Rapid pathogenic microorganism detection method and kit thereof

Abstract

The invention discloses a rapid pathogenic microorganism detection method adaptive to a Nanopore sequencing platform and a kit thereof. Detection of pathogenic microorganisms can be completed within 4-6 hours. According to the invention, a specific primer is designed for a detection target, and the enrichment of nucleic acid is realized through two rounds of PCR amplification; a universal sequence is added to the 5'end of a specific primer, a first-round PCR product does not need to be purified and is directly used for second-round amplification, phosphorylation is added to the tail end of a primer with a universal tag sequence during second-round amplification, the generated amplification product can be directly subjected to TA connection of a sequencing joint, the library building step is greatly simplified, and the experiment time is shortened. According to the method, the problem of human genome DNA pollution in pathogenic microorganism detection is solved, the whole fragment can be directly detected without splicing amplified long fragment data by using a Nanopore sequencing platform with a long read length, and the average length of data sequenced by the method is 1.6 k and is increased by more than 10 times compared with that of a second-generation platform; the accuracy and the stability of pathogenic microorganism judgment can be greatly improved.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a rapid detection method for pathogenic microorganisms and a kit thereof. Background technique [0002] Microorganisms that are pathogenic to humans and animals are called pathogenic microorganisms, also known as pathogens, including viruses, bacteria, rickettsia, mycoplasma, chlamydia, spirochetes, fungi, and actinomycetes. These pathogenic microorganisms can cause infections, allergies, tumors, dementia and other diseases, and are also one of the main factors that endanger food safety. Diseases such as SARS, highly pathogenic avian influenza, and West Nile virus infection that have emerged in recent years are highly contagious and often cause worldwide pandemics. Therefore, the detection of pathogens must be fast and accurate. Conventional etiological detection methods are cumbersome to operate, the detection cycle is long, and the requirements for the technical l...

AI Technical Summary

Problems solved by technology

[0006] For the detection of pathogenic microorganisms, the current general method is to use the second-generation sequencing platform for metagenomic sequencing. This platform has long sequencing time, short sequencing read length, large amount of human genome DNA contamination in the sequencing results, high sequencing data volume and poor sequencing results. more difficult questions

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