Other Applications

The real-time PCR-based focus on pathogen genotype is having a significant impact on the detection of viruses, bacteria, and parasites in diagnostic microbiology.[23] The biggest application of real-time technology is probably in virology, where these assays have been used to investigate the role of viruses in a range of human diseases. The ability to multiplex has allowed the reliable measurement of different viral nucleic acid targets within a single sample, and the discrimination of multiple viral genotypes within a single reaction tube. A major application is in the detection of viral load, and real-time assays have become very useful as indicators of the extent of active infection, host/virus interaction, and the efficacy of antiviral treatment. Other uses include the assessment of viral gene therapy vectors before their use in clinical trials and the study of new and emerging viruses and clinical symptoms experienced by patients.[24]

A major benefit lies in the rapidity with which results can be obtained. This is of major importance when detecting bacterial pathogens, as it allows a specific and timely application of antibiotics. Real-time assays are ideal for distinguishing between different serotypes of a single bacterial species,[25] for detecting and monitoring drug resistance among clinical isolates,[26] for detecting pathogens in food,[27] and not least for identifying microbes used as agents of biological warfare.[28]

The worldwide approval of a large number of genetically modified organisms (GMOs) among countries, and the associated labeling requirements, has resulted in the development of real-time PCR-based methods for the detection of the presence of GMO in food or food additives.[29] Genetically modified organism detection through PCR relies on parallel amplification of the transgene and of an endogenous reference gene that provides a control both for the lack of inhibition and for the ability to amplify the target DNA in the sample. Additionally, for quantitative analyses, amplification of the reference gene provides an estimation of the total amount of target DNA present in the sample. Targeting the DNA is particularly appropriate, because of the high stability of this molecule under the extreme conditions used during processing of some food products.

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