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Method and kit for multiple detection of drug resistance sites of neisseria gonorrhoeae

A technology of Neisseria gonorrhoeae and multiple detection, applied in the field of multiple detection of Neisseria gonorrhoeae drug-resistant loci and its kit, can solve the problems of increased cost, expensive fluorescently labeled primers or probes, etc.

Pending Publication Date: 2020-01-03
USTAR BIOTECHNOLOGIES (HANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods require the use of expensive fluorescently labeled primers or probes, which greatly increases the cost of these methods in application

Method used

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  • Method and kit for multiple detection of drug resistance sites of neisseria gonorrhoeae
  • Method and kit for multiple detection of drug resistance sites of neisseria gonorrhoeae
  • Method and kit for multiple detection of drug resistance sites of neisseria gonorrhoeae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1, the establishment of multiple detection Neisseria gonorrhoeae drug-resistant loci method

[0065] 1. Select 48 isolates of Neisseria gonorrhoeae with clear information on drug resistance sites, and establish multiple detection methods;

[0066] 2. Use commercial kit method to extract strain genomic DNA;

[0067] 3. Using the genomic DNA of Neisseria gonorrhoeae isolates with clear drug-resistant site information as a template, prepare an HRM amplification reaction system under the guidance of the above-mentioned special primer set. The system is 20 μl, including: 10 μL of EvaGreen Master Mix, Assay The optimum amplification concentration of each primer in (Table 1), sample genomic DNA 2μl, ddH 2 O to make up to 20 μl.

[0068] 4. Perform amplification reaction and HRM analysis on QuantStudio 6 Flex Real-Time PCR System. The conditions of the amplification reaction are: incubate at 95°C for 10 minutes, then anneal at 95°C for 15 seconds, and extend at 60...

Embodiment 2

[0075] Example 2. Comparison of the method for multiple detection of Neisseria gonorrhoeae drug-resistant loci and whole genome sequencing

[0076] 1. Select 218 isolates of Neisseria gonorrhoeae whose whole genome information was obtained by whole genome sequencing, and compare multiple detection methods;

[0077] 2. Using the genomic DNA of 218 Neisseria gonorrhoeae isolates as a template, prepare the HRM amplification reaction system under the guidance of the above-mentioned special primer set. Amplification concentration (Table 1), sample genomic DNA 2μl, ddH 2 O to make up to 20 μl.

[0078]3. Perform amplification reaction and HRM analysis on QuantStudio 6 Flex Real-Time PCR System. The conditions of the amplification reaction are: incubate at 95°C for 10 minutes, then anneal at 95°C for 15 seconds, and extend at 60°C for 1 minute. Slowly heat up at a rate of 95°C, and collect fluorescence signals continuously;

[0079] 4. The results showed that the multiple detecti...

Embodiment 3

[0084] Example 3, Application of multiple detection kit for Neisseria gonorrhoeae drug resistance site

[0085] 1) Use the sample collection tube provided by the kit of the present invention to collect patient secretions or urine samples.

[0086] 2) For urine samples, centrifuge at 8,000rpm for 10 minutes, discard the supernatant, and add an appropriate amount of crude extraction reagent Lysis buffer to the sample collection tube; for swab samples of secretions, add an appropriate amount of crude extraction reagent Lysis buffer to the sample collection tube, Stir and soak the swab in Lysis buffer for 5 minutes.

[0087] 3) Place the sample collection tube in a metal bath or water bath, heat at 95°C for 10 minutes, and let stand at room temperature to complete the extraction of genomic DNA from the sample. (The above steps can be used to complete the extraction of genomic DNA with other nucleic acid extraction kits or methods)

[0088] 4) Using the sample genomic DNA obtaine...

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Abstract

The invention belongs to the technical field of molecular biology detection, and relates to a detection method for drug resistance sites, in particular to a method and a kit for multiple detection ofthe drug resistance sites of neisseria gonorrhoeae. A provided specific primer for the multiple detection of the drug resistance sites of the neisseria gonorrhoeae selects drug-resistance-related genes of two drug combinations (ceftriaxone and azithromycin) as target genes for detection, and includes penA, ponA, porB, mtrR, and 23S rRNA. Based on high resolution melting curve analysis technique, the DNA demulsification process is monitored in real time through high resolution melting of PCR products, the mutation condition of the genes is analyzed according to the characteristic change of a melting curve, and thus a basis is provided for determining the drug-resistant condition of the neisseria gonorrhoeae.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, relates to a method for detecting drug-resistant sites, in particular to a method for multiple detection of drug-resistant sites of Neisseria gonorrhoeae and a kit thereof. Background technique [0002] Gonorrhea is the second most common bacterial sexually transmitted infection (Sexually transmitted infection, STI) in the world caused by Neisseria gonorrhoeae (NG), with an annual incidence of 87 million new cases worldwide. At present, there is no effective vaccine against Neisseria gonorrhoeae, and effective antibacterial therapy is still the main means of treatment and control of gonorrhea. Extended-spectrum cephalosporins (ESCs) are considered to be the last option for empirical treatment of gonorrhea. With the wide application of cephalosporin antibiotics, the drug resistance of Neisseria gonorrhoeae to cephalosporins also began to appear slowly, and gonorrhea gonorrhea ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6827C12Q1/04C12N15/11C12R1/36
CPCC12Q1/689C12Q1/6827C12Q2600/16C12Q2600/106C12Q2549/119C12Q2527/107
Inventor 彭俊平修乐山
Owner USTAR BIOTECHNOLOGIES (HANGZHOU) CO LTD
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