Quantitative standard substance applied to qPCR (quantitative Polymerase Chain Reaction) accurate quantification of Illumina platform next generation sequencing sample and duplication method thereof

Abstract

The invention discloses a quantitative standard substance applied to qPCR (quantitative Polymerase Chain Reaction) accurate quantification of an Illumina platform next generation sequencing sample anda duplication method thereof. The duplication method comprises the following steps: (1) performing PCR amplification by using a linker primer P1 and a linker primer P2 and taking a standard substancein a commercialized kit as a template, wherein the nucleotide sequence of the linker primer P1 is 5'-AATGATACGGCGACCACCGAGA-3', and the nucleotide sequence of the linker primer P2 is 5'-CAAGCAGAAGACGGCATACGAG-3'; (2) purifying with AMPure XP bead at the end of a reaction, performing Qubit quantification at the end of purification, initially diluting step by step by 10 times after Qubit concentration measurement, performing a quantitative experiment on the diluted sample, picking up a sample which is closest to the commercialized standard substance in concentration to serve as a duplicated standard substance 1, and diluting the duplicated standard substance with enzyme-free water step by step by 10 times to obtain second to sixth duplicated standard substances; (3) preserving the duplicated standard substances in a solution containing a buffering system and a stabilizer. The quantitative standard substance is accurate in quantification, and is convenient to use; compared with the commercialized kit, the quantitative standard substance has the advantage that the experiment cost is lowered greatly.

Description

technical field [0001] The invention relates to next-generation sequencing (NGS), in particular to a quantitative standard for qPCR to accurately quantify NGS samples on the Illumina platform and a copying method thereof. Background technique [0002] With the advancement of science and technology, the biotechnology industry is also constantly developing and innovating. The requirements of existing scientific research and medical applications are getting higher and higher. The original Sanger generation sequencing technology can no longer meet its needs. The second-generation sequencing technology (NGS) has greater advantages for genome sequencing because of its low cost, high speed, and high throughput. In order to obtain high-quality nucleic acid sequencing data, we need to analyze and evaluate the quality of the sequencing library, which is particularly important. Therefore, it is necessary to carry out accurate quantitative detection and analysis on the DNA sequencing l...

AI Technical Summary

Problems solved by technology

However, there are few reports at home and abroad on the stability of standard products and the impact of cryopreservation and resuscitation on them.

In the process of repeated freezing and thawing, the volume change that occurs when the solid-liquid state transitions will cause mechanical shearing of the enzyme, and the structural integrity of the enzyme will be destroyed, thereby affecting its stability and ultimately affecting the accuracy of qPCR quantification

Fluorescent quantitative PCR kits involved in clinical testing will be repeatedly frozen and thawed during use, and experimenters often worry that this will affect the quantitative results

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