Multiplex PCR primer design method and device

Abstract

The present invention relates to a method and device for designing multiple PCR primers. The method includes: acquiring mutation information of a tumor sample, selecting a preset number of monitoring sites according to the mutation information to form a monitoring site set; Design at least one candidate primer pair with reference to the corresponding target sequence on the genome, and select one candidate primer pair for each monitoring site to generate an evaluation primer pair set corresponding to the monitoring site set; the evaluation primer pair set comes from different candidate primer pairs. Primer-dimer evaluation is performed for each pair of primers, and qualified candidate primer pairs are screened as target multiplex PCR primers according to the primer-dimer evaluation results of all primers in the evaluation primer pair set. The multiplex PCR primer design method of the present invention can design multiple PCR primers for the mutation sites of tumor patients, so as to achieve the purpose of stably detecting ctDNA mutation information with an abundance of ≥0.02%.

Description

technical field [0001] The invention relates to the technical field of bioinformatics, in particular, to a method and device for designing multiple PCR primers. Background technique [0002] Minimal Residual Disease (MRD) refers to the clinical state in which there are still a small number of tumor cells or small lesions in the patient during or after treatment, also known as Molecular Residual Disease or Measurable Residual Disease (Measurable Residual Disease). Residual Disease). [0003] MRD is applicable to both solid and hematologic tumors, and is more commonly used to represent the persistence of malignant cells below the standard detection limit in the bone marrow of patients with hematologic tumors. After tumor treatment, residual tumor cells in the body continue to proliferate, leading to disease recurrence. MRD may occur after treatment because not all cancer cells respond to treatment, or because tumor cells have developed resistance to the drugs used, and the p...

AI Technical Summary

Problems solved by technology

[0009] Generally, the amplicon size used in multiplex PCR design is about 300bp, but the fragment size of ctDNA is 90-150bp. In order to effectively detect MRD, the fragment size including the primer sequence during primer design can only be about 100bp or less.

Primer3 is the most widely used PCR primer design software. Usually, primer3 can only detect the hairpin structure and TM value between a pair of primers, and cannot check the primer dimer and non-specific amplification between multiple PCR primers. , while primer dimers and non-specific amplification are the key to the success of multiplex PCR and the detection of LOD (limit of detection)

Images