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Detection primer and kit for MTB

A technology for detecting primers and kits, which is applied in the determination/inspection of microorganisms, microorganisms, and methods based on microorganisms, etc. It can solve the problems of different amplification efficiencies, inability of target DNA molecules, affecting PCR efficiency and measurement response, etc., to achieve high Specificity and accuracy, improved detection efficiency, and excellent amplification efficiency

Inactive Publication Date: 2020-04-17
上海润达榕嘉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many deficiencies in this technology: 1. The difference in the background between the calibrator and the sample easily affects the efficiency of PCR and the measurement response; 2. The target DNA molecule with a low copy number cannot be detected by amplification; 3. The PCR amplification efficiency of the sample may be Different from the amplification efficiency of the calibrator; 4. Impurities or DNA degradation introduced into the DNA solution during DNA extraction affect the PCR dynamic amplification process
Therefore, fluorescent quantitative PCR is difficult to achieve the detection and absolute quantification of low copy value MTB

Method used

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  • Detection primer and kit for MTB
  • Detection primer and kit for MTB
  • Detection primer and kit for MTB

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Primer sequences for detection of MTB amplification

[0037] It includes the sequence shown in SEQ ID NO.: 1-3, and is the forward and reverse primers and MGB probe primers for amplifying MTB.

[0038]The base distribution of forward and reverse primers designed by the present invention is random, there is no continuous G or C base aggregation at the 3' end of the primer, and there is no self-complementary overlapping sequence at the 3' end, which can avoid the generation of hairpin structure and primer dimer . Related literature (Harkins K M, Buikstra J E, Campbell T, et al. Screening ancient tuberculosis with qPCR: challenges and opportunities [J]. Philosophical Transactions of the Royal Society B: Biological Sciences, 2015, 370(1660): 20130622.) reported primers As shown in SEQ ID NO.: 5-7, its reverse primer has a self-complementary sequence, which is easy to form a hairpin structure, which is not conducive to PCR amplification.

[0039] Forward primer reported in...

Embodiment 2

[0048] Testing MTB detection kits based on digital PCR technology

[0049] Including: Tissue DNA Extraction Kit (Extraction Kit from Capgemini, Cat. No. 158467); 70% Ethanol; Isopropanol; Chip Kit (Chip Kit from Hanghang Gene Company, Cat. No. V1); Detection System PCR Reaction Solution, positive control substance, negative control substance. The detection system PCR reaction solution includes: 2x Multiplex PCR Plus Buffer; dNTPMix; HotStar Taq Plus DNA Polymerase; forward and reverse primers and MGB probe primers for amplifying MTB shown in SEQ ID NO.: 1-3. In this article, 2x Multiplex PCR Plus Buffer, dNTP Mix; HotStar Taq PlusDNA Polymerase, and ROX dye are commercial routine reagents, and the corresponding brands can be directly retrieved. Experimenters can make corresponding commercial adjustments according to their own needs or self-preparation adjustments according to prescriptions Element.

Embodiment 3

[0051] A method for detecting MTB based on digital PCR technology

[0052] (1) Genomic DNA extraction from whole blood samples:

[0053] 1) Take 600 μL of blood and add 900 μl of red blood cell lysate, mix by inversion 10 times, place at room temperature for 1 min, during this period, invert and mix several times, centrifuge at 16,000 x g for 1 min, absorb the supernatant, leave the white blood cell precipitate, add 600 μl of cell lysate, Shake for 10s to thoroughly suspend and mix the white blood cells.

[0054] 2) Add 18 μl proteinase K solution, mix well, and incubate at 58 for 2 hours.

[0055] 3) Add 200 μl of protein precipitation solution, shake and mix at high speed for 20 seconds.

[0056] 4) Centrifuge at 16000x g for 1 min, leave the supernatant, and discard the precipitate.

[0057] 5) Add the supernatant from the previous step to a 1.5ml centrifuge tube filled with 600μl isopropanol in advance, and gently invert up and down for 50 times to mix well. At this tim...

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Abstract

The invention provides a detection primer for MTB, and belongs to the technical field of gene detection. The detection primer comprises a forward primer, a reverse primer and an MGB probe primer for amplifying an MTB gene. A digital PCR technology is adopted to detect the MTB, and the designed primers and the MGB probe can expand to be suitable for other digital PCR systems; and the detection primer can detect a low-concentration MTB nucleic acid sample, has very high specificity and sensitivity and a high primer amplification efficiency, greatly improves the detection rate of the MTB, and provides a reliable method for clinical detection of the MTB.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to primers and kits for detecting human MTB by digital PCR. Background technique [0002] Mycobacterium tuberculosis (MTB), abbreviated as tubercle bacilli, is the pathogenic bacterium that causes tuberculosis. It can invade all organs of the body, but tuberculosis is the most common. Tuberculosis is still an important infectious disease. According to WHO statistics, about 1 out of every 3 people in the world is infected with Mycobacterium tuberculosis. In some developing countries, the carrier rate of Mycobacterium tuberculosis among adults is as high as 80%, and about 5% to 10% of the carriers may be infected. develop active tuberculosis. Due to the prevalence of AIDS in the past two decades, Mycobacterium tuberculosis carriers infected with HIV are 30 to 50 times more likely to develop active tuberculosis than those without HIV infection because the virus destroys the b...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12N15/11C12R1/32
CPCC12Q1/689C12Q1/6851C12Q2531/113
Inventor 钱学庆秦炜宋成林贺笑非吴小虎胡雄敏崔丹杨觐瑜赵垠莹侯隽杰
Owner 上海润达榕嘉生物科技有限公司
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