Probe and primer combination for digital PCR amplification and design method of probe and primer combination

Abstract

The invention discloses a design method of a probe and primer combination for digital PCR amplification, the probe and primer combination for digital PCR amplification, a digital PCR amplification method and an application thereof. The problem that a digital PCR fluorescent probe and primer design is limited by a base sequence is solved, a novel problem and primer design thought and a novel probeand primer combination are provided, a two-step method comprising purification after primary amplification and then secondary amplification is not needed, and conventional one-step operation is only required, so that the possibility that a sample is polluted is reduced, and an amplified fluorescent signal is better.

Description

technical field [0001] The present invention relates to the field of digital PCR, in particular to a probe primer combination for digital PCR amplification, a design method for a probe primer combination for digital PCR amplification, a digital PCR amplification method and its application. Background technique [0002] Digital PCR (dPCR) is a modification of the conventional polymerase chain reaction (PCR) method that can be used to directly quantify and clonally amplify nucleic acids (including DNA, cDNA, methylated DNA or RNA). One difference between dPCR and traditional PCR is the method used to measure the amount of nucleic acid. Both PCR and dPCR perform one reaction per individual sample, dPCR also performs a single reaction within a sample, but the sample is separated into a large number of partitions and the reaction is performed separately in each partition. This separation allows sensitive measurement of nucleic acid amounts. dPCR has proven effective for studyi...

AI Technical Summary

Problems solved by technology

The disadvantages of this technology include: a) real-time detection cannot be realized; b) the analysis of the map results is difficult, and it is easy to misjudge; c) PCR products need post-processing, which is easy to pollute the detection environment

Also, sometimes the signal of a probe will be low, requiring a lot of effort to optimize

[0008] The present invention aims to solve the problem that the design of digital PCR fluorescent probes is limited by the base sequence, and provides a new combination of probes and primers. It does not require a two-step method of purification and re-amplification after one amplification, and only one conventional step is required.

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