Real-time fluorescence PCR method for detecting multiple genes or different targets with primer associated universal probe

Abstract

The invention discloses a real-time fluorescence PCR method for detecting multiple genes or different targets with a primer associated universal probe. The method comprises the following steps: firstly, relying on a composite sequence primer with a special structure, and introducing a combined position of a universal probe and a universal amplification primer at the tail end of an amplified product in the process of initial amplification so as to realize different targets; using the same probe for real-time fluorescence detection. The method has the advantage that the production process is simple and easy to control, the absolute usage amount of the probe is decreased, and the reagent cost is reduced. In a single detection, different items can share one probe, so that the production process is simplified and is convenient to control. In multiple detections, different targets realize specific real-time fluorescence detection by using the same probe without different probes, so that the absolute usage amount of the probe is directly reduced, the problems of poor detection curves and low sensitivity caused by a high multi-probe fluorescent background are solved, and besides, the reagent cost is also reduced by using the same probe.

Description

Technical field [0001] The present invention involves real -time fluorescent PCR amplification detection methods, especially the real -time fluorescence PCR method that uses a primer to detect multiple genes or different targets. Background technique [0002] Polymerase chain reaction (PCR) is an in vitro nucleic acid amplification technology developed in the mid -1980s. It has outstanding advantages such as sensitivity, high yields, fast, simple, repetitive, easy to automate, and can be in it.A test tube will expand to 100,000 or even one million times the purpose of the purpose of the purpose or a DNA fragment to be studied in a few hours.The basic principles of PCR technology are similar to the natural replication process of DNA, and its specificity depends on the oligonucleotide primer that complement each other at both ends of the target sequence.PCR is composed of degeneration-annealing-extended three basic reactions steps: ① Draphic degeneration of template DNA: After the ...

AI Technical Summary

Problems solved by technology

When detecting more than 6 targets in a single tube, more than 2 targets need to share one channel, and these targets need to be labeled with the same fluorescent dye corresponding to each probe, and each probe has a certain background signal, and the background signal of multiple probes is accumulated , it will appear that the background signal of this channel is too high, which seriously affects the linearity of the amplification curve, and the synthesis of multiple probes is expensive

[0008] Using the fluorescent dye melting curve analysis method to identify the PCR product and analyze the target type in the product, as described in the patent application number CN102559868A, using the closed-tube analysis technology, which reduces the risk of product contamination and detection cost, but prolongs the analysis time , high technical requirements, highly subjective results

[0009] To sum up, although there are a variety of detection technologies for nucleic acid sequence recognition and quantification at home and abroad, there are still certain limitations in industrial production cost control, process simplicity, and technical compatibility in single and multiple detection. , so it is necessary to develop a general method that can use the same probe for real-time fluorescence detection of different targets

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