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A kind of detection kit and method for Mycobacterium tuberculosis

A technology of mycobacterium tuberculosis and a kit, applied in the biological field, can solve the problems of false positives and very high requirements for the target sequence, and achieve the effects of good accuracy, shortened detection time, and high detection efficiency

Active Publication Date: 2020-08-11
宁波胤瑞生物医学仪器有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The results of this kit do not require electrophoresis and are easy to observe by eye, and are very suitable for the rapid detection of Mycobacterium tuberculosis. However, this method also has many problems: First, because the primer design of LAMP needs to select 6 regions on the target sequence (If you use loop primers to improve efficiency, you need an additional pair) So it has very high requirements for the target sequence itself, that is, not all pathogens have suitable sequences to design primers
Second, because the amplification efficiency of LAMP technology is very high, it is also prone to false positives due to contamination

Method used

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  • A kind of detection kit and method for Mycobacterium tuberculosis
  • A kind of detection kit and method for Mycobacterium tuberculosis
  • A kind of detection kit and method for Mycobacterium tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1 A kind of kit for detecting Mycobacterium tuberculosis

[0059] The kit includes the following parts: individually packaged following reagents: Primer Probe Master Mix, Reaction Master Mix, Positive Quality Control, Negative Quality Control, Sterile Water.

[0060] 1. Primer probe master mix:

[0061] Detect the upstream primer TB-DNA-F of Mycobacterium tuberculosis DNA, the sequence is SEQ ID NO: 1 (TB-DNA-F): 5'---CACATCGATCCGGTTCAGCG---3', the concentration is 400nM;

[0062] Detect the downstream primer TB-DNA-R of Mycobacterium tuberculosis DNA, the sequence is SEQ ID NO: 2 (TB-DNA-R): 5'---GTCATAGGAGCTTCCGACCG---3', the concentration is 400nM;

[0063] The fluorescent probe TB-DNA-P for detecting Mycobacterium tuberculosis DNA has a sequence of SEQ ID NO: 3(TB-DNA-P): 5'---CTCGCCGAGGCAGGCATCCA---3', and the 5' end is connected with The fluorescent reporter group FAM, the 3' end is connected with the quencher group BHQ-1, the concentration is 200nM; ...

Embodiment 2

[0078] Embodiment 2 utilizes the method that the kit provided by the invention detects Mycobacterium tuberculosis

[0079] Test sample: artificially synthesized standard containing the target fragment of TB;

[0080] Sample source: Shanghai Sangong Company.

[0081] Include the following steps:

[0082] 1. Preparation of DNA templates with different concentrations:

[0083] A 300bp DNA fragment containing the target fragment of Mycobacterium tuberculosis DNA was artificially synthesized by Shanghai Sangong Company, and ligated into a vector plasmid, then transferred to E. The enzyme cuts the plasmid linearly, and quantifies the double-stranded DNA with Qubit3.0;

[0084] Another human normal T lymphocyte line H9 was cultured, its DNA was extracted, quantified using Qubit3.0, and then diluted to a concentration of 10 ng / μL.

[0085] Prepare Mycobacterium tuberculosis DNA copy number system with final concentrations of 1 and 10 1 、10 2 、10 3 、10 4 、10 5 and 10 6 Copy t...

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Abstract

The invention provides a mycobacterium tuberculosis detection kit and method and belongs to the technical field of biology. The mycobacterium tuberculosis detection kit comprises the following independent-packaged reagents: a primer probe premix, a reaction premix, a positive quality control and a negative quality control. The method for detecting mycobacterium tuberculosis by using the kit comprises the following steps: preparing a digital PCR mixed solution, carrying out a PCR amplification reaction, carrying out signal collection on a product of the PCR amplification reaction, and judging whether a sample to be detected contains a mycobacterium tuberculosis DNA template or not and the content of the mycobacterium tuberculosis DNA template in the sample to be detected according to typesof fluorescence signals. Through detecting the mycobacterium tuberculosis by using the kit, the efficiency is high, the accuracy is good, the detection time can be greatly shortened, a standard curveis not required to be made, the specificity and coverage of detection are obviously improved, and the kit has relatively high stability.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection kit and method for mycobacterium tuberculosis. Background technique [0002] Mycobacterium tuberculosis (M.tuberculosis) is referred to as Mycobacterium tuberculosis (tuberclebacilli, TB). As an ancient disease, tuberculosis caused by Mycobacterium tuberculosis infection has plagued humans for thousands of years. The bacteria can invade various tissues and organs of the whole body, but the most common infection is the lungs. Respiratory transmission between people is the main mode of infection of the disease, and the source of infection is tuberculosis patients who have contact with bacteria. The clinical manifestations of tuberculosis are diverse and can affect various organ systems throughout the body. The lesions are often very hidden, which brings great obstacles to the diagnosis and treatment of tuberculosis. Therefore, early and accurate diagnosis and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12R1/32
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2563/107C12Q2521/531C12Q2545/113
Inventor 刘一博林强金鑫浩隋硕任鲁风张未来俞育德于军
Owner 宁波胤瑞生物医学仪器有限责任公司