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Primer combination and application for nanopore sequencing library construction of a respiratory pathogen

A technology of nanopore sequencing and primer combination, which is applied in the field of detection of respiratory pathogens, can solve the problems of long circulation period, cumbersome operation process, and many operation steps, and achieve the effect of accurate identification and identification, cumbersome operation process and simple operation steps

Active Publication Date: 2021-10-26
GUANGZHOU DARUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods can only detect one or a limited number of pathogens each time, the operation process is cumbersome, the detection time is long, and there are also disadvantages of low positive rate in culture.
[0004] Existing next-generation sequencing technology can be used to identify pathogenic microorganisms of unknown origin, such as Chinese patent CN111394486A "Pathogen Detection and Identification Method for Infectious Diseases in Children Based on Metagenome Sequencing". The difficulty of the sequencer; the investment in the sequencer is large, and the data analysis can only be performed on high-performance computing equipment after the sequencing is completed; there are many operation steps, and the cycle from sample to result is long, which is not conducive to the rapid and accurate identification of pathogens
[0005] Chinese patent CN111041130A composition, kit and method for detecting pathogens, using single-channel multiplex real-time fluorescent PCR technology to detect multiple pathogens, but only for specific 15 kinds of pathogens, but unable to detect unknown pathogens

Method used

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  • Primer combination and application for nanopore sequencing library construction of a respiratory pathogen
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  • Primer combination and application for nanopore sequencing library construction of a respiratory pathogen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 A primer combination for library construction based on nanopore sequencing

[0051] Including sequence (1) to sequence (5), each sequence is composed of segment a sequence, segment b sequence and segment c sequence from the 5' end to the 3' end;

[0052] Wherein the sequence (1) to sequence (5) is consistent with the b-section sequence, the nucleotides of the sequence are as shown in any one of SEQ ID NO: 1-108;

[0053] The nucleotides of sequence (1) to segment c of sequence (5) are shown in sequence as SEQ ID NO: 109-113;

[0054] The sequence nucleotides of sequence (1), sequence (3) and sequence (4) in sequence (a) are shown in SEQ ID NO: 114, and the sequence nucleotides in sequence (2) and sequence (5) in sequence (a) are as shown in SEQ ID NO : 115.

[0055] The sequence is detailed in Table 1:

[0056] Table 1

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]According to the above rules, there will be a total of 108 different pr...

Embodiment 2

[0107] Example 2 A method for building a library based on nanopore sequencing

[0108] Operation process such as figure 1 As shown, the extracted and purified nucleic acid is a library after two steps of PCR amplification, product purification, and sequencing adapter connection, which can be directly used on the machine for real-time data analysis. The specific experimental steps are as follows:

[0109] 1. DNA extraction and purification

[0110] The nucleic acid extraction kit (DP710-T6B) of Tiangen Biochemical Technology Co., Ltd. was used for extraction and purification.

[0111] (1) Lysis and digestion of alveolar lavage fluid samples

[0112] Samples Take 500 μl of the sample and place it in a 2ml EP tube, add 500 μl of lysate GHH-A and 50 μl of Proteinase K respectively, vortex and shake to mix, and bathe in 65°C water bath for 20 minutes.

[0113] (2) Glass bead mill

[0114] 1) Take 1000 μl of the lysed and digested sample and place it on a bead mill for grinding ...

Embodiment 3

[0174] Example 3 Nanopore sequencing of alveolar lavage fluid samples

[0175] 1. Experimental method

[0176] Using the method in Example 2, the nanopore sequencing of 24 cases of alveolar lavage fluid samples, the samples were numbered 1-24, and the samples numbered 1-12 used the primer combination 1 in Example 1 for the first multiplex PCR Amplification, samples numbered 13-24 were amplified using primer combination 2 in Example 1 for the first multiplex PCR amplification.

[0177] During the second PCR amplification, samples numbered 1-12 were added to BP1-BP12 in turn, and samples numbered 13-24 were added to BP1-BP12 in turn.

[0178] 2. Experimental results

[0179] The causative pathogens detected in each sample are shown in Table 2.

[0180] Table 2:

[0181]

[0182]

[0183] The fragment distribution of nanopore sequencing reads is as follows figure 2 As shown, the peaks at 600bp and 1500bp correspond to the amplified bands of fungal ITS and bacterial 16...

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Abstract

The invention discloses a combination of primers for constructing a library by nanopore sequencing of a respiratory pathogen and an application thereof. Utilize the invented primer combination for library construction for nanopore sequencing library construction of respiratory pathogens. This method only needs 2 steps of PCR amplification and adapter connection to achieve the purpose of library construction. In the second step of PCR amplification, only It needs to supplement primers without adding enzymes, and the operation steps are simple; it has the characteristics of rapid library construction, and then can be sequenced and analyzed in real time; simultaneously detects bacterial metagenomes and fungal metagenomes in samples.

Description

technical field [0001] The invention relates to the technical field of detection of respiratory pathogens, and more specifically, to a primer combination for library construction based on nanopore sequencing and its application. Background technique [0002] Respiratory tract infection is a common clinical disease and one of the most serious diseases that endanger public health. Respiratory tract infections can be caused by various pathogens such as bacteria and fungi. Pathogen detection is the basis for precise treatment of respiratory tract infections and a prerequisite for effective disease prevention and control. [0003] Traditional pathogen detection methods include smear microscopy, immunological detection, microbial culture, and molecular detection. However, these methods can only detect one or a limited number of pathogens each time, the operation process is cumbersome, the detection time is long, and there are also disadvantages of low positive rate in culture. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12Q1/6869C40B50/06C12Q1/689C12Q1/6895C12Q1/04C12N15/11
CPCC12Q1/6806C12Q1/6869C12Q1/689C12Q1/6895C12Q2600/16C40B50/06C12Q2531/113C12Q2537/143C12Q2525/191C12Q2563/185C12Q2535/122C12Q2565/631
Inventor 吴英松陆才瑞杨学习李明李贻林欧阳国军韩博炜
Owner GUANGZHOU DARUI BIOTECH
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