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Real-time fluorescent nucleic acid constant temperature amplification detection kit for bird-mycobacterium avium complex, and specific primer and probe thereof

A constant temperature amplification detection technology of Mycobacterium avium, applied in the biological field, achieves the effect of simple equipment, high specificity and accurate detection results

Active Publication Date: 2017-07-11
SHANGHAI RENDU BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problems faced by the application of SAT technology in the detection of different types of pathogens are different, and it is necessary to specifically analyze the characteristics of pathogens for special design.
At present, there is no research report on the real-time fluorescent nucleic acid constant temperature amplification detection technology for MAC

Method used

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  • Real-time fluorescent nucleic acid constant temperature amplification detection kit for bird-mycobacterium avium complex, and specific primer and probe thereof
  • Real-time fluorescent nucleic acid constant temperature amplification detection kit for bird-mycobacterium avium complex, and specific primer and probe thereof
  • Real-time fluorescent nucleic acid constant temperature amplification detection kit for bird-mycobacterium avium complex, and specific primer and probe thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, designing primers and probes dedicated to real-time fluorescent nucleic acid constant temperature amplification detection of Mycobacterium avium

[0069] In the nucleic acid detection of Mycobacterium avium (MAC), the main amplified target sequences include MPT64, katG gene, insertion sequence, rpoB gene and 16S rRNA, etc., combined with literature reports and the technical characteristics of the detection target of the present invention as RNA , the inventors analyzed and determined 16S rRNA as the target sequence for real-time fluorescent nucleic acid constant temperature amplification. On this basis, the inventor has obtained a high-specificity, high-sensitivity special primer pair and corresponding detection probe through a large number of screening and verification, using this specific primer pair can be used for Mycobacterium avium and Mycobacterium intracellulare The nucleic acid is simultaneously amplified, which has the advantages of high specific...

Embodiment 2

[0082] Example 2, preparation of a kit dedicated to real-time fluorescent nucleic acid constant temperature amplification detection of Mycobacterium avium

[0083] The Mycobacterium avium (MAC) real-time fluorescent nucleic acid constant temperature amplification detection kit of the present invention mainly comprises: T7 primer, nT7 primer, Mycobacterium avium detection probe, Mycobacterium intracellular detection probe, M-MLV Reverse transcriptase and T7 RNA polymerase.

[0084] The T7 primer, nT7 primer and detection probe exist in the MAC amplification detection liquid, and the MAC reverse transcriptase and T7 RNA polymerase exist in the SAT enzyme liquid.

[0085] Since the SAT amplification is easily affected by various factors, the amplification fails, causing reagent users to make wrong judgments and draw erroneous conclusions. Therefore, a control can also be set in the reagent of the present invention to eliminate the distortion of the detection results.

[0086] Th...

Embodiment 3

[0116] Embodiment 3, the specific detection of the real-time fluorescent nucleic acid constant temperature amplification detection kit of Mycobacterium avium

[0117]Use the real-time fluorescent nucleic acid constant temperature amplification detection kit of Mycobacterium avium of the present invention to specifically detect microorganisms that are similar in classification to Mycobacterium tuberculosis and non-tuberculosis Mycobacteria, have similar symptoms, and are prone to cross-reactions, 1 -Sample No. 9 is Mycobacterium tuberculosis, Mycobacterium chelonis, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium toads, Mycobacterium scrofula, Mycobacterium fortuitum, Mycobacterium gordonii, and smegmatis Mycobacteria (all strains were provided by Shanghai Pulmonary Hospital).

[0118] The specific detection method includes the following steps:

[0119] 1 Reagent preparation

[0120] 1.1 Balance the temperature of all reagents to room temperature (18°C-26°C) be...

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Abstract

The invention relates to a real-time fluorescent nucleic acid constant temperature amplification (SAT) detection kit for bird-mycobacterium avium complex, and a specific primer and a probe thereof. The specific region of 16S rRNA is adopted as a detection target. The specific primer pair and the probe have the advantages of high specificity and high sensitivity, and the kit can detect the bird-MAC RNA in a clinic sample isolate strain, has the characteristics of high specificity, high sensitivity, low pollution and fastness in detection, and plays a role in the clinic diagnosis of the bird-MAC.

Description

technical field [0001] The invention belongs to the molecular biological detection of bacteria in the field of biotechnology, in particular to a real-time fluorescent nucleic acid constant temperature amplification detection of Mycobacterium avium based on nucleic acid extraction technology and real-time fluorescent nucleic acid constant temperature amplification detection technology (SAT) Kit and its special primers and probes. Background technique [0002] Mycobacterium avium (MAC) is the abbreviation of Mycobacterium avium-Mycobacterium intracellulare complex. The most frequently isolated species belonged to nontuberculous mycobacteria. [0003] Nontuberculous mycobacteria (NTM) refers to Mycobacterium tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, and Mycobacterium microti) and other branches other than Mycobacterium leprae bacilli. Originally known as atypical mycobacterium, its characteristics are different from Mycob...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/02C12N15/11C12R1/325C12R1/32
CPCC12Q1/6844C12Q1/689C12Q2600/166C12Q2527/101C12Q2545/107C12Q2561/113C12Q2563/107
Inventor 崔振玲胡忠义赵国玲于明辉居金良
Owner SHANGHAI RENDU BIOTECH
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