CN118834934A - Lysate for releasing mycobacterium tuberculosis nucleic acid and application thereof - Google Patents

Abstract

The present invention discloses a lysate for releasing nucleic acid of Mycobacterium tuberculosis and its application, the lysate comprises an enzyme component and a buffer component; wherein the enzyme component comprises lysozyme, colorless peptidase, lytic enzyme, mutanolysin, chitinase, and the buffer component comprises Tween-20, potassium chloride, Tris-HCl, EDTA; the enzyme component and the buffer component are mixed evenly during use. Compared with the commonly used lysate of Mycobacterium tuberculosis, the lysate of the present invention does not require high-temperature heating, mechanical vibration, ultrasound and other additional steps during use, does not require equipment assistance, is easy to operate and has low energy consumption, has low dependence on professional environment and professionals, and does not damage nucleic acid substances, and is more friendly to downstream nucleic acid amplification and detection; the enzyme component in the lysate provided by the present invention has a mild and efficient nucleic acid release effect for the cell wall of Gram-positive bacteria, which is not only applicable to Mycobacterium tuberculosis, but also has universality for Gram-positive bacteria.

Description

Lysis solution for releasing nucleic acid of Mycobacterium tuberculosis and its application

Technical Field

The invention relates to the field of biomedicine, and in particular to a lysis solution for releasing nucleic acid of Mycobacterium tuberculosis and application thereof.

Background Art

Tuberculosis (TB) is an infectious disease caused by infection with Mycobacterium tuberculosis (MTB). MTB can enter the human body through a variety of routes and infect a variety of tissues and organs, but the most common way of infection is through the inhalation of droplets and dust containing MTB through the respiratory tract. Every year, more than 10 million people worldwide develop pulmonary tuberculosis, and the morbidity and mortality of tuberculosis are higher than other infectious diseases.

Molecular point-of-care diagnosis (POCT) is one of the important frontier areas in the development of modern medicine. It has the characteristics of fast speed, high specificity and high sensitivity. It is the key to quickly and accurately detect tuberculosis to guide treatment and prevention and control. In this technology, the Mycobacterium tuberculosis in the sample needs to be treated with nucleic acid release for subsequent nucleic acid amplification and detection. However, as a typical Gram-positive bacterium, Mycobacterium tuberculosis contains a large amount of cross-linked peptidoglycan and lipids in its cell wall, which makes it difficult to break the bacterial wall. Its nucleic acid release often requires more drastic operating conditions, such as 100°C thermal lysis, high-speed vibration mechanical lysis, ultrasonic lysis, etc. These methods not only require equipment assistance and have large energy consumption, but also the drastic lysis method also has a destructive effect on the released genomic nucleic acid, which is easy to affect the downstream nucleic acid amplification reaction. Therefore, the efficient and mild Mycobacterium tuberculosis nucleic acid release method can effectively reduce the equipment dependence of tuberculosis molecular diagnosis and improve the detection rate, and has a good application in the rapid screening of tuberculosis at the grassroots level and on-site detection in extreme environments.

Therefore, it is necessary to improve the existing technology to provide a more reliable solution.

Summary of the invention

The technical problem to be solved by the present invention is to provide a lysis solution for releasing nucleic acid of Mycobacterium tuberculosis and its application in view of the deficiencies in the above-mentioned prior art. The nucleic acid release process does not require any heating, mechanical vibration and the like, and can efficiently and gently release nucleic acid of Mycobacterium tuberculosis without the assistance of additional equipment.

To achieve the above-mentioned purpose, the technical scheme adopted by the present invention is: in the first aspect of the present invention, a lysis solution for releasing nucleic acid of Mycobacterium tuberculosis is provided, comprising an enzyme component and a buffer component; wherein the enzyme component comprises lysozyme, colorless peptidase, lytic enzyme, mutanolysin, and chitinase, and the buffer component comprises Tween-20, potassium chloride, Tris-HCl, and EDTA; when used, the enzyme component and the buffer component are mixed evenly.

Preferably, the concentration of each component in the enzyme component is: lysozyme 20-200 μg/mL, colorless peptidase 50-600 μg/mL, mutanolysin 100-1000 μg/mL, lytic enzyme 100-500 μg/mL, and chitinase 20-100 μg/mL, based on the final concentration in the lysate obtained after the enzyme component and the buffer component are evenly mixed.

Preferably, based on the final concentration in the lysate obtained after the enzyme component and the buffer component are uniformly mixed, the concentration of each component in the buffer component is: Tween-20 0.05wt%-0.4wt%, potassium chloride 0.05-1M, Tris-HCl 50-500mM, EDTA 25-250mM.

Preferably, the pH value of the lysate is 7.4-8.0.

Preferably, the concentration of each component is as follows, based on the final concentration in the lysate obtained after the enzyme component and the buffer component are uniformly mixed: lysozyme 50 μg/mL, colorless peptidase 100 μg/mL, mutanolysin 200 μg/mL, lytic enzyme 200 μg/mL, chitinase 50 μg/mL, Tween-20 0.1wt%, potassium chloride 0.1M, Tris-HCl 100mM, EDTA 50mM.

The second aspect of the present invention provides a use of the above-mentioned lysis solution in the release of Mycobacterium tuberculosis nucleic acid.

Preferably, the lysis temperature of the lysis solution for releasing nucleic acid from Mycobacterium tuberculosis is 25-40°C.

Preferably, the lysis solution is used for nucleic acid release from a Mycobacterium tuberculosis suspension sample, and the application method is:

Take a Mycobacterium tuberculosis suspension sample and mix it with the lysis solution in a volume ratio of 0.25-1:1.5-6, then mix it at 25-40°C and let it stand for 30-60 minutes to complete the nucleic acid release.

Preferably, the lysis solution is used for nucleic acid release from a tongue swab sample containing Mycobacterium tuberculosis pathogen cells, and the application method is:

Immerse the tongue swab sample containing Mycobacterium tuberculosis pathogen cells in the lysis solution, rotate the tongue swab in the lysis solution for 15-30 circles, then discard the tongue swab, and let the lysis solution stand at 25-40°C for 30-60 minutes to complete the release of nucleic acid.

Preferably, the lysis solution is used for nucleic acid release from sputum samples containing Mycobacterium tuberculosis pathogen cells, and the application method is:

1) Add 2-4 times the volume of 4% sodium hydroxide solution to the sputum sample containing Mycobacterium tuberculosis pathogen cells, shake and mix, and then stand for liquefaction for 30 minutes;

2) taking 500 μL of the liquefied sample obtained in step 1) into a 1-2 mL container, centrifuging and discarding the supernatant;

3) adding 1 mL of saline to the sample obtained in step 2) to resuspend the precipitate;

4) Centrifuge again, discard the supernatant, and add 0.05-2 mL of lysis buffer;

5) Mix well and let stand for 30-60 min at 25-40°C to complete nucleic acid release.

The beneficial effects of the present invention are:

The present invention provides a lysis solution for releasing nucleic acid of Mycobacterium tuberculosis. Compared with the commonly used lysis solution of Mycobacterium tuberculosis, the lysis solution of the present invention does not require high-temperature heating, mechanical vibration, ultrasound and other additional steps during use, does not require equipment assistance, is easy to operate, has low energy consumption, and has low dependence on professional environment and professionals.

The enzyme components in the lysate provided by the present invention have a mild and efficient nucleic acid release effect on the cell wall of Gram-positive bacteria, and are not only applicable to Mycobacterium tuberculosis, but also have universal applicability to Gram-positive bacteria;

The lysate provided by the present invention is mild and efficient in the process of nucleic acid release, has no violent reaction, will not damage nucleic acid substances, and is more friendly to downstream nucleic acid amplification and detection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a comparison curve of nucleic acid release effects of three lysates used in Example 1;

FIG2 is a comparison of CT values of three lysates used in Example 1 after nucleic acid release;

FIG3 shows the effect of the lysis solution of Example 1 on nucleic acid release of 8 clinical samples of Mycobacterium tuberculosis.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

The main sources of raw materials are as follows:

7H9 powder was from Haibo Biotechnology (#HB9233), glycerol (66258P), Tween-20 (89190EA), potassium chloride (80636BC), Tris-HCl (E8026), and EDTA (E8032) were from Adamas life, H37Ra attenuated strain was from Beina Biotechnology Co., Ltd. (263834), lysozyme (L8402), colorless peptidase (A3547), chitinase (C8241), and mutanolysin (M9901) were from Sigma Aldrich, lytic enzyme was from Solebao Biotechnology Co., Ltd. (L8141), and TaqManqPCR mix and primer probe sequence synthesis were from Shanghai Shenggong Biotechnology Co., Ltd.

Example 1

S1. Mycobacterium tuberculosis bacterial culture

Preparation of 7H9 liquid culture medium: Take 4.7 g 7H9 powder, 10 mL 50% glycerol and 5 mL 50% Tween-20 and dissolve them in PBS to make up to 1 L.

Inoculation: Inoculate the attenuated Mycobacterium tuberculosis strain H37Ra into the liquid culture medium and seal it.

Culture: Culture at 37°C with shaking for 4-5 weeks until obvious colonies appear.

S2. Lysis buffer preparation

A lysis buffer was prepared in which the final concentrations of the components were as follows: lysozyme 50 μg/mL, colorless peptidase 100 μg/mL, mutanolysin 200 μg/mL, lytic enzyme 200 μg/mL, chitinase 50 μg/mL, Tween-20 0.1 wt%, potassium chloride 0.1 M, Tris-HCl 100 mM, EDTA 50 mM, dissolved in 1 mL of sterile water and mixed well for later use.

S3. Mycobacterium tuberculosis (H37Ra) nucleic acid release

Take 20 μL of the bacterial solution in step S1 and mix it with 120 μL of the lysis solution in step S2. After mixing, place it at room temperature for 1 hour to complete the nucleic acid release.

4. Evaluation of nucleic acid release effect

In order to evaluate the effect of the lysis solution of the present invention, a commercial thermal lysis solution (Shengxiang Biotechnology Co., Ltd.) and an ultrasonic lysis solution (Zeye Bio, ZY6SL1036) for lysing Mycobacterium tuberculosis were also used to release nucleic acids from Mycobacterium tuberculosis suspensions of the same concentration, and a Mycobacterium tuberculosis qPCR detection kit was designed to detect and compare the lysis effects of three different lysis solutions on combined mycobacteria.

Primer probe design for H37Ra nucleic acid amplification:

Upstream primer: 5’-GCGATACGCAGGGCGTCGGT-3’

Downstream primer: 5’-ACGCGCAGACTCTAGGCACC-3’

Probe: 5’FAM-TGCGCCACCTCGGCCACACACA-BHQ3’

Reaction system:

TaqMan qPCR mix (2×) 12.5μL Upstream primer (10 μM) 2μL Downstream primer (10 μM) 2μL Probe (10 μM) 1μL Template (cleavage product) 2.5μL water 5μL

Reaction procedure:

Step 1: UNG enzyme reaction, 50℃/2min;

Step 2: Taq enzyme activation, 95°C/5 minutes;

Step 3: denaturation, 95°C/15 seconds;

Step 4: Annealing, 57°C/30 seconds, collect fluorescence.

Step 3 and step 4 are repeated 45 times.

The nucleic acid is released from the same H37Ra bacterial solution using three lysates. The nucleic acid release method of the lysate prepared in Example 1 is as shown in step S3 above;

The method of releasing nucleic acid from H37Ra bacterial solution using commercial pyrolysis solution (Shengxiang Bio-Pyrolysis Agent) is as follows: 20 μL bacterial solution is mixed with 100 μL pyrolysis agent and then heated at 100°C for 15 min;

The method of using ultrasonic lysis solution (Zeye Biological Ultrasonic Lysis Agent) to release nucleic acid from H37Ra bacterial solution is as follows: 20 μL of bacterial solution is mixed with 100 μL of lysis agent and then maintained at 40 kHz and 100% power for 20 minutes;

The bacterial solution after release was taken as a qPCR sample and tested using the above steps and system. The results are shown in Figures 1 and 2. It can be seen that the lysis solution provided by the present invention has basically the same nucleic acid release effect as the commercial thermal lysis agent, but the lysis solution does not require heating operation. The lysis solution provided by the present invention has better effect than the ultrasonic lysis solution.

Example 2

The lysis solution in Example 1 was used to release nucleic acid from 8 clinical samples of Mycobacterium tuberculosis tongue swabs (6 positive and 2 negative), the swabs were immersed in 1 mL of lysis solution, the swabs were discarded after 30 rotations, the lysis solution was placed in a 30°C environment for 1 hour, and the solution after release was taken as a qPCR sample and tested using the detection steps and system in Example 1. The test results are shown in Figure 3, and it can be seen that the Mycobacterium tuberculosis nucleic acid in the 6 positive samples was successfully released and the detection was completed.

Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims (10)

1. A lysis solution for releasing nucleic acid of Mycobacterium tuberculosis, characterized in that it comprises an enzyme component and a buffer component; wherein the enzyme component comprises lysozyme, colorless peptidase, lytic enzyme, mutanolysin, and chitinase, and the buffer component comprises Tween-20, potassium chloride, Tris-HCl, and EDTA; when used, the enzyme component and the buffer component are mixed evenly. 2. The lysate for releasing nucleic acid from Mycobacterium tuberculosis according to claim 1, characterized in that, based on the final concentration in the lysate obtained after the enzyme component and the buffer component are evenly mixed, the concentration of each component in the enzyme component is: lysozyme 20-200 μg/mL, colorless peptidase 50-600 μg/mL, mutanolysin 100-1000 μg/mL, lyticase 100-500 μg/mL, and chitinase 20-100 μg/mL. 3. The lysate for releasing nucleic acid from Mycobacterium tuberculosis according to claim 2, characterized in that, based on the final concentration in the lysate obtained after the enzyme component and the buffer component are evenly mixed, the concentration of each component in the buffer component is: Tween-20 0.05wt%-0.4wt%, potassium chloride 0.05-1M, Tris-HCl 50-500mM, EDTA 25-250mM. 4. The lysis solution for releasing nucleic acid of Mycobacterium tuberculosis according to claim 1, characterized in that the pH value of the lysis solution is 7.4-8.0. 5. The lysate for releasing nucleic acid of Mycobacterium tuberculosis according to claim 1, characterized in that, based on the final concentration in the lysate obtained after the enzyme component and the buffer component are evenly mixed, the concentration of each component is: lysozyme 50 μg/mL, colorless peptidase 100 μg/mL, mutanolysin 200 μg/mL, lytic enzyme 200 μg/mL, chitinase 50 μg/mL, Tween-20 0.1wt%, potassium chloride 0.1M, Tris-HCl 100mM, EDTA 50mM. 6. Use of the lysis solution according to any one of claims 1 to 5 in the release of nucleic acid from Mycobacterium tuberculosis. 7. The use according to claim 6, characterized in that the lysis temperature of the lysis solution for releasing nucleic acid of Mycobacterium tuberculosis is 25-40°C. 8. The use according to claim 7, characterized in that the lysis solution is used to release nucleic acid from a Mycobacterium tuberculosis suspension sample, and the application method is: Take a Mycobacterium tuberculosis suspension sample and mix it with the lysis solution in a volume ratio of 0.25-1:1.5-6, then mix it at 25-40°C and let it stand for 30-60 minutes to complete the nucleic acid release. 9. The use according to claim 8, characterized in that the lysate is used to release nucleic acid from a tongue swab sample containing Mycobacterium tuberculosis pathogen cells, and the use method is: Immerse the tongue swab sample containing Mycobacterium tuberculosis pathogen cells in the lysis solution, rotate the tongue swab in the lysis solution for 15-30 circles, then discard the tongue swab, and let the lysis solution stand at 25-40°C for 30-60 minutes to complete the release of nucleic acid. 10. The use according to claim 7, characterized in that the lysate is used to release nucleic acid from a sputum sample containing Mycobacterium tuberculosis pathogen cells, and the use method is: 1) Add 2-4 times the volume of 4% sodium hydroxide solution to the sputum sample containing Mycobacterium tuberculosis pathogen cells, shake and mix, and then stand for liquefaction for 30 minutes; 2) taking 500 μL of the liquefied sample obtained in step 1) into a 1-2 mL container, centrifuging and discarding the supernatant; 3) adding 1 mL of saline to the sample obtained in step 2) to resuspend the precipitate; 4) Centrifuge again, discard the supernatant, and add 0.05-2 mL of lysis buffer; 5) Mix well and let stand for 30-60 min at 25-40°C to complete nucleic acid release.