Method, device and application for identifying multi-copy regions in microbial target fragments

Abstract

The present invention provides a method for identifying a multi-copy region in a microbial target segment, which at least includes the following steps: S100: Find a candidate multi-copy region: perform an internal comparison on the microbial target segment, and search for a sequence corresponding to a sequence whose similarity satisfies a preset value The region of the candidate multi-copy region is used as a candidate multi-copy region, and the similarity refers to the product of the coverage rate and the matching rate of the sequence to be tested; S200: Verify that the multi-copy region is obtained: obtain the median value of the copy number of the candidate multi-copy region; if the candidate multi-copy region When the median value of the copy number is greater than 1, it is recorded as a multi-copy region. Compared with the literature database, the identification method of the multi-copy region in the microbial target fragment of the present invention has high accuracy and high sensitivity, and can identify the undiscovered multi-copy region; it can search for repetitive sequences in motifs with incomplete assembly; compared with 16srRNA More comprehensive than that, not all 16srRNA has multiple copies.

Description

technical field [0001] The invention relates to the field of bioinformatics, in particular to a method, device and application for identifying a multi-copy region in a microbial target segment. 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 species-sp...

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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

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