Method for identifying mycobacterium tuberculosis

A technology of Mycobacterium tuberculosis and Mycobacterium tuberculosis, which is applied in the fields of biochemical equipment and methods, material stimulation analysis, microbial measurement/testing, etc., can solve the problem of high cost and low accuracy of Mycobacterium tuberculosis, and can only identify tuberculosis disease and other problems

Active Publication Date: 2013-04-03
SHANGHAI PULMONARY HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, Mycobacterium tuberculosis can undergo mutations such as morphology, colony, virulence, immunogenicity, and drug resistance, and there are still many deficiencies in current medical technology, and there may still be some unconfirmed Mycobacterium tuberculosis. Therefore, therefore, Mycobacterium tuberculosis Judgment is of great significance to the early prevention of tuberculosis and the discovery of new species
[0004] The traditional identification method is smear microscopy. The specimen is directly smeared and stained with acid-fast. If acid-fast positive bacteria are found, it can be initially determined as Mycobacterium tuberculosis, but the sensitivity is not strong and the accuracy is not high; animal experiments determine The method of Mycobacterium tuberculosis is costly; the tuberculin skin test (PPD) and the T cell immune interferon gamma release method (IGRA) are both cell-based immunodiagnostic methods. Although the method has been widely used, it can only identify Tuberculosis disease, currently not used to identify Mycobacterium tuberculosis

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Step 1, design primers

[0051] A pair of homologous primers were designed with SEQ ID No.2 as the target as follows:

[0052] Forward primer: 5′-gatccccgatagcatcaaga-3′

[0053] Reverse primer: 5′-attggcaagacgaaaacgag-3′

[0054] Step 2, PCR amplification

[0055] Extract clinical strain NDA as template DNA, and use the homologous primers designed in step 1 for PCR amplification. The amplification system is as follows:

[0056] 10×Taq PCR buffer 2μL

[0057] 1U Taq DNA polymerase 1U

[0058] MgCl 2 2.5mM

[0059] dNTP solution 0.2mM

[0060] Forward primer 0.2μM

[0061] Reverse primer 0.2μM

[0062] Template DNA 10ng

[0063] wxya 2 O was added to a total volume of 20 μL

[0064] Amplification conditions: 94°C, 5min—(94°C, 1min—65°C, 30s—72°C, 1min)×35 cycles—72°C, 10min

[0065] Step 3, Gel Electrophoresis

[0066] The amplified product in step 3 was subjected to 2% gel electrophoresis and EB staining.

[0067] Both gel electro...

Embodiment 2

[0071] Step 1, design primers and molecular probes

[0072] Design a pair of homologous primers and molecular probes with SEQ ID No.2 as the target,

[0073] Forward primer: 5′-gatccccgatagcatcaaga-3′

[0074] Reverse primer: 5′-attggcaagacgaaaacgag-3′

[0075] Molecular Probe: 5′-6FAM-tcgggtttgctgtcctagtc-MGB-NFQ-3′

[0076] Step 2, PCR amplification

[0077] Extract DNA from clinical specimens as template DNA, use the homologous primers and molecular probes designed in step 1 to amplify and analyze by real-time PCR instrument, the amplification system is as follows:

[0078] Reagent: TaqMan Universal PCR Master Mix 1μL

[0079] Molecular Probes 20nM

[0080] Forward primer 500 nM

[0081]Reverse primer 500 nM

[0082] Template DNA 10ng

[0083] Total volume 15 μL

[0084] Amplification conditions: 95°C, 10min—(92°C, 15s—65°C, 1min)×40 cycles

[0085] Step 3, Identify Mycobacterium tuberculosis

[0086] After performing analysis with a PCR instrument in step 3, if ...

Embodiment 3

[0088] Step 1, design primers and molecular probes

[0089] Design a pair of homologous primers and molecular probes with SEQ ID No.2 as the target,

[0090] Forward primer: 5′-gatccccgatagcatcaaga-3′

[0091] Reverse primer: 5′-attggcaagacgaaaacgag-3′

[0092] Molecular Probe: 5′-6FAM-tcgggtttgctgtcctagtc-MGB-NFQ-3′

[0093] Step 2, PCR amplification

[0094] Extract RNA from clinical specimens, reverse transcribe the extracted RNA into cDNA as template DNA, and use the homologous primers and molecular probes designed in step 1 to amplify and analyze by real-time PCR instrument. The amplification system is as follows:

[0095] Reagent: TaqMan® Universal PCR Master Mix 1μL

[0096] Molecular Probes 20nM

[0097] Forward primer 500 nM

[0098] Reverse primer 500 nM

[0099] Template DNA 10ng

[0100] Total volume 15 μL

[0101] Amplification conditions: 95°C, 10min—(92°C, 15s—65°C, 1min)×40 cycles

[0102] Step 3, Identify Mycobacterium tuberculosis

[0103] After pe...

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Abstract

The invention provides a method for identifying mycobacterium tuberculosis. According to the invention, mycobacterium tuberculosis coding gene Rv1508c sequence is adopted as a gene sequence to be detected; and detection is carried out with primers (and probes) designed with the sequence as a detection target. The method provided by the invention has the advantages of good accuracy and high sensitivity. With the method, a novel scheme is provided for the identification of mycobacterium tuberculosis. The method can be used in biological strain identification, tuberculosis infection identification, and researches in respects such as efficacy and mycobacterium tuberculosis drug resistance in drug development processes.

Description

technical field [0001] The invention relates to a method for identifying mycobacterium tuberculosis, in particular to a method for determining mycobacterium tuberculosis by detecting the sequence of the coding gene Rv1508c of mycobacterium tuberculosis. Background technique [0002] Mycobacterium tuberculosis (M.tuberculosis), commonly known as Mycobacterium tuberculosis, is the pathogenic bacterium that causes tuberculosis. It can invade various organs of the body, but tuberculosis is the most common. Tuberculosis is still an important infectious disease, and it is estimated that 1 / 3 of the world's population is infected with Mycobacterium tuberculosis. According to WHO reports, about 8 million new cases occur every year, and at least 3 million people die from the disease. In some developing countries, the carrier rate of Mycobacterium tuberculosis among adults is as high as 80%, and about 5% to 10% of them are carriers. Can develop active tuberculosis. Due to the prevale...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
Inventor 秦莲花胡忠义郑瑞娟
Owner SHANGHAI PULMONARY HOSPITAL
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