Real-time fluorescent nucleic acid isothermal amplification detection kit for Mycobacterium tuberculosis and its special primers and probes

A technology for the detection of Mycobacterium tuberculosis and constant temperature amplification, which is applied in the field of biomedical detection, can solve the problems of mutual interference, lower detection sensitivity, interference, etc., and achieve the effect of high detection sensitivity

Active Publication Date: 2022-07-08
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The SAT technology has been patented and authorized by the applicant (ZL200810111479.0). 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.
The nucleic acid amplification detection of Mycobacterium tuberculosis using SAT technology has been patented by the applicant and obtained authorization (ZL201110137694.X), but in the detection method of this patent document, the pretreated sample is added in one step The primers and probes for detecting Mycobacterium tuberculosis may cause mutual interference between different primers and probes, as well as the interference of more impurities in the sample, resulting in a reduction in detection sensitivity; in addition, the patent document is using Before using SAT technology to amplify and detect Mycobacterium tuberculosis nucleic acid, the sample is pretreated with NaOH solution, which will lead to the death of Mycobacterium tuberculosis and RNA degradation, resulting in false negative results and reduced detection sensitivity; and Mycobacterium tuberculosis is sensitive to NaOH Some tolerance, which increases the risk of infection for operators

Method used

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  • Real-time fluorescent nucleic acid isothermal amplification detection kit for Mycobacterium tuberculosis and its special primers and probes
  • Real-time fluorescent nucleic acid isothermal amplification detection kit for Mycobacterium tuberculosis and its special primers and probes
  • Real-time fluorescent nucleic acid isothermal amplification detection kit for Mycobacterium tuberculosis and its special primers and probes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Design of special primers and probes for the detection of Mycobacterium tuberculosis by isothermal amplification of real-time fluorescent nucleic acid ), primers and probes are designed according to the principle of primer and probe design, so as to carry out real-time fluorescent nucleic acid isothermal amplification detection of Mycobacterium tuberculosis.

[0059] In this example, a total of multiple sets of primers and probes were designed, wherein the following three sets of primers and probes (group 1, group 2 and group 3) were selected for the positive control and negative control of Mycobacterium tuberculosis (without Mycobacterium tuberculosis nucleic acid) system, such as normal saline) for detection and verification, so as to screen out primers and probe sets with good specificity, sensitivity and repeatability for the detection of Mycobacterium tuberculosis.

[0060] Group 1:

[0061] Specific capture probe: caatattccccactgctgcctcccgtaggatttaaaaa...

Embodiment 2

[0084] Example 2: Real-time fluorescent nucleic acid isothermal amplification detection kit for Mycobacterium tuberculosis

[0085] The kit for the detection of Mycobacterium tuberculosis nucleic acid provided in this example is based on the principle of constant temperature synchronous amplification detection of RNA nucleic acid to detect Mycobacterium tuberculosis nucleic acid (a segment of gene conserved region of 16S rRNA, and the corresponding DNA sequence is as shown in SEQ ID NO: 1). Based on the primers and probes shown in group 1 determined in the examples, the kit specifically includes the following components:

[0086] (1) Nucleic acid extraction solution: used for extracting and purifying Mycobacterium tuberculosis nucleic acid in samples, containing 250-800 mM HEPES, 50-500 mg / L magnetic beads (solid support), 1-50 μM specific capture probe (SEQ ID NO: 2), 25-150 pmol / mL extraction probe (SEQ ID NO: 3);

[0087] (2) Detection solution a: it contains 10-50 mM Tris...

Embodiment 3

[0096] Embodiment 3: The method of real-time fluorescent nucleic acid isothermal amplification to detect Mycobacterium tuberculosis

[0097] The method of this embodiment detects Mycobacterium tuberculosis nucleic acid based on the principle of RNA constant temperature synchronous amplification detection. It uses the kit provided in the above embodiment 2 to detect whether the sputum sample obtained from the subject contains Mycobacterium tuberculosis nucleic acid. Specifically The operation steps are:

[0098] 3.1. Sample processing

[0099] Take 1 mL of sputum sample, add 1 mL of sample pretreatment solution (lithium dodecyl sulfate (LLS) 8%, 15 mM Tris-HCl, 0.3 mM EDTA), and vortex for 1 min to obtain a mixed solution. Heat treatment at 90°C for 15min, and then ultrasonically treat for 300w for 15min to obtain the pretreated sample.

[0100] 3.2. Nucleic acid extraction

[0101] (1) Take 1 mL of the pretreated sample into an EP tube, add 250 μL of nucleic acid extraction...

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Abstract

The invention discloses a real-time fluorescent nucleic acid constant temperature amplification detection kit for Mycobacterium tuberculosis and special primers and probes thereof, belonging to the technical field of biomedical detection. The kit provided by the present invention includes nucleic acid extraction solution, detection solution a, detection solution b, SAT enzyme solution, etc., wherein the nucleic acid extraction solution contains an optimally designed specific capture probe extraction probe, which can be used for pretreated samples. Effective enrichment, extraction and purification of the target nucleic acid in the test solution, combined with the step-by-step addition of detection solution a, detection solution b and SAT enzyme solution, can achieve high-sensitivity detection of Mycobacterium tuberculosis.

Description

technical field [0001] The invention belongs to the technical field of biomedical detection, in particular to a detection kit for Mycobacterium tuberculosis and special primers and probes thereof, in particular to the use of specific target capture technology and real-time fluorescent nucleic acid constant temperature amplification detection technology (Simultaneous Amplification and Detection Technology) Test, SAT) kit for detecting Mycobacterium tuberculosis (Tuberculousbacillus, TB) nucleic acid in samples and its dedicated primers and probes. Background technique [0002] Tuberculosis caused by Mycobacterium tuberculosis infection of human body is an infectious disease that seriously affects human life and health. It can invade susceptible organisms through respiratory tract, digestive tract or skin damage. Mycobacterium tuberculosis not only infects humans, but also mammals. In addition to the transmission between humans and animals by aerosols in the air, some animal p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6844C12Q2600/166C12Q2521/119C12Q2521/107C12Q2531/119C12Q2563/107C12Q2527/125C12Q2545/113C12Q2561/113C12Q2523/301Y02A50/30
Inventor 居金良崔振玲葛俊楠沈晓宁
Owner SHANGHAI RENDU BIOTECH
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