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

A pathogenic microorganism and multiple amplification technology, which is applied in the field of multiple amplification of pathogenic microorganism identification combined with high-throughput sequencing methods and kits, can solve the problems of cumbersome operation, increase of patient medical expenses, cumbersomeness, etc., and improve the efficiency of library construction , shorten the detection cycle, and simplify the operation process

Inactive Publication Date: 2021-06-25
GUANGZHOU SAGENE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

Method used

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

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Experimental program
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Effect test

Embodiment 1

[0070] Embodiment 1 The flow process of pathogenic microorganism identification of the present invention

[0071] Method of the present invention specifically comprises the steps:

[0072] 1. Sample processing:

[0073] (1) Collection and transportation: collect clinical samples from patients, and adopt different collection and transportation methods according to different sample types.

[0074] (2) Pretreatment: According to the requirements of the sample type, pre-extraction treatment is carried out. If it is a sample of alveolar lavage fluid, it needs to be treated with wall breaking, and if it is a blood sample, it needs to be treated with plasma separation.

[0075] (3) Nucleic acid extraction: Use the DNA and RNA co-extraction kit developed by Sage (Yuesui Machinery Equipment 20200771), and extract DNA / RNA according to the instructions. The concentration and purity of the extracted nucleic acid were tested using nanodrop2000 / qubit3.0.

[0076] 2. Before amplification:...

Embodiment 2

[0100] Example 2 Multiple Amplification Reference Strain Research

[0101] 1. Pathogen composition: According to the previously accumulated data and some published literature or research reports, the present invention selects 20 common pathogenic microorganisms of respiratory tract and blood infection as research objects, and the types are shown in the following table:

[0102]

[0103] 2. Nucleic acid preparation of reference strains: 20 standard reference strains of pathogenic microorganisms were purchased from the Guangdong Provincial Culture Collection Center, and the corresponding strains were inoculated on corresponding petri dishes in an ultra-clean bench, and cultured at their respective suitable temperatures for 12-24 h . Pick a single colony, inoculate in 5 ml liquid medium, and culture overnight. Take 1ml of bacterial liquid and use QIAamp DNA MicrobiomeKit to extract nucleic acid according to the instructions. The extracted nucleic acid is tested for concentrat...

Embodiment 3

[0129] Embodiment 3 multiple amplification clinical sample research

[0130] In order to illustrate that the sensitivity of the method of the present invention is higher than that of traditional clinical methods, the following comparative experiments were carried out.

[0131] 1. Clinical sample collection:

[0132] The alveolar lavage fluid (BALF) and blood samples of 10 critically ill patients were collected from the ICU of the cooperative hospital. The patients were between 35 and 80 years old, 10 males and 10 males, and the SOFA score was between 1 and 13 points.

[0133] 2. Sample processing:

[0134] All 20 samples were identified using traditional clinical methodologies: BALF and blood culture submission, antibody testing and qPCR testing. Another sample of the same patient carries out the method pre-treatment of the present invention:

[0135] 1) Alveolar lavage fluid: Take 0.5 ml of sample and first pass through a shaker + glass beads to break the wall for 30 minut...

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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...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6895C12Q1/686C12Q1/6869
CPCC12Q1/686C12Q1/6869C12Q1/689C12Q1/6895C12Q1/701C12Q2537/143C12Q2535/122
Inventor 林东旭朱方何袁光孝李新慧张陈陈陈杰
Owner GUANGZHOU SAGENE BIOTECH
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