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Primer group and kit for rapidly identifying respiratory tract microorganisms based on nanopore sequencing and application of primer group

A nanopore sequencing and kit technology, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, recombinant DNA technology, etc., and can solve problems such as sequence analysis that cannot be pathogenic

Active Publication Date: 2021-03-16
GUANGZHOU DARUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The common point of the above patents is that the method of multiple fluorescent PCR or gene chip is used for detection, but the sequence of the pathogen cannot be analyzed. If the concentration of the pathogen is relatively low and close to the detection limit of the kit, the result at this time has certain uncertainty.

Method used

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  • Primer group and kit for rapidly identifying respiratory tract microorganisms based on nanopore sequencing and application of primer group
  • Primer group and kit for rapidly identifying respiratory tract microorganisms based on nanopore sequencing and application of primer group
  • Primer group and kit for rapidly identifying respiratory tract microorganisms based on nanopore sequencing and application of primer group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] The design of embodiment 1 respiratory tract microorganism detection primer

[0083] According to bibliographical reports and epidemiological trend, the present embodiment designs the antimicrobial agent for 14 kinds of respiratory microorganisms (Streptococcus pneumoniae, Staphylococcus aureus (methicillin resistance), Klebsiella pneumoniae, Pseudomonas aeruginosa, Baumannii Detection of Acinetobacter, Stenotrophomonas maltophilia, Candida albicans, Haemophilus influenzae, Legionella pneumophila, Enterococcus faecium, Chlamydia psittaci, Cryptococcus gattii, Aspergillus fumigatus, Pneumocystis jirovecii) primers.

[0084] The detection objects designed in this embodiment include common microorganisms such as bacteria, fungi, and viruses. The above-mentioned microorganisms were identified by designing different target regions, among which (1) bacteria: 4 pairs of primers were designed to amplify the V1-V3, V4-V5, V6-V7 and V8 regions of 16s rRNA respectively, and the s...

Embodiment 3

[0088] Embodiment 3 Respiratory tract microorganism detection kit and its application method

[0089] 1. Composition

[0090]The nucleotide sequence is as shown in the primer set of SEQ ID NO: 1-20, and the primer set is made into multiple PCR primer mixture, and the concentration of each primer is 400nM-1μM, and the concentration of each primer is shown in Table 3;

[0091] table 3:

[0092]

[0093]

[0094] Library construction reagents: multiplex PCR amplification enzyme, ligation reaction mixture, eluent, positive quality control, negative quality control and internal standard, wherein the positive quality control is the target amplified fragment containing the primer set Cloning plasmid, negative quality control product is human genomic DNA, internal standard is MS2 phage DNA, multiplex PCR amplification enzyme contains Taq enzyme (0.5U / μl) and dNTPs (0.8mM), ligation reaction mixture is DNA ligase (1U / μl) μl) and buffer mixture, the eluent is 1×TE;

[0095] Lin...

Embodiment 4

[0134] The detection of embodiment 4 respiratory tract microbial samples

[0135] Collection of Streptococcus pneumoniae, Staphylococcus aureus (methicillin resistant), Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Candida albicans, Haemophilus influenzae Bacillus, Legionella pneumophila, Enterococcus faecium, Chlamydia psittaci, Cryptococcus gattii, Aspergillus fumigatus, Pneumocystis jirovecii each 1 case; use the kit of Example 2 to detect.

[0136] The results are shown in Table 5, all of which were determined to be corresponding microorganisms.

[0137] table 5:

[0138] Numbering pathogen number of reads 1 Streptococcus pneumoniae (5) 2 Methicillin-resistant Staphylococcus (7) 3 Klebsiella pneumoniae (2) 4 Pseudomonas aeruginosa (1) 5 Acinetobacter baumannii (2) 6 Stenotrophomonas maltophilia (3) 7 Candida albicans (1) 8 Haemophilus influenzae (1...

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Abstract

The invention discloses a primer group for detecting respiratory tract microorganisms based on a nanopore sequencing method. The nucleotide sequences of the primer group are as shown in SEQ ID NO:1-20. The microorganisms are streptococcus pneumoniae, staphylococcus aureus (resisting to methicillin), klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter baumannii, stenotrophomonas maltophilia, candida albicans, haemophilus influenzae, legionella pneumophila, enterococcus faecium, chlamydia psittaci, cryptococcus gattii, aspergillus fumigatus and pneumocystis jiroveci. According to the invention, sequencing optimization is carried out on different types of samples of the respiratory tract microorganisms, so that the detection method, the kit and the like are suitable for various typesof respiratory tract samples; and a targeted amplification method is adopted, so that the detection requirements of sputum, alveolar lavage and other sample types can be met. In addition, parallel sequencing can be performed, so that the flux of the detected samples is increased, the sequencing time is shortened, and the contradiction between the flux of detected pathogenic species and the cost and time is further relieved.

Description

technical field [0001] The present invention relates to the technical field of detection of respiratory tract microorganisms, and more specifically, to a primer set, kit and application thereof for rapid identification of respiratory tract microorganisms based on nanopore sequencing. Background technique [0002] The disease caused by pathogenic microorganisms invading the respiratory tract and multiplying is called respiratory tract infection. There are many kinds of pathogenic microorganisms in respiratory tract infection, the symptoms of infection are similar, it is difficult to distinguish clinically, and co-infection of multiple pathogenic microorganisms often occurs. Since precise diagnosis and treatment urgently require the right medicine, the identification of multiple pathogenic microorganisms is required. [0003] The most direct way to identify the type of pathogenic microorganism is to detect the nucleic acid of the pathogenic microorganism. At present, the com...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12Q1/6895C12Q1/6893C12Q1/689C12Q1/14C12Q1/10C12Q1/04C12N15/11
CPCC12Q1/6869C12Q1/6895C12Q1/6893C12Q1/689C12Q2600/16C12Q2565/631C12Q2531/113C12Q2537/143C12Q2525/191Y02A50/30
Inventor 周其伟刘欢杨学习李明
Owner GUANGZHOU DARUI BIOTECH
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