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Optimum Design Method of Pathogen Species-Specific PCR Primers

A technology for optimizing design and primer design, applied in the field of bioinformatics analysis, can solve problems such as affecting the sensitivity and specificity of primer design, affecting the performance of multiplex PCR combination reactions, and being inapplicable, so as to avoid strain-specific problems.

Active Publication Date: 2021-07-09
JIANGSU SIMCERE MEDICAL DEVICE CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Method used

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  • Optimum Design Method of Pathogen Species-Specific PCR Primers
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  • Optimum Design Method of Pathogen Species-Specific PCR Primers

Examples

Experimental program
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Effect test

Embodiment 1

[0059] The optimal development of embodiment 1 specific primer design method

[0060] The present invention has finally established and developed a specific primer design method through a large number of bioinformatics analysis and optimization experiments. The specific steps of the scheme are as follows:

[0061] 1. Construction of the background comparison library:

[0062] 1. Obtain the reference genomes of all microbial species in NCBI RefSeq, and divide K-mer for each genome to form a unique K-mer set. The value of K is 18-20, preferably 20.

[0063] 2. Select the K-mer with a frequency >= 2 from the unique K-mer set at the microbial species level as the candidate K-mer set. The value of K is 18-20, preferably 20.

[0064] 3. The human genome is divided into unique K-mer collections according to method 1, and combined with the candidate K-mer collections at the microbial species level in method 2 to construct a K-mer db and obtain a background comparison library. The ...

Embodiment 2

[0076] Example 2 Bacteria / fungus / virus species-specific PCR primer design and evaluation

[0077] 1. Take the bacteria Mycoplasma pneumoniae / fungus Aspergillus flavus / virus EBV as examples to design PCR primers respectively:

[0078] 1. Screen n target species genomes from the NCBI RefSeq / GenBank library. The value of n is 10, see figure 2 .

[0079] 2. Divide the screened genomes into K-mers to obtain unique K-mer sets. The value of K is 20.

[0080] 3. Use meryl software to build a K-mer db for the unique K-mer set in method 2, extract K-mers with a frequency greater than n*p from it, and use it as a K-mer set shared by the species level, and construct a species-shared K -mer comparison library. The value of K is 20, the value of n is 10, and the value of p is 0.8.

[0081] 4. Use the NCBI RefSeq / GenBank optimal reference genome screening rules to screen the reference genome of the target species, divide the K-mer, and retain the position information of all K-mers, an...

Embodiment 3

[0114] The wet test verification of embodiment 3 species-specific PCR primers

[0115] This example is further validated by the wet test of Aspergillus flavus species-specific PCR primers (F: 5'-CCCTCTTGCCTGTTCCAGAG-3' (SEQ ID NO.1), R: 5'-CATGGGTGGGTGCTCTTCAT-3' (SEQ ID NO.2)) As an example, to illustrate the effectiveness of the specific primers designed based on the ideas of the present invention.

[0116] 1) Reagent consumables

[0117] Enzyme-free sterile water: ThermoFisher, Nuclease-Free Water (not DEPC-Treated) (Cat. No.: AM9937); Qubit Fluorometer DNA Detection Kit: Qubit 1X dsDNA HS Assay Kit (Cat. No.: Q33231); PCR amplification enzyme: GXL DNA Polymerase (R050A).

[0118] 2) Primer verification

[0119] The nucleic acid extracted from the Aspergillus flavus standard was used as a template for PCR reaction verification, and at the same time, gDNA and Zymo bacteria DNA templates were added in parallel to the PCR reaction system to simulate real clinical samples f...

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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,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G16B20/00G16B45/00
CPCG16B20/00G16B45/00
Inventor 梁相志李振中周水莲何祥鹏潘吾思胥慧郭昊李诗濛任用
Owner JIANGSU SIMCERE MEDICAL DEVICE CO LTD
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