Construction method of sequencing library for pathogenic microorganism detection

Abstract

The invention discloses a construction method of a sequencing library for pathogenic microorganism detection. The method comprises the following steps: S1, extracting DNA and RNA from a sample, and carrying out reverse transcription on tbe RNA; S2, adding a double-strand synthesis reaction solution and a double-strand synthetase into step S1 to make RNA / cDNA composite double strands form double-strand cDNA; S3, purifying; S4, adding a DNA interruption repair reaction solution and a DNA interruption repair enzyme mixture into the purified product obtained in step S3, and performing fragmentation, terminal repair and A tail addition on the product at the same time; S5, directly adding a linker, a linker reaction liquid and ligase into the fragmented solution without purifying, and performinglinker connection to obtain a linker connection product; S6, purifying; S7, carrying out library amplification; and S8, purifying the amplification product in step S7 to obtain the original DNA / RNA sequencable library. The method is short in time; and when the RNA is subjected to one-strand synthesis and two-strand synthesis, only RNA is acted, DNA existing in a system is not interfered, and after RNA is converted into double-strand cDNA, RNA and double-strand DNA are fragmented together, so that the effect of co-building the library in one process is truly achieved.

Description

technical field [0001] The invention belongs to the technical field of high-throughput sequencing, and in particular relates to a method for constructing a sequencing library for pathogenic microorganism detection. Background technique [0002] Infectious diseases are mostly local or systemic inflammation or organ dysfunction caused by pathogenic microorganisms such as bacteria, viruses and fungi and their products, which are relatively harmful and have a high mortality rate. At present, the incidence of infectious diseases in the world has increased, and pathogenic microorganisms are showing a trend of diversification and complexity. Severe Acute Respiratory Syndrome (SARS), Novel Coronavirus Pneumonia (NCP), New Mutant Creutzfeldt-Jakob Disease, H7N9 Avian Influenza and other new infectious diseases continue to emerge. The pathogenic microorganisms of classic infectious diseases such as HIV, multi or broad-spectrum drug-resistant Mycobacterium tuberculosis, and Mycoplasma...

AI Technical Summary

Problems solved by technology

Since infection samples often come from complex samples such as pleural effusion, alveolar lavage fluid, cerebrospinal fluid, and blood, the quality of the extracted nucleic acid is not high, and the microbial content is low, which often fails to meet the requirements for library construction.

Moreover, DNA and RNA are operated in two separate processes, and there are many processes and purifications, resulting in a large loss of samples.

[0007] In the patent CN202010111731.9, the Tn5 transposase method is used for the DNA / RNA co-construction library scheme. The Tn5 method needs to re-prepare the transposase complex for different sequencing platforms, and needs to re-test the conditions, and has specific requirements for the starting amount. requirements, and the Tn5 transposase method has a great preference, and the interruption efficiency is also low, especially for cDNA. These are often criticized by the industry. According to statistics, Tn5 has 9 sites on both sides of the insertion site. There is a clear preference for the left and right bases

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