Method and device for obtaining species-specific consensus sequences of microorganisms and application

Abstract

The invention provides a method for obtaining species-specific consensus sequences of microorganisms, which at least comprises the following steps: S100, searching for candidate consensus sequences: clustering specific sequences of target strains belonging to the same strain based on a clustering algorithm to obtain a plurality of candidate species-specific consensus sequences; S200, verifying andobtaining a species-specific consensus sequence of primary screening; judging whether the candidate species-specific consensus sequences meet the following conditions: 1) the plant seed coverage degree meets a preset value; 2) the effective copy number meets a preset value; if the candidate species-specific consensus sequences meet all the conditions, determining that the candidate species-specific consensus sequences are species-specific consensus sequences. The method is high in sensitivity; a repetitive sequence can be found in an incompletely assembled motif; obtained species-specific consensus sequences are accurate, and the subspecies level can be identified; the conservative property of identified consensus sequences is high, and the maximum value of the plant seed coverage is achieved as much as possible with the least consensus sequences; and all logic modules have multiple verifications, so that the accuracy is high.

Description

technical field [0001] The invention relates to the field of bioinformatics, in particular to a method, device and application for obtaining a species-specific consensus sequence of microorganisms. Background technique [0002] Because the DNA concentration of pathogenic microorganisms in biological samples is mostly very low, close to the detection limit. However, when using traditional PCR or real-time PCR detection, the detection sensitivity is often lacking. Other methods such as two-step nested PCR can be used to improve sensitivity, but the method is time-consuming, costly and has poor accuracy. Therefore, it is crucial to improve the detection sensitivity. One way is to find a suitable template region when designing primers, usually plasmids and 16S rRNA are used. [0003] However, the choice of plasmids for primer design creates some problems: not all microorganisms contain species-specific plasmids, and some microorganisms do not have plasmids. First, the specie...

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Problems solved by technology

[0003] However, the choice of plasmids for primer design creates some problems: not all microorganisms contain species-specific plasmids, and some microorganisms do not have plasmids

First, the species-specificity of plasmid DNA is uncertain, and the sequences on the plasmids of some species are highly similar to the sequences on the plasmids of other species, so plasmid-based PCR detection will have a high risk of false positive or false negative results, and many clinical laboratories still Additional PCR primer pairs are required for confirmatory experiments

Secondly, plasmids are not universal. Some species do not have plasmids, so plasmids cannot be used to detect this species, let alone design primers on plasmids to improve detection sensitivity.

For example, it has been reported that approximately 5% of N. gonorrhoeae strains were undetectable due to lack of plasmid

[0004] Similarly, there are some problems in choosing the rRNA gene region as the template for detecting PCR: although the rRNA gene exists in the genome of all microbial species, and often has multiple copies, it can improve the detection sensitivity

In addition, some rRNA gene sequence changes are not suitable for detection

For example, among closely related species or even between strains of different subtypes of the same species, rRNA genes cannot meet the requirements of species-specificity or even subspecies-specificity because their sequences are too conserved

[0005] On the other hand, if an outbreak of an epidemic is caused by a microorganism with an unknown sequence, the database of pathogenic microorganisms will continue to update the data, which may cause the original probe primer design to fail to cover the pathogenic microorganisms of the epidemic, thereby affecting the quality of nucleic acid detection reagents

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