PCR Real Time (qPCR) procedure — OPERON скачать в хорошем качестве

PCR Real Time (qPCR) procedure — OPERON

Quantitative PCR (qPCR) or real time PCR is a form of polymerase chain reaction (PCR) used to amplify and simultaneously detect the amplification product of nucleic acids. This is achieved by incorporating fluorescent molecules, which associate with the amplified nucleic acids, so that the increase in fluorescence is proportional to the increase in the amount of amplified DNA/RNA molecules in the reaction. It is a simple and reliable technique that can be applied to different fields in modern biology and related sciences. Real-time PCR can be used for the diagnosis of diseases, such as infectious diseases, cancer and genetic abnormalities. The introduction of quantitative PCR assays in microbiology laboratories has improved the diagnosis of infectious diseases and is also used as a tool to detect new emerging diseases. The real-time PCR technique can be performed using DNA and RNA from different sources such as tissues and microorganisms, including peripheral blood, skin, hair, saliva and bacteria or viruses. Only trace amounts of DNA/RNA are needed to generate sufficient copies to be detected by the fluorescence generated. For this reason, the PCR technique is very sensitive, so it is extremely important to maintain good laboratory practices, especially with regard to hygiene, order and safety in any of the phases of the technique (extraction of nucleic acids, preparation of the reactions and detection of the amplified material), in order to avoid possible contamination that may affect the test result. To minimise this problem, there are certain conditions that must be controlled, such as: Separate working areas to separate amplicon and amplicon-free areas. Separate sets of equipment that should not be shared between areas (micropipettes, micropipette racks, gowns, gloves, markers, microtubes, centrifuges, refrigerators, etc.). Maintenance of a unidirectional flow of work from the cleanest to the dirtiest areas. Air flows with positive and negative pressures depending on the area. PCR quality materials (micropipette tips, microtubes, etc.), free of exogenous DNA, DNAases and RNAases. Micropipette tips with filter to reduce the risk of contamination by aerosol formation. Cleaning of the surface used to prepare PCR reactions with ethanol (70 %) and, if possible, decontamination with ultraviolet radiation before and after each preparation (15 to 30 min). The video shows an example of how a PCR should be performed correctly, showing the different zones for the different activities.