Amplifying and sequencing primer for subtyping and drug resistance detection of helicobacter pylori, and reagent kit

A technology of Helicobacter pylori and sequencing primers, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., which can solve the problems of multiple mutation types and incomplete detection of Helicobacter pylori, and avoid Complicated and difficult, reducing manpower and material resources, and the effect of multiple experimental information

Pending Publication Date: 2020-07-28

THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV

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AI-Extracted Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the defects of many types of Helicobacter pylori mutations and incomplete detection in the existing molecular diagnostic technology, and provide a set of optimized primers for PC...

Abstract

The invention discloses an amplifying and sequencing primer for subtyping and drug resistance detection of helicobacter pylori, and a reagent kit. The reagent kit comprises reaction liquid A for PCR amplification, a reaction tube for coating primer pairs, and primers H1, H2, H3, H4, H5, H6, H7, H8, H9, H10 and H11 for sequencing. The amplifying and sequencing primer for subtyping and drug resistance detection of helicobacter pylori, and the reagent kit disclosed by the invention can amplify all 11 objective sequences of the helicobacter pylori under the same PCR reaction condition at one time,and then the sequencing primers are used for performing sequencing according to a set procedure. A PCR reaction system has fluorescent dye, an amplification result can be judged directly through theelevation position of a fluorescent value, and inconvenience and pollution caused by electrophoresis can be avoided. The primers are directly coated in the reaction tube, so that tedious configurationduring primer use can be avoided. The reagent kit can be used for detecting virulence subtypes and drug resistance genes of the helicobacter pylori. The amplifying and sequencing primer and the reagent kit disclosed by the invention can be used for detecting clinical biopsy specimens and paraffin-embedded specimens and can provide bacterium subtypes and drug resistance information for helicobacter pylori treatment.

Application Domain

Microbiological testing/measurementDNA/RNA fragmentation

Technology Topic

FluorescenceVirulence +10

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Examples

Experimental program(5)

Example Embodiment

[0062] Example 1

[0063] A set of specific primers for the amplification and sequencing of Helicobacter pylori, including UreA amplification and sequencing primers are SEQ ID NO. 1 and SEQ ID NO. 2, and 16S rRNA amplification and sequencing primers are SEQ ID NO. 3 and SEQ ID NO. 4, rdxA amplification and sequencing primers are SEQ ID NO. 5 and SEQ ID NO. 6, 23S rRNA amplification and sequencing primers are SEQ ID NO. 7 and SEQ ID NO. 8, gyrA The amplification and sequencing primers are SEQ ID NO. 9 and SEQ ID NO. 10, the amplification and sequencing primers of PBP1 are SEQ ID NO. 11 and SEQ ID NO. 12, and the amplification and sequencing primer of cagA is SEQ ID NO. 13 And SEQ ID NO.14, the amplification and sequencing primers of vacAs are SEQ ID NO.15 and SEQ ID NO.16, and the amplification and sequencing primers of vacAm are SEQ ID NO.17 and SEQ ID NO.18, the amplification of vacAi The amplification and sequencing primers are SEQ ID NO. 19 and SEQ ID NO. 20, and the amplification and sequencing primers of vacAii are SEQ ID NO. 21 and SEQ ID NO. 22.

Example Embodiment

[0064] Example 2

[0065] A kit for the amplification and sequencing of Helicobacter pylori, consisting of the following reagents:

[0066] Reagent A: Consists of 2mL buffer solution, including Green I dye, 0.2mM dNTPs (including dATP, dCTP, dGTP, dTTP), 80mM Tris-HCl, 80mMKCl, 40mM(NH4)2SO4, 6mM MgSO4, 100U TaqDNA polymerase;

[0067] Reagent H1: A 30μL aqueous solution of sequencing primers for UreA, containing 4μM SEQ ID NO.1;

[0068] Reagent H2: 30μL aqueous solution of sequencing primers for 16S rRNA, containing 4μM SEQ ID NO.3;

[0069] Reagent H3: 30μL aqueous solution of sequencing primers for rdxA, containing 4μM SEQ ID NO.5;

[0070] Reagent H4: a 30μL aqueous solution of sequencing primers for 23S rRNA, containing 4μM SEQ ID NO.7;

[0071] Reagent H5: 30μL aqueous solution of sequencing primers for gyrA, containing 4μM SEQ ID NO.9;

[0072] Reagent H6: 30μL aqueous solution of sequencing primers for PBP1, containing 4μM SEQ ID NO.11;

[0073] Reagent H7: 30μL aqueous solution of cagA sequencing primers, containing 4μM SEQ ID NO.13;

[0074] Reagent H8: 30μL aqueous solution of sequencing primers for vacAs, containing 4μM SEQ ID NO.15;

[0075] Reagent H9: 30μL aqueous solution of sequencing primers for vacAm, containing 4μM SEQ ID NO.17;

[0076] Reagent H10: 30μL aqueous solution of sequencing primers for vacAi, containing 4μM SEQ ID NO.19;

[0077] Reagent H11: 30μL aqueous solution of sequencing primers for vacAii, containing 4μM SEQ ID NO.21;

[0078] PCR reaction tube coated with primers:

[0079] Reaction tube No. 1: Contains a mixture of amplification primers for UreA, containing 1.5×10 -2 nmol SEQ IDNO.1 and 1.5×10 -2 nmol SEQ ID NO.2;

[0080] No. 2 reaction tube: contains a mix of amplification primers for 16S rRNA, containing 1.5×10 -2 nmol SEQ IDNO.3 and 1.5×10 -2 nmol SEQ ID NO.4;

[0081] No. 3 reaction tube: contains a mixture of amplification primers for rdxA, containing 1.5×10 -2 nmol SEQ IDNO.5 and 1.5×10 -2 nmol SEQ ID NO.6;

[0082] No. 4 reaction tube: contains a mixture of amplification primers for 23S rRNA, containing 1.5×10 -2 nmol SEQ IDNO.7 and 1.5×10 -2 nmol SEQ ID NO. 8;

[0083] No. 5 reaction tube: contains a mixture of amplification primers for gyrA, containing 1.5×10 -2 nmol SEQ IDNO.9 and 1.5×10 -2 nmol SEQ ID NO.10;

[0084] No. 6 reaction tube: contains a mixture of amplification primers targeting PBP1, containing 1.5×10 -2 nmol SEQ IDNO.11 and 1.5×10 -2 nmol SEQ ID NO. 12;

[0085] No. 7 reaction tube: contains a mixture of amplification primers for cagA, containing 1.5×10 -2 nmol SEQ IDNO.13 and 1.5×10 -2 nmol SEQ ID NO. 14;

[0086] No. 8 reaction tube: contains a mix of amplification primers for vacAs, containing 1.5×10 -2 nmol SEQ IDNO.15 and 1.5×10 -2 nmol SEQ ID NO. 16;

[0087] No. 9 reaction tube: contains a mix of amplification primers for vacAm, containing 1.5×10 -2 nmol SEQ IDNO.17 and 1.5×10 -2 nmol SEQ ID NO.18;

[0088] No. 10 reaction tube: contains a mixture of amplification primers for vacAi, containing 1.5×10 -2 nmol SEQ IDNO.19 and 1.5×10 -2 nmol SEQ ID NO.20;

[0089] No. 11 reaction tube: contains a mix of amplification primers for vacAii, containing 1.5×10 -2 nmol SEQ IDNO.21 and 1.5×10 -2 nmol SEQ ID NO.22.

Example Embodiment

[0090] Example 3

[0091] The kit of the present invention is used to perform real-time FQ PCR to amplify all the sequences of Helicobacter pylori. First, calculate the required amount of each reagent according to the number of specimens in the experiment. The experimental steps are as follows:

[0092] 1. Amplification reaction solution configuration: configure the reaction solution (1 test specimen) in order, add 120μL of water, 150μL of reagent A, and 30μL of DNA sample, total 300μL; mix well.

[0093] 2. Add 25μL of amplification reaction solution to each tube in sequence and close the lid.

[0094] 3. Amplify according to the PCR program designed in the experiment: after pre-denaturation at 95°C for 3 minutes, denaturation at 94°C for 1 minute, annealing at 60°C for 1 min, extension at 72°C for 35 seconds, the fluorescence value obtained during the extension period, 35 cycles, and finally stored at 25 ℃; Take it out in time for sequencing.

[0095] 4. The fluorescence value takes off in 22-28 cycles, and the simultaneous take-off of each group is considered as specific amplification.

[0096]

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Amplifying and sequencing primer for subtyping and drug resistance detection of helicobacter pylori, and reagent kit

AI-Extracted Technical Summary

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Abstract

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Example Embodiment

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Description & Claims & Application Information

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