Method of detecting microorganisms and application thereof

A technology of microorganisms and detection probes, which is applied in the field of detection of microorganisms, can solve the problem that there is no non-amplification method to identify microorganisms, and achieve the effect of simple method, high efficiency and short time

Inactive Publication Date: 2018-09-11
INNER MONGOLIA HUAXING KANGWEI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the existing PCR and IA all need complex enzymatic reactions to increase the number of nucleic acids to achieve the purpose of detection and analysis. In this field, there is no record of using non-amplification methods to identify microorganisms.

Method used

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  • Method of detecting microorganisms and application thereof
  • Method of detecting microorganisms and application thereof
  • Method of detecting microorganisms and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1) Utilize the conserved sequence of Mycobacterium tuberculosis as shown in SEQ ID NO:1, design the specific detection probe of Mycobacterium tuberculosis, and carry out FTIC mark at the 5' end of specific detection probe sequence, the designed The specific detection probe is shown in SEQ ID NO: 2, and the FTIC label is carried out at the 5' end of the sequence: 5'FTIC-TACGGTGCCCGCAAAAGTGTGG3';

[0045] 2) prepare 10 respectively 2 、10 3 、10 4 、10 5 、10 6 copies / mL of Mycobacterium tuberculosis genomic DNA sample;

[0046] 3) Mix the specific detection probe, the random detection probe labeled with 5' end biotin, Buffer and genomic DNA, the final concentration of the specific detection probe is 0.02 μM, and the final concentration of the random detection probe is 10 μM;

[0047] 4) After mixing, heat to 95°C for 5 minutes, and cool to room temperature naturally;

[0048] 5) Drop all the mixture on the commercially purchased nucleic acid test strips, and read the r...

Embodiment 2

[0051] 1) Utilize the conserved sequence of Mycobacterium tuberculosis as shown in SEQ ID NO:1, design the specific detection probe of Mycobacterium tuberculosis, and carry out FTIC mark at the 5' end of specific detection probe sequence, the designed The specific detection probe is shown in SEQ ID NO:3, and the FTIC label is carried out at the 5' end of the sequence: 5'FTIC-GAGTGCTGGGCTGGAAGA3';

[0052] 2) prepare 10 respectively 3 copies / mL of Mycobacterium tuberculosis genomic DNA sample and 10 4 copies / mL of nontuberculous mycobacteria (NTM) genomic DNA;

[0053] 3) Mix the specific detection probe, the random detection probe labeled with 5' end biotin, Buffer and genomic DNA, the final concentration of the specific detection probe is 0.02 μM, and the final concentration of the random detection probe is 10 μM;

[0054] 4) After mixing, heat to 95°C for 5 minutes, and cool to room temperature naturally;

[0055] 5) Drop all the mixture on the commercially purchased nucl...

Embodiment 3

[0058] 1) Utilize the conserved sequence of Mycobacterium tuberculosis as shown in SEQ ID NO:1, design the specific detection probe of Mycobacterium tuberculosis, and carry out FTIC mark at the 5' end of specific detection probe, the specificity of design The detection probe sequence is shown in SEQ ID NO: 2, and the FTIC label is carried out at the 5' end of the sequence: 5'FTIC-TACGGTGCCCGCAAAAGTGTGG3';

[0059] 2) prepare 10 respectively 6 、10 5 、10 4 、10 3 、10 2 、10 1 copies / mL of Mycobacterium tuberculosis genomic DNA;

[0060] 3) Coating the specific capture probe on the surface of the microtiter plate or PCR tube;

[0061] 4) Mix Buffer and genomic DNA in a microtiter plate or PCR tube, heat to 95°C for 5 minutes, and cool to room temperature naturally;

[0062]5) Wash to remove unbound nucleic acid, add 200ul of commercially purchased DNA dye Accublue dye to the holes of the microplate or PCR tube respectively, incubate for 20 minutes, and detect in BIOTEC Syner...

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Abstract

The invention provides a method of detecting microorganisms and belongs to the technical field of quick diagnosis of nucleic acid. The method includes the steps of: 1) extracting nucleic acid from a sample to obtain a nucleic acid template; 2) mixing the nucleic acid template with a staining group and PCR buffer to obtain a mixed sample; 3) heating the mixed sample for degeneration or performing areaction with a non-specific nucleic acid dye to obtain a stained sample; 4) measuring color value and according to change of shade of the color, measuring the content of the nucleic acid and determining negative or positive of the microorganisms; herein, the staining group is a modified probe or a non-specific nucleic acid dye, wherein the probe includes a specific detection probe and a random detection probe; the non-specific nucleic acid dye includes: methyl blue, methyl green, xanthylium, SYBR Green, Ultrapure ethidium bromide, or Accublue fluorescein. The method is free of amplificationof an enzymatic reaction and can form a detection result within 5-10 min, is high in sensitivity and has good specificity.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid rapid diagnosis, and in particular relates to a method for detecting microorganisms and an application thereof. Background technique [0002] Since the discovery of polymerase chain reaction (PCR) and constant temperature amplification, researchers have devoted themselves to applying its powerful amplification principle to improve the sensitivity of its detection. In theory, it can detect a single copy of nucleic acid, making nucleic acid detection Technology has come a long way. After real-time fluorescent quantitative PCR detection, new technologies such as qPCR have emerged in an endless stream. Nucleic acid PCR / isothermal amplification nucleic acid detection has been widely used in the fields of medicine, biology, animals and plants, and environmental detection, and has achieved very good results. [0003] PCR is a molecular biology technique for amplifying and amplifying specific DNA f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6816C12Q1/6834C12Q1/689C12Q1/04C12R1/32C12R1/01
CPCC12Q1/6816C12Q1/6834C12Q1/689C12Q2563/107C12Q2563/173C12Q2563/131
Inventor 温永俊江明韩慧敏宣铁民
Owner INNER MONGOLIA HUAXING KANGWEI BIOTECH CO LTD
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