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A kind of primer and probe and its kit for on-site rapid detection of Mycobacterium tuberculosis complex

A technology for Mycobacterium tuberculosis and Mycobacterium nucleatum, which is applied in biochemical equipment and methods, determination/inspection of microorganisms, DNA/RNA fragments, etc., can solve the problems of high false positive rate, low sensitivity and poor specificity, etc. Achieve the effect of convenient use, high sensitivity and simple detection

Active Publication Date: 2018-02-23
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Smear staining microscopic examination is simple and fast, but the method has low sensitivity and poor specificity
Immunological diagnosis has poor specificity and high false positive rate due to the crossover between existing antigens or antibodies and other microorganisms
Molecular biology diagnosis is fast and sensitive, and specific DNA fragments can be used to distinguish the species in the Mycobacterium tuberculosis complex. However, molecular biology methods represented by PCR technology have high requirements for equipment and technical personnel, and cannot Rapid diagnosis on site
Although constant isothermal amplification technology (LAMP) can be used for on-site detection, its reaction time is generally within 1 hour, and there are problems such as errors of human eyes in the judgment of results.

Method used

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  • A kind of primer and probe and its kit for on-site rapid detection of Mycobacterium tuberculosis complex
  • A kind of primer and probe and its kit for on-site rapid detection of Mycobacterium tuberculosis complex
  • A kind of primer and probe and its kit for on-site rapid detection of Mycobacterium tuberculosis complex

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1. Design and screening of primers and probes

[0042] At present, there are no specific rules for the design of RPA-LFD primers and probes. Only after RPA reaction and detection with lateral flow chromatography (LFD) can the primers and probes that can be used for clinical detection be screened.

[0043] The length of the RPA primer is generally 30-35nt, and if the primer is too short, it will seriously affect the activity of the recombinase. Longer primers do not necessarily improve amplification performance, but rather increase the likelihood of secondary structure formation. The length of the LF probe is generally 46-52nt, and the nfo ribozyme is about 30 bases away from the 5' end of the probe, and about 16-22 bases away from the 3' end.

[0044] In the experiment, it is necessary to design multiple pairs of primers and probes from both ends of the target sequence for optimization and screening. The substitution or increase or decrease of individual bas...

Embodiment 2

[0050] Example 2. Establishment of the detection method for Mycobacterium tuberculosis complex RPA-LFD

[0051] 1. Experimental steps

[0052] (1) Extraction of bacterial genomic DNA

[0053] Take 1 mL of the bacterial liquid, and extract the total bacterial DNA according to the instruction of Tiangen Biochemical Technology (Beijing) Co., Ltd. Bacterial Genomic DNA Extraction Kit (catalogue number: DP302).

[0054] (2) Establishment of the Mycobacterium tuberculosis complex RPA-LFD reaction system

[0055] The RPA reaction system is 50 μL:

[0056]

[0057] Sample DNA or crude lysate and ddH to be tested 2 O 13.2 μL

[0058] Add the above mixture into the TwistAmp nfo reaction tube, fully dissolve, then add 2.5 μL of magnesium acetate solution, and place the reaction tube in a 38°C water bath for 25 minutes. After the reaction, mix 2 μL with 98 μL of LFD detection buffer, place the LFD within 5 minutes to observe the results, if the detection band and the control band ...

Embodiment 3

[0065] Embodiment three, clinically diagnosed as the detection of tuberculosis sample

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Abstract

The invention discloses a primer and a probe for on-site rapid detection of mycobacterium tuberculosis complex and a kit thereof. The forward primer sequence is shown as SEQ ID No.1, the reverse primer sequence is shown as SEQ ID No.2, and the probe sequence is shown as SEQ ID No.3. The invention provides the PA-LFD primer and the probe for on-site distinctive, sensitive, simple and rapid detection of the mycobacterium tuberculosis complex (mycobacterium tuberculosis, mycobacterium bovis, African mycobacteria and Canna mycobacteria) and the kit containing the primer and the probe.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for identifying Mycobacterium tuberculosis complex using Recombinase Polymerase Amplification and Lateral Flow Dipstick (RPA-LFD) detection technology and related Primers and probes and detection kits. Background technique [0002] The pathogenic bacteria in the Mycobacterium tuberculosis complex (Mycobacterium tuberculosis complex, MTC) that can cause tuberculosis in humans and animals include Mycobacterium tuberculosis (M.tuberculosis), Mycobacterium bovis (M.bovis), Mycobacterium africa (M.africanum), Mycobacterium canneri (M.canettii), etc. Tuberculosis is a zoonotic infectious disease that seriously endangers the health of humans and animals. The number of deaths due to tuberculosis is about 2 million to 3 million worldwide every year. According to the statistics of the World Health Organization, my country is one of the 22 countries with severe tuberculos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/6844C12Q2522/101C12Q2537/1376C12Q2521/507
Inventor 何洪彬赵贵民王洪梅
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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