Optimum Design Method of Pathogen Species-Specific PCR Primers

Abstract

The invention provides a method for optimally designing pathogenic species-specific PCR primers. The primer optimization design method uses the K-mer method to identify the specific interval of the species, and the primer design is based on the specific fragment, which ensures the species specificity of the primers and avoids the strain specificity caused by the use of a single genome.

Description

technical field [0001] The invention relates to the field of bioinformatics analysis, in particular to a method for designing and optimizing specific PCR primers. Background technique [0002] Polymerase Chain Reaction (PCR), as one of the most basic molecular biology experiments, can amplify the copy number of target DNA by millions of times in vitro, so it is widely used in genetic engineering and genetic diagnosis , target sequence enrichment and other fields. The pros and cons of the primer design is one of the important parameters affecting the PCR test. [0003] In clinical microbial diagnosis, multiplex PCR detection, Illumina and third-generation Nanopore high-throughput platforms have become important detection methods for detecting unknown infections in clinical samples. And in recent years, there has been a trend to improve the detection accuracy by targeting the enrichment of pathogenic regions by PCR method. Among these detection methods, designing sensitive,...

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Problems solved by technology

However, both methods have their limitations: 1. The high similarity of 16S rRNA gene sequences between different microorganisms

Even at the genus level, many researchers have reported resolution issues with 16S rRNA gene sequences, so 16S rRNA designed primers are not suitable for identification of microorganisms at the strain / species level in the complex environment of infected samples

2. Merge primer design based on multiple sequence alignment, 1) requires a large amount of background knowledge research and accumulation of target microorganisms in the early stage; 2) duplication of work, for each target species, it is necessary to repeatedly construct multiple sequence alignment and homology conservation Regional screening; 3) The level of sequence conservation obtained will seriously affect the sensitivity and specificity of primer design; 4) Annexation primers cannot directly evaluate the free energy of the interaction between primers, which will affect the wet test for multiplex PCR combination reaction performance

Therefore, the design of degenerate primers based on multiple sequence alignment is not suitable for multiple PCR reactions in large batches of infected samples for targeted enrichment

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