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Oligonucleotide combination, method and kit used for mycobacterium strain identification

An oligonucleotide and mycobacterial technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc., can solve the problem of inability to identify specific species of mycobacteria, insensitivity and accuracy verification , the inability to detect multiple mycobacterial species/subspecies, etc., to achieve the effect of fast detection, convenient detection and wide clinical application.

Active Publication Date: 2019-08-02
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this kit or method cannot identify mycobacteria to a specific species, and cannot detect multiple mycobacteria species / subspecies at the same time. In addition, the sensitivity and accuracy have not been verified

Method used

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  • Oligonucleotide combination, method and kit used for mycobacterium strain identification
  • Oligonucleotide combination, method and kit used for mycobacterium strain identification
  • Oligonucleotide combination, method and kit used for mycobacterium strain identification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, the design of primer and probe

[0069] The oligonucleotide combination provided in the present invention includes 28 primers and probes as shown in the following list 1: SEQ ID NO: 1-28, which can specifically detect tuberculosis mycobacteria and non-tuberculous mycobacteria, specifically including Mycobacterium tuberculosis (TB), Mycobacterium kansasii (MK), Mycobacterium avium (MA), Mycobacterium fortuitously (MF), Mycobacterium abscessus subsp. abscessus (MAA), Mycobacterium abscessus subsp. ), Mycobacterium intracellulare (MIN), etc.

[0070] Table 1

[0071]

[0072]

[0073] Wherein, F and R are forward and reverse primer pairs of amplification primers, IC-F and IC-R are internal standard primer pairs, P is a detection probe, and P1 and P2 are fluorescent amplification primer pairs, wherein, The fluorescent amplification primer pair P1 and P2 also have the structure of the C3 interarm, the fluorescent amplification primer pair MAA and TB P2 ...

Embodiment 2

[0074] Embodiment 2, the extraction process of sample DNA

[0075] The detection sample of the present invention is sputum. Use the nucleic acid extraction / purification reagent based on the magnetic bead method (product number S1006, Hunan Shengxiang Biotechnology Co., Ltd.) to extract DNA, and perform the following operations in the sample processing room:

[0076] 1. Take an appropriate amount of 1.5 mL sterilized centrifuge tubes, mark the negative control, positive control and samples to be tested respectively, and add 300 μL DNA extraction solution 1 to each tube;

[0077] 2. Add 200 μL of the sample to be tested or negative control and positive control to each tube; cover the tube cap, shake and mix for 10 seconds, and centrifuge briefly;

[0078] 3. Add 100 μL of DNA extraction solution 2-mix to each tube (mix well and then draw), shake and mix for 10 seconds, then let stand at room temperature for 10 minutes;

[0079] 4. After instantaneous centrifugation, place the ...

Embodiment 3

[0083] Embodiment 3, PCR amplification process

[0084] Take 5 μL of the eluent prepared in Example 2 above, and prepare according to the reaction system described in Table 2 below.

[0085] Table 2

[0086] Component Volume / concentration for each reaction Mg 2+

2-6mM dNTPs (100mM) 0.1-0.4mM Taq enzyme (5U / μL) 2-10U SEQ ID NO:1-16 100-500nM SEQ ID NO:17-20 50-250nM SEQ ID NO:21-24 50-250nM SEQ ID NO:25-28 50-250nM eluent 5μL PCR buffer Make up to 50μL

[0087] The PCR amplification program is shown in Table 3 below.

[0088] table 3

[0089]

[0090]

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Abstract

The invention relates to the field of biological detection of molecules, in particular to the field of identification of mycobacteria, and provides an oligonucleotide combination. The oligonucleotidecombination comprises oligonucleotide for detecting mycobacterium fortuitum and oligonucleotide for detecting mycobacterium intracellulare, and selectively comprises at least one group, which can specifically detect mycobacterium tuberculosis and non-tuberculous mycobacteria and can identify specific species / subspecies, of the rest three groups, and meanwhile, two kinds of mycobacteria can be detected through a channel, so that the number of mycobacteria detected by fluorescence PCR is increased. The invention further provides a kit containing the oligonucleotide combination and a method usedfor identifying the mycobacteria.

Description

technical field [0001] The invention belongs to the field of molecular biological detection, more specifically, to the field of mycobacterium identification. Background technique [0002] Mycobacterium (Mycobacterium) is a class of slender slightly curved, sometimes branched or filamentous bacteria. There are many types of mycobacteria, mainly including mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria. [0003] Mycobacterium tuberculosis (M.tuberculosis, TB), commonly known as Mycobacterium tuberculosis, is the pathogenic bacteria that causes tuberculosis. It can invade all organs of the body, but tuberculosis is the most common. Tuberculosis is still an important infectious disease. According to the WHO, about 8 million new cases occur each year and at least 3 million people die from the disease. Before the founding of the People's Republic of my country, the mortality rate reached 200-300 people / 100,000, ranking first among the causes of death ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/686C12Q2600/166C12Q2563/107C12Q2527/107C12Q2561/101C12Q2547/101C12Q2545/101
Inventor 戴立忠孙青芝任小梅谭德勇毛君杰李倩程星邓中平
Owner SANSURE BIOTECH INC
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