Real-time fluorescent PCR (polymerase chain reaction) method and application

Abstract

The invention relates to a real-time fluorescent PCR (polymerase chain reaction) method and application. The method only requires one pair of upstream primer and downstream primer and one detection probe, wherein the upstream primer and the detection probe are respectively subjected to single marking; the upstream primer comprises three regions, namely a specific primer sequence 3, a specific detection sequence 2 and an additional sequence 13; the downstream primer is designed according to general primer design; and the detection probe comprises a specific detection sequence 5. The specific detection sequence 2 and an extension sequence of the upstream primer form an unique neck ring structure, the structure is formed by guiding of the specific detection sequence 2, and the detection probe is hybridized at adjacent position. The method provided by the invention has good specificity and strong selectivity, probe and primer design are simple and flexible, purification is easy, yield is high, the method can be used for monitoring amplification product quantity in real time to realize qualitative and quantitative analysis on a target sequence, also can be applicable to melting curve analysis, is especially applicable to detection of SNP (Single Nucleotide Polymorphism) or mutational site and has strong single base difference distinguishing capability.

Description

technical field [0001] The invention relates to a PCR method, in particular to a real-time fluorescent PCR method. Background technique [0002] Since the polymerase chain reaction (PCR) was proposed in 1985, it has developed at an astonishing speed. At present, PCR technology has been widely used in both basic life science research and clinical diagnosis. Detection techniques that rely on PCR are mainly divided into two categories, one is heterogeneous detection technology, and the other is homogeneous detection technology. Heterogeneous detection techniques include agarose gel electrophoresis, capillary electrophoresis, chip hybridization and other detection methods, while homogeneous detection techniques mainly refer to real-time fluorescence detection techniques. Real-time fluorescent PCR detection technology is to monitor the formation of products in real time during the PCR amplification process. It is fast and convenient without opening the tube, and can obtain the i...

AI Technical Summary

Problems solved by technology

This method has fast reaction speed and high signal-to-noise ratio, but the design and synthesis of Scorpion probes are complicated and costly

[0005] CN101033486B discloses a fluorescent PCR detection method, using upstream and downstream primers and a probe for detection, the probe is designed to be complementary to the upstream primer, and no amplification product appears When the fluorescent group on the upstream primer and the quenching group on the probe are close to the fluorescence and is quenched, when the amplification product appears, the fluorescent group on the upstream primer and the quenching group on the probe release the fluorescent signal separately , so as to realize real-time fluorescent PCR. The specificity of this method is poor, and the fluorescent signal can still be detected when non-specific amplification occurs.

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