Multi-amplification and high-throughput sequencing method and kit for identifying pathogenic microorganisms

Abstract

The invention discloses a multi-amplification and high-throughput sequencing method and kit for identifying pathogenic microorganisms, and the method comprises the following two steps: in the first-step amplification, an upstream long primer pool is used for single-end amplification; in the second-step amplification, a downstream long primer pool, a primer P1 and a primer P2 are added into a reaction system, the three primers form a primer for the second-step amplification, and then quality control, purification, high-throughput sequencing and data analysis are carried out. According to the method, the pathogenic microorganisms which cannot be identified in a traditional clinical method are detected in a human respiratory tract alveolar lavage fluid sample and a blood or plasma sample by using a two-step multiple amplification and next-generation sequencing method for the first time, the sensitivity is high, the detection limit is 50 pathogenic genome copies per milliliter, the method is rapid and simple, the sequencing data volume is small, and the detection cost is reduced.

Description

technical field [0001] The invention relates to the technical field of clinical detection of infectious diseases, in particular to a multiple amplification combined with high-throughput sequencing method and kit for identification of pathogenic microorganisms. Background technique [0002] There are many types of pathogenic microorganisms that can infect humans, most of which are viruses, bacteria, fungi, parasites, mycoplasma, chlamydia, etc., which widely exist in the human body. Infection through the respiratory tract is the most common way, and has a high case fatality rate. According to the 2016 statistics released by the World Health Organization (WHO) in 2018, the lower respiratory tract infection ranks fourth among the top ten causes of death in the world, and ranks first among infectious diseases, causing a total of about 3 million deaths. [0003] The screening of infectious pathogens mainly relies on the separation and culture method and the automated microbial i...

AI Technical Summary

Problems solved by technology

Although mNGS technology has many advantages, it also has several defects and flaws: 1. Low sensitivity. Due to the limitation of the sequencing method itself, the detection sensitivity of mNGS has been criticized. There will be missed detection of internal bacteria and RNA viruses

2. Host influence, mNGS is the detection of all nucleic acids in clinical samples, so a large number of human-derived host nucleic acids have a greater impact on the interference of the method, and it is necessary to use de-hosting technology to remove human-derived nucleic acids, but often removing the host will also affect the pathogen Nucleic acid has an impact, which ultimately affects the detection accuracy

3. The operation time is long and cumbersome. Before mNGS metagenomic sequencing, the samples need to be processed on the machine for sequencing, such as wall breaking, extraction, host removal, interruption, end repair, adapter connection, and multiple purifications. The TAT cycle is cumbersome to operate, and every time an experimental link is added, a part of the pathogenic nucleic acid will be lost, which will directly affect the detection rate.

4. The cost is high. mNGS requires a basic 20M reads of sequencing data to ensure the accuracy of the test results, so the sequencing cost accounts for a large proportion of the total cost, plus the cost of the multiple experimental links mentioned above

The high cost also directly leads to the high cost of testing services in the market, increases the medical expenses of patients, and also affects the large-scale popularization and use of technology

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