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Mycobacterium tuberculosis nucleic acid detection kit by utilizing RNA isothermal amplification

A technology of Mycobacterium tuberculosis and detection kit, which is used in the determination/inspection of microorganisms, material excitation analysis, fluorescence/phosphorescence, etc. High, specific effect

Active Publication Date: 2011-09-14
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0026] The technical problem to be solved by the present invention is to overcome the shortcomings of prior art specificity, low sensitivity, and difficult control of experimental pollution, and to provide a synchronous amplification detection technology (SAT) for releasing bacterial nucleic acid RNA and constant temperature nucleic acid by ultrasonically disrupting bacterial cells. ) kit for detecting Mycobacterium tuberculosis (TB)

Method used

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  • Mycobacterium tuberculosis nucleic acid detection kit by utilizing RNA isothermal amplification
  • Mycobacterium tuberculosis nucleic acid detection kit by utilizing RNA isothermal amplification
  • Mycobacterium tuberculosis nucleic acid detection kit by utilizing RNA isothermal amplification

Examples

Experimental program
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Effect test

Embodiment 1

[0050] The extraction of TB nucleic acid RNA, the specific steps are:

[0051] 1. Take 1.5 ml of sputum, add 1-2 times the volume of 4% NaOH to a sterile sample tube depending on the properties of the sputum specimen, vortex for 1 min to fully homogenize the sputum, and place at room temperature for 15-20 min.

[0052] 2. Sample processing tube (1.5ml centrifuge tube, the number is: the number of samples to be tested + 2), respectively mark the number of the sample to be processed and the positive and negative controls;

[0053] 3. Take 1 ml of the liquefied sputum sample into the sample processing tube, centrifuge at 13,000 rpm for 5 min, discard the supernatant; add 1 ml of normal saline to resuspend and wash, centrifuge at 13,000 rpm for 5 min, discard the supernatant, and add 50 μL of TB diluent to resuspend.

[0054] 4. Put 50 μl of the processed sample, 50 μl of positive control, and 50 μl of negative control into an ultrasonic processor for sonication for 15 minutes, an...

Embodiment 2

[0061] SAT nucleic acid amplification detection

[0062] 1. Put the clean micro-reaction tube containing the amplification detection solution into a constant temperature preheating device, keep at 60°C for 10 minutes, and keep at 42°C for 5 minutes; at the same time, preheat the SAT enzyme solution to 42°C;

[0063] 2. Add 10μl of pre-warmed enzyme solution to the micro-reaction tube (do not touch the sample tip to the micro-reaction tube, if there is contact, be sure to replace the tip), cover the tube after adding the enzyme, and shake at 1200 rpm for 15 seconds to mix well. ;

[0064] 3. Quickly transfer the micro-reaction tube to a suitable constant temperature fluorescence detection instrument, react at 42°C for 40 minutes, and set the fluorescence detection every 1 minute, for a total of 40 detections;

[0065] 4. After the reaction, take out the micro-reaction tube directly and soak it in 10% 84 disinfectant. Take the micro-reaction tube carefully and do not open the r...

Embodiment 3

[0074] Preparation and assembly of components of TB kit

[0075] Kit Components:

[0076] TB diluent: sterilized DEPC aqueous solution containing 0.1-1% RNAse inhibitor;

[0077] TB reaction solution: containing dNTPs and NTPs, mainly including Tris 10~50mM, MgCl 2 10~40mM, dNTP 0.5~5mM, NTP 1~10mM, PVP40 1~10%, KCl 5~40mM;

[0078] SAT enzyme solution: containing T7 RNA polymerase 200~2000U / reaction, M-MLV reverse transcriptase 400~4000U / reaction, 2~10mM HEPES pH7.5, 10~100mM N-acetyl-L-cysteine, 0.04~0.4 mM zinc acetate, 10~100mM trehalose, 40~200mM Tris-HCl pH 8.0, 40~200mM KCl, 1~10mM EDTA, 2~20%(v / v)Triton X-100, 10~40%(v / v )glycerol;

[0079] TB detection solution: contains TB specific primers and specific fluorescent probes, both primers and probes are dissolved in TE solution;

[0080] TB T7: aatttaatac gactcactat agggagacac caacaagctg ataggccgcg g;

[0081] TB nT7: gcaagtcgaa cggaaaggtc tc;

[0082] TB Probe: cguccggaua ggaccacggg acg.

[0083] The primer probe...

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Abstract

The invention relates to a Mycobacterium tuberculosis nucleic acid detection kit by utilizing RNA isothermal amplification. The kit contains TB (total bilirubin) diluent, TB reaction solution, TB detection solution, SAT enzyme solution, TB positive control and TB negative control. After Mycobacterium tuberculosis is liquefied, dosing culture and non-dosing culture are performed, the Mycobacteriumtuberculosis cultured by the two methods has differences, co-immunopricipitation is utilized or NaOH is used to treat the two types of Mycobacterium tuberculosis bacterial liquid, satisfiability problem (SAT) detection is performed on the treated bacterial liquid, and the drug resistance of Mycobacterium tuberculosis is analyzed according to the detection result. The method can be used to analyzethe drug resistance of Mycobacterium tuberculosis easily and effectively, and the method is simple and time-saving.

Description

technical field [0001] The invention relates to the field of in vitro molecular biological detection of Mycobacterium nucleatum TB, in particular to a Mycobacterium tuberculosis nucleic acid detection kit utilizing RNA constant temperature amplification. Background technique [0002] Mycobacterium tuberculosis (M. Tuberculosis, TB) is the causative bacterium of human and animal tuberculosis (tuberculosis), and belongs to the genus Mycobacterium of the family Actinobacteriaceae. It can be divided into human type, bovine type, bird type, mouse type, cold-blooded animal type and African type discovered in recent years. Humans are mainly caused by human type, bovis is less common, ornithobacillus is rarer, atypical mycobacteria can also cause similar tuberculosis-like lesions, but they are rare. The first three types have been fully confirmed after the London International Tuberculosis Conference in 1901 due to their early discovery, and are called classic tuberculosis. The of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
Inventor 方亮于明辉居金良
Owner SHANGHAI RENDU BIOTECH
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