Pathogen molecule detection method based on nanopore sequencing

Abstract

The invention belongs to the technical field of biology, and discloses a pathogen molecule detection method based on nanopore sequencing. The pathogen molecule detection method based on nanopore sequencing comprises the following steps: obtaining a throat swab sample or a nasopharyngeal swab sample; carrying out nucleic acid extraction and purification on the obtained throat swab sample or nasopharyngeal swab sample; preparing cDNA (complementary deoxyribonucleic acid); amplifying and purifying the cDNA; preparing a nanopore library and carrying out sequencing; and carrying out bioinformatics analysis to obtain a pathogen molecule detection result. According to the invention, pathogenic molecule detection can be directly carried out on the basis of the clinical samples, the sequencing speed is high, and real-time monitoring can be carried out; and meanwhile, the sensitivity is high and the detection limit is low. According to the invention, with the addition of a universal joint and the one-step PCR amplification during the reverse transcription construction of the nanopore library, the unbiased amplification of all the sequences in the clinical samples can be achieved, so that the pathogenic molecule composition and the abundance in the samples are truly reflected, and more importantly, the detection sensitivity and the detection accuracy are substantially improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for detecting pathogenic molecules based on nanopore sequencing. Background technique [0002] Present: Influenza is a major global public health threat because of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Genomic virus sequencing offers the potential for diagnostic testing of influenza viruses and provides insights into transmission, evolution, and drug resistance while detecting other viruses. [0003] Most existing clinical diagnostic tests for influenza virus rely on the detection of viral antigen or PCR amplification of respiratory viral nucleic acid; antigen tests are usually rapid but less sensitive; and PCR is more time-consuming but more sensitive. But regardless of test results, most clinical diagnostic devices report non-quantitative (binary) diagnostic results, and data routinely generated for influenza diagnosis have limi...

AI Technical Summary

Problems solved by technology

[0003] Most existing clinical diagnostic tests for influenza viruses rely on the detection of viral antigens or PCR amplification of respiratory viral nucleic acid; antigen tests are usually rapid but less sensitive; and PCR is more time-consuming but more sensitive

But regardless of test results, most clinical diagnostic devices report non-quantitative (binary) diagnostic results, and data routinely generated for influenza diagnosis have limited ability to provide insights into epidemiological links, vaccine efficacy, or antiviral drug susceptibility

[0004] Through the above analysis, the problems and defects of the existing technology are: the existing pathogenic molecular detection method has low sensitivity and long detection time, and at the same time, the data used for influenza diagnosis can provide epidemiological links, vaccine efficacy or antiviral drug sensitivity. Sexual insights are limited

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