Real-time fluorescent nucleic acid isothermal amplification detection kit for mycobacterium tuberculosis as well as special primer and probe thereof

A technology for the detection of mycobacterium tuberculosis and constant temperature amplification, which is applied in the field of biomedical detection, can solve problems such as mutual interference, interference, and detection sensitivity reduction, and achieve high detection sensitivity

Active Publication Date: 2021-10-19
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 as well as special primer and probe thereof
  • Real-time fluorescent nucleic acid isothermal amplification detection kit for mycobacterium tuberculosis as well as special primer and probe thereof
  • Real-time fluorescent nucleic acid isothermal amplification detection kit for mycobacterium tuberculosis as well as special primer and probe thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Design of special primers and probes for real-time fluorescent nucleic acid constant temperature amplification detection of Mycobacterium tuberculosis The inventor disclosed the detection sequence according to ZL201110137694.X (its nucleotide sequence is shown in SEQ ID NO: 1 ), carry out primer and probe design according to primer and probe design principle, to carry out real-time fluorescent nucleic acid constant temperature amplification detection to Mycobacterium tuberculosis.

[0059] This embodiment has designed multiple groups of primers and probes altogether, wherein selects following three groups of primers and probes (group 1, group 2 and group 3) to Mycobacterium tuberculosis positive control and negative control (do not contain Mycobacterium tuberculosis nucleic acid system, such as physiological saline) for detection verification, so as to screen out primers and probe sets with good specificity, sensitivity and reproducibility for the detection of...

Embodiment 2

[0084] Embodiment 2: Real-time fluorescent nucleic acid constant temperature 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 RNA nucleic acid constant temperature simultaneous amplification detection to detect a gene conservation region of Mycobacterium tuberculosis nucleic acid (16S rRNA, the corresponding DNA sequence is shown in SEQ ID NO: 1 Shown) the kit, on the basis of the primers and probes shown in Group 1 determined in the examples, specifically includes the following components:

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

[0087] (2) Detection solution a: it contai...

Embodiment 3

[0096] Embodiment 3: Real-time fluorescent nucleic acid isothermal amplification method for detecting Mycobacterium tuberculosis

[0097] The method of this embodiment is based on the principle of RNA constant temperature synchronous amplification detection to detect Mycobacterium tuberculosis nucleic acid, which uses the kit provided in Example 2 above 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 lauryl 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 15 minutes, and then ultrasonic treatment at 300w for 15 minutes to obtain the pretreated sample.

[0100] 3.2. Nucleic acid extraction

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

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Abstract

The invention discloses a real-time fluorescent nucleic acid isothermal amplification detection kit for mycobacterium tuberculosis as well as a special primer and a probe thereof, and belongs to the technical field of biomedical detection. The kit provided by the invention comprises a nucleic acid extracting solution, a detection solution a, a detection solution b, an SAT enzyme solution and the like, the nucleic acid extracting solution comprises an optimally designed specific capture probe extraction probe, target nucleic acid in a pretreated sample can be effectively enriched, extracted and purified, and the detection solution a, the detection solution b and the SAT enzyme solution which are added step by step are combined, and the high-sensitivity detection on the mycobacterium tuberculosis can be realized.

Description

technical field [0001] The invention belongs to the technical field of biomedical detection, in particular to a detection kit for Mycobacterium tuberculosis and its special primers and probes, in particular to the use of specific target capture technology and real-time fluorescent nucleic acid constant temperature amplification detection technology (Simultaneous Amplification and Test, SAT) kit for detecting Mycobacterium tuberculosis (Tuberculous bacillus, TB) nucleic acid in samples and its special primers and probes. Background technique [0002] Tuberculosis caused by Mycobacterium tuberculosis infection 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 products such as meat, milk, animal fur, e...

Claims

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Application Information

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Patent Type & Authority Applications(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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