzyme was detected with this probe... 

Kim, K. H. Min, J. H. Lee, I. H. Kim, A. G. Method for highly sensitive nucleic acid detection using nanopore and non-specific nucleic acid-binding... 

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