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Composition and kit for detecting mycobacterium tuberculosis and/or brucella and application of composition

A technology of Mycobacterium tuberculosis and Brucella, applied in the field of genetic engineering, can solve the problems of low sensitivity and long reaction time, and achieve high sensitivity, low detection cost and good detection effect

Pending Publication Date: 2020-05-19
SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to overcome the defects of low sensitivity and long reaction time in the detection of Mycobacterium tuberculosis and / or Brucella by single-channel fluorescent PCR method or fluorescent PCR, the present invention provides a Composition, kit and application thereof for detecting Mycobacterium tuberculosis and / or Brucella, which detect amplification products in a closed tube state, avoiding false positives caused by contamination of amplification products; probe hybridization is specific It is more reliable; it can realize the detection of Mycobacterium tuberculosis and / or Brucella alone or at the same time, which greatly shortens the detection time and reduces the detection cost; there is no need for follow-up treatment after PCR, and the operation is simpler, faster, and safe. Pollution

Method used

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  • Composition and kit for detecting mycobacterium tuberculosis and/or brucella and application of composition
  • Composition and kit for detecting mycobacterium tuberculosis and/or brucella and application of composition
  • Composition and kit for detecting mycobacterium tuberculosis and/or brucella and application of composition

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Experimental program
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Effect test

Embodiment 1

[0070] This embodiment is a preferred design of specific primers and probes for detecting Mycobacterium tuberculosis and Brucella.

[0071] At the nucleotide level, the homology between Mycobacterium bovis and Mycobacterium tuberculosis genome is greater than 99.95%. IS6110-RFLP (IS6110 insertion element based Restriction fragmentlength polymorphism) is a typing method based on the restriction fragment length polymorphism of the IS6110 insertion element, because the insertion site and copy number of the IS6110 insertion element are different in different strains , so that different gel electrophoresis patterns can be obtained. IS6110-RFLP is considered the "gold standard" for tuberculosis molecular epidemiological investigations and is widely used in studies all over the world. The disadvantage of this method is that the isolates are required to have a high copy number of the IS6110 insertion element, the resolution of low-copy isolates is low, and the experimental results ar...

Embodiment 2

[0078] This embodiment is the establishment and optimization of a preferred reaction system containing combination 1 in Example 1 for detecting Mycobacterium tuberculosis and Brucella.

[0079] 1. Sample preparation: synthesize the IS6110 gene of Mycobacterium tuberculosis insert sequence and the Brucella BCSP31 gene in the target amplified region, connect to the PET30a vector, transform into BL21(B) recipient bacteria, select positive clones, and construct a tuberculosis-containing clone. The positive plasmids of the amplified regions of Mycobacterium and Brucella are used as positive controls for the detection of Mycobacterium tuberculosis and Brucella; , Streptococcus as a specific reference; deionized water was used as a negative control, and TRIZOL was used to extract the DNA of the above-mentioned positive control and specific reference, and the negative control was set aside.

[0080] 2. Optimization of the concentration of primers and probes: In the case of other compo...

Embodiment 3

[0086] This embodiment is a preferred establishment of a standard curve using combination 1 in embodiment 1.

[0087] After measuring the concentration of Mycobacterium tuberculosis and Brucella positive plasmid DNA with a spectrophotometer, use DEPC-H 2 O Make a 10-fold serial dilution of the plasmid standard so that the copy number in each 5 μL detection volume is 3.1×10 7 , 3.1×10 6 , 3.1×10 5 , 3.1×10 4 , 3.1×10 3 copy / μL, set 3 repetitions, and perform amplification. After the reaction, the standard curve was automatically obtained using the Vii fluorescent quantitative PCR analysis software. The results showed that the FAM detection channel for the detection of Mycobacterium tuberculosis established with primers (Mycobacterium tuberculosis TB-F1 and Mycobacterium tuberculosis TB-R1) and probe (Mycobacterium tuberculosis TB-P1) showed a typical S-type Amplification curve, the exponential area is more obvious, the initial template concentration of the standard and the...

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Abstract

The invention relates to a composition for detecting mycobacterium tuberculosis and / or brucella. The composition includes primers and probes for detecting mycobacterium tuberculosis and / or brucella, wherein the primers include the sequences shown in SEQ ID NO.1-SEQ ID NO.4, and the probes include the sequences shown in SEQ ID NO.5-SEQ ID NO.6. The invention further relates to a kit comprising thecomposition and application of the composition. The kit performs amplification product detection in a closed-tube state, so that false positives caused by contamination of an amplification product isavoided; the probe hybridization specificity is higher; the detection of the two bacteria of mycobacterium tuberculosis and / or brucella can be simultaneously achieved, the detection time is greatly shortened, and the detection cost is reduced; and after PCR is carried out, no subsequent processing is required, the operation is simpler and faster, safe and pollution-free, the detection is high in specificity, sensitive, fast and safe, and the detection effect on samples containing trace amounts of mycobacterium tuberculosis and / or brucella genomes is good.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a composition, a kit and an application thereof for detecting Mycobacterium tuberculosis and / or Brucella. Background technique [0002] Tuberculosis is a zoonotic disease caused by infection with strains of Mycobacterium tuberculosis complex. OIE lists it as a disease that must be notified, and it is a second-class animal infectious disease in my country and listed as an object of quarantine and culling. Mycobacterium tuberculosis complex includes Mycobacterium tuberculosis (M.tb), Mycobacterium bovis (M.bovis), BCG (Mycobacterium bovis bacillus Calmette-Guérin, BCG), Mycobacterium microti (Mycobacterium microti), Mycobacterium africanum, among which Mycobacterium tuberculosis and Mycobacterium bovis are the most important pathogens causing diseases in mammals. These subspecies have the same 16Sr RNA sequence, and the genomes are highly similar, reaching 99.9%. Myc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6851C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 杨德全赵洪进王建鞠厚斌杨显超王可萱葛菲菲卢军李鑫吴秀娟夏炉明沈海潇朱九超
Owner SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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