Two-step detection method of PCR (Polymerase Chain Reaction) of emulsion

Abstract

The invention discloses a two-step detection method of PCR (Polymerase Chain Reaction) of an emulsion, which belongs to the technical fields of biomedicine and clinical medicine. The method specifically comprises the steps of preparing a water-in-oil emulsion first, and then loading a PCR tube, filled with the water-in-oil emulsion, into a PCR instrument for amplification for the first step; then centrifuging an amplified product, and discarding an upper oil phase, so as to prepare an oil-in-water emulsion, loading into the PCR tube for amplification for the second step, and performing agarose gel electrophoresis detection after amplification is ended. According to the two-step detection method, the yield is further increased, and the gene detection sensitivity is increased; the two-step detection method has the characteristics of interference resistance and high specificity of the PCR of the emulsion and has the characteristic of high yield of the PCR of the emulsion in a conventional PCR method, and additionally, the processes of diethyl ether demulsification, purification and the like are not applied, so that the effects of simplicity, convenience and economy are achieved.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and clinical medicine, and relates to a two-step emulsion PCR method, in particular to a two-step emulsion PCR method for amplifying trace target gene fragments. Background technique [0002] Polymerase chain reaction (PCR) is a molecular biology technique that amplifies specific DNA fragments in vitro. Its biggest feature is that it can greatly increase the amount of DNA. However, when a small amount of target fragment is amplified, especially when there are similar interference fragments, the specificity of amplification is poor, and the target fragment is often not amplified. Existing improved PCR methods such as nested PCR and limiting dilution semi-nested PCR (Anticancer Res. 2007; 27(3B):1459-1463) and so on. Nested PCR uses two sets of primers for two-step PCR to amplify specific DNA fragments. The first pair of PCR primers amplifies fragments similar to ordinary PCR. The second pair ...

AI Technical Summary

Problems solved by technology

However, before the second step of amplification, this method needs to perform operations such as ether demulsification and kit purification on the first step of the emulsion PCR product. These operations are not only cumbersome, but also the recovery efficiency cannot be guaranteed, and the product is easily lost, especially in the first step. When the amount of one-step emulsion PCR product is very small, the product may not be recovered

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