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Sample treatment reagent and kit for detecting mycobacterium tuberculosis nucleic acid and nucleic acid amplification method of mycobacterium tuberculosis

A Mycobacterium tuberculosis treatment method technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/testing, etc., can solve the problems of being too expensive and unable to routinely use the burden of tuberculosis, and achieve easy access, The effect of reducing the risk of human infection and low price

Pending Publication Date: 2020-04-07
GUANGZHOU KINGMED DIAGNOSTICS GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these commercial tests are still too expensive to be routinely used, especially in most developing countries with a high burden of TB

Method used

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  • Sample treatment reagent and kit for detecting mycobacterium tuberculosis nucleic acid and nucleic acid amplification method of mycobacterium tuberculosis
  • Sample treatment reagent and kit for detecting mycobacterium tuberculosis nucleic acid and nucleic acid amplification method of mycobacterium tuberculosis
  • Sample treatment reagent and kit for detecting mycobacterium tuberculosis nucleic acid and nucleic acid amplification method of mycobacterium tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 A sample processing reagent for detecting Mycobacterium tuberculosis nucleic acid

[0052] In this embodiment, a sample processing reagent for detecting Mycobacterium tuberculosis nucleic acid comprises the following components: digestion solution, Tris-HCl solution and lysate.

[0053] The digestive juice is an aqueous solution containing the following concentration components: dithiothreitol 13.33g / L; sodium chloride 104g / L; potassium chloride 2.67g / L; disodium hydrogen phosphate 14.93g / L; dihydrogen phosphate Potassium 2.67g / L; The pH value of described digestive juice is 7.4.

[0054] The concentration of the Tris-HCl solution is 0.1±0.01mol / L, and the pH value is 7.4.

[0055] The lysate is an aqueous solution containing the following concentration components: NaOH 0.1w / v%; SDS 0.025w / v%.

Embodiment 2

[0056] Example 2 A sample processing method for detecting Mycobacterium tuberculosis nucleic acid

[0057] In this embodiment, a sample processing method for detecting Mycobacterium tuberculosis nucleic acid, using the sample processing reagent described in Embodiment 1 to process the sputum sample, includes the following steps:

[0058] (1) Add 100 μL of digestive solution to 1 mL of sputum sample, vortex to mix, and then incubate at 65°C for 10 minutes;

[0059] (2) The mixed solution obtained in step (1) was centrifuged at 13200g for 10 minutes, and the supernatant was discarded;

[0060] (3) Add 100 μL Tris-HCl solution to the precipitate obtained in step (2), vortex and shake to mix, then centrifuge at 13200g for 10 minutes, and discard the supernatant;

[0061] (4) Add 100 μL of lysate to the precipitate obtained in step (3), and then incubate at 65° C. for 45 minutes;

[0062] (5) Add 100 μL of Tris-HCl solution to the mixture obtained in step (4), mix well to obtain ...

Embodiment 3

[0063] Embodiment 3 A kind of test kit for detecting Mycobacterium tuberculosis nucleic acid

[0064] This embodiment is a kit for detecting Mycobacterium tuberculosis nucleic acid, comprising the following components: the sample processing reagent described in Example 1, a primer pair 1 for IS6110; a primer pair 2 for IS6110; a probe primer for IS6110; Quality control template; probe primers for quality control template; QuantiNova Probe PCR Master Mix and DEPC water.

[0065] The primer pair 1 for IS6110 includes IS6110-FW1 shown in SEQ ID NO.1 and IS6110-RV1 shown in SEQ ID NO.2; the primer pair 2 for IS6110 includes the sequence shown in SEQ ID NO.3 IS6110-FW2 and IS6110-RV2 shown in SEQ ID NO.4; the probe primer sequence for IS6110 is shown in SEQ ID NO.5, the 5' end of the probe primer is modified with the fluorescent group FAM, and the 3' end Modified with quenching group BHQ1;

[0066] The quality control template sequence is shown in SEQ ID NO.6; the probe primer se...

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Abstract

The invention provides a sample treatment reagent and a kit for detecting mycobacterium tuberculosis nucleic acid, and a nucleic acid amplification method of mycobacterium tuberculosis. The sample treatment reagent comprises digestive juice, a Tris-HCl solution and a lysate, wherein the digestive juice is an aqueous solution containing the following concentration components: 13-14 g / L of dithiothreitol, 100-105 g / L of sodium chloride, 2-3 g / L of potassium chloride, 14-15.5 g / L of disodium hydrogen phosphate and 2-3 g / L of potassium dihydrogen phosphate; the lysate is an aqueous solution containing the following concentration components: 0.08-0.15 w / v% of NaOH and 0.02-0.03 w / v% of SDS. The sample treatment reagent for detecting mycobacterium tuberculosis nucleic acid can extract mycobacterium tuberculosis DNA from a sputum sample more quickly and effectively, interference of human-derived DNA is effectively removed, mycobacterium tuberculosis is completely inactivated in the treatmentprocess, the risk of personnel infection is effectively reduced, and the sample treatment reagent is safer and more efficient. The kit for detecting the mycobacterium tuberculosis nucleic acid has good sensitivity, specificity and accuracy, and can realize rapid detection of the mycobacterium tuberculosis nucleic acid.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a sample processing reagent and kit for detecting mycobacterium tuberculosis nucleic acid and a nucleic acid amplification method for mycobacterium tuberculosis. Background technique [0002] Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTBC), has been a global public health challenge for decades. In 2016, an estimated 10.4 million new cases of tuberculosis were diagnosed worldwide, resulting in 1.7 million deaths. Traditional tuberculosis diagnosis fundamentally relies on acid-fast bacilli (AFB) smear microscopy and mycobacterial culture on selective solid media, both of which have limitations. First, the sensitivity of the AFB smear is low (30-40%) and its accuracy is often limited. Second, while culture-based methods have high accuracy and have long been considered the gold standard for laboratory TB diagnosis, the long turnaround time ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/6806C12Q1/04C12N15/10C12N15/11C12R1/32
CPCC12Q1/689C12Q1/6844C12Q1/6806C12N15/1003
Inventor 韩琦陈敬贤刘勇严婷于世辉任永昌马超杰
Owner GUANGZHOU KINGMED DIAGNOSTICS GRP CO LTD
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