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Multi-detection method for drug-resistant sites of neisseria gonorrhoeae

A technology for multiple detection of Neisseria gonorrhoeae, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of inapplicability of large-scale sample monitoring and restrictions on the wide application of WGS

Pending Publication Date: 2020-07-10
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the cost and technology limit the wide application of WGS in some areas with limited resources, and it is also not suitable for the monitoring of large-scale samples.

Method used

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  • Multi-detection method for drug-resistant sites of neisseria gonorrhoeae
  • Multi-detection method for drug-resistant sites of neisseria gonorrhoeae
  • Multi-detection method for drug-resistant sites of neisseria gonorrhoeae

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Embodiment 1

[0101] Embodiment 1, detection method

[0102] In the method for highly sensitive detection and / or identification of Neisseria gonorrhoeae drug-resistant loci provided by the embodiments of the present invention, the 19 Neisseria gonorrhoeae drug-resistant loci to be detected include: 16S rRNA C1192, 23S rRNA C2611T, 23S rRNAA2059G, gyrA D95 / A, gyrA S91F, parC D86N, parC S88, mtrR deletion-A, mtrR G45D, penAD345-insertion, penA G542S, penA G545S, penA A501T / V, penA P551S / L, ponA L421P, porB A121(DN) / G, porB G120D / (KNR), rpsE T24P and penA A311V.

[0103] Implementation of the present invention comprises the steps:

[0104] 1) Primer design: First, download the drug-resistant gene sequence of each Neisseria gonorrhoeae that has been fully annotated as a reference sequence from the GenBank database (https: / / www.ncbi.nlm.nih.gov / genbank / ). Nucleic acid sequence BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) was performed on the sequence and NCBI's nr database, and the result...

Embodiment 2

[0125] Embodiment 2, test kit

[0126] The application of the kit provided by the invention for detecting 19 drug-resistant sites of Neisseria gonorrhoeae comprises the following steps:

[0127] 1. Nucleic acid extraction: Use the nucleic acid extraction kit to extract the secretion or urine sample from the test.

[0128] 2. Multiplex PCR reaction: Cooperate with the special amplification reaction system, after a round of multiplex PCR, realize the gene amplification of multiple items. First react at 45°C for 2 minutes. The purpose of this step is to exert the action of UNG enzyme to digest dUTP, and then inactivate UNG at 94°C for 4 minutes (this step also achieves the effect of pre-denaturation), and then start multiplex PCR amplification. The reaction conditions were denaturation at 95°C for 30 seconds, annealing at 56.5°C for 30 seconds, and extension at 72°C for 1 minute. A total of 45 cycles were performed; the final extension was at 72°C for 5 minutes, and stored at 4°...

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Abstract

The invention provides a method for detecting mutation of drug-resistant sites of 19 neisseria gonorrhoeae. The method utilizes a multiplex PCR-mass spectrometry method to detect the mutation of the drug-resistant sites of the 19 neisseria gonorrhoeae. The detection method comprises the following steps: 1) designing primers for different drug-resistant sites; 2) carrying out multiplex PCR amplification reaction; 3) treating shrimp alkaline phosphatase; 4) carrying out single base extension reaction; 5) desalting and purifying resin; and 6) carrying out mass spectrometric detection.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, relates to a method for detecting multiple drug-resistant sites, in particular to a method for detecting drug-resistant sites of Neisseria gonorrhoeae by multiplex PCR-mass spectrometry. Background technique [0002] Neisseria gonorrhoeae (Neisseria gonorrhoeae, NG) is a sexually transmitted pathogen that seriously affects public health, with a global new infection rate as high as 87 million cases. High morbidity not only increases the global health economic cost, but also leads to serious drug resistance. Accompanied by the rapid development and spread of drug resistance of gonorrhea, antibiotics (sulfonamides, penicillins, early cephalosporins, tetracyclines, macrolides, and quinolones) that were widely recommended in the past have been withdrawn from the recommended first-line treatment for gonorrhea. At present, the first-line empirical drug recommended by the World Healt...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/689C12Q1/04C12N15/11
CPCC12Q1/6858C12Q1/689C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2565/627C12Q2521/525C12Q2533/101
Inventor 彭俊平郭军华李雅梅
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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