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PCR in detection of Listeria monocytogenes

Polymerase chain reaction (PCR) is one of the most important techniques in molecular biology. This method enables the in vitro synthesis of specific DNA fragments resulting in billions of copies in very little time. Real-time PCR is an advanced form of PCR enabling real-time monitoring of target DNA amplification. The basic principle of real-time PCR is the detection of fluorescence emitted by fluorophore which increases as a reaction proceeds. Today, real-time PCR has widespread applications, e.g. in medical diagnostics, DNA cloning for sequencing, DNA fingerprinting (used in forensic sciences and paternity testing) as well as in the identification of foodborne pathogens. Listeria monocytogenes is a Grampositive, non- spore forming, facultative anaerobic rod that grows between -0.4 and 50 °C. It is catalase positive and oxidase negative and expresses P-haemolysin which produces zones of clearing on blood agar. L. monocytogenes is widely present in plants, soil, and surface water samples, and has also been found in silage, sewage, slaughterhouse waste, milk of normal and mastitic cows, and human and animal feces. Classical microbiological analysis, according to the ISO method, is very time- consuming and can last up to 6 days. PCR’s advantages over classical microbiology are: high specificity and sensitivity, low possibility of false-positive results, less time spent. However, there are also a few disadvantages: the high cost of analysis and the high risk of contamination. For now, PCR is mainly used as a confirmation method or, due to its speed, as a screening method.

DNA analyses ; fluorescence ; molecular biology ; real-time PCR

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