CN114717228A

CN114717228A - Method for rapidly extracting genome of sputum sample by using paramagnetic particle method

Info

Publication number: CN114717228A
Application number: CN202210541280.1A
Authority: CN
Inventors: 申洪杰; 任文玉
Original assignee: Guangzhou Diao Gene Technology Co ltd
Current assignee: Guangzhou Diao Gene Technology Co ltd
Priority date: 2022-05-17
Filing date: 2022-05-17
Publication date: 2022-07-08

Abstract

The invention relates to a method for rapidly extracting a genome of a sputum sample by using a magnetic bead method, which is characterized in that the method comprises the following steps of extracting by using a reagent combination, wherein the reagent combination comprises a first reagent and a second reagent, the first reagent comprises DTT (dithiothreitol), guanidinium isothiocyanate, sodium acetate, Tris-HCl, EDTA, NaCl, triton and SDS; the second reagent comprises Tris, EDTA, NaCl and PEG 2000. The invention also provides a corresponding extraction method, the liquefaction and the genome extraction of the sputum sample are completed in one reaction tube by combining the reagent combination with the magnetic bead method, the operation steps are obviously simplified, the operation time is shortened, and the experimental error caused by the operation is reduced due to the reduction of the operation complexity; in addition, the reagent used by the extraction method has low cost, saves the cost of nucleic acid extraction, and has great advantages in the application in the fields of clinic and scientific research, etc.

Description

Method for rapidly extracting sputum sample genome by using paramagnetic particle method

Technical Field

The invention relates to the technical field of microbial molecular biology detection, in particular to a method for rapidly extracting a sputum sample genome by using a paramagnetic particle method.

Background

Respiratory viral infections are transmissible, typically by salivary droplet, and in addition, direct or indirect intimate contact with the patient increases the probability of respiratory viral transmission. One of the major causes of high mortality and morbidity in the world is viral infection of the lower respiratory tract. With the rise and development of molecular diagnosis technology, the technology is widely applied to the screening and diagnosis process of various diseases, the molecular diagnosis technology monitors the diseases by detecting target DNA/RNA, and has the advantages of reliable detection result, large detection flux and the like.

In the detection of respiratory infectious viruses, molecular diagnosis technology provides a convenient, rapid, accurate and efficient diagnosis mode. Generally, when a molecular diagnosis technology is used for detecting respiratory system diseases, most detection samples are sputum samples which contain various pathogenic microorganisms, various inflammatory cells, necrotic exfoliated mucosal epithelial cells, tumor cells and the like, and the sputum samples have the advantages of convenience in sampling, simple and practical sampling method and large sampling amount. However, sputum samples also have the characteristics of high viscosity, complex sample components and the like, and certain obstacles are formed to molecular diagnosis. Therefore, the sputum sample is liquefied before detection, so that the viscosity of the sample is reduced, and interfering components in the sample are removed, so that the reliability of the detection result is improved. NaOH method, DTT (dithiothreitol) method and protease method are commonly used as methods for liquefying a sputum sample. The DTT liquefied sputum mainly destroys disulfide bonds in the sputum, so that protein components in the sputum are degraded, and genetic materials in the sputum are released.

With the continuous development of genetic material extraction technology, the magnetic bead method is favored by molecular diagnosis technology due to the characteristics of rapidness, simple operation, capability of extracting genome in large batch, safety, no toxicity and the like. In the extraction of respiratory system diseases, the experimental operation of extracting genome by magnetic bead method is generally divided into the steps of liquefaction, cracking, combination, elution, washing and the like, and the operation needs to be carried out in different reaction tubes, so the operation is complicated and time-consuming.

Chinese patent document CN107904232A discloses a method for rapidly extracting nucleic acid from sputum, which uses organic solvent harmful and uses many kinds of reagents in the operation of liquefying sputum sample, the operation steps are complicated, and the liquefaction of the sputum and the extraction of genome need to be completed separately. Chinese patent document CN108265105A discloses a method for pre-treating sputum for rapid nucleic acid detection, in which a magnetic bead method is used to extract genome from a sputum sample, but in this technique, liquefaction of the sputum sample and extraction of genome are performed in separate reaction tubes, which requires many steps and is likely to cause pollution.

Therefore, those skilled in the art hope to develop a new method for extracting genome from sputum sample to solve the problems of complex reagent components, difficult operation, tedious steps, long time consumption, easy pollution, etc. in the prior art.

Disclosure of Invention

The invention aims to provide a method for rapidly extracting a sputum sample genome by using a paramagnetic particle method, which can realize the liquefaction of a sputum sample and the extraction of the sputum sample genome in the same reaction tube, so that the nucleic acid extraction of the sputum sample becomes convenient and rapid.

To this end, in a first aspect, the present invention provides a reagent combination comprising a first reagent and a second reagent; the first reagent comprises 1-5% (w/w) Dithiothreitol (DTT), 1-5M Guanidinium Isothiocyanate (GITC), 10-500mM sodium acetate, 5-500mM Tris-HCl, 1-200mM EDTA, 0.2-5M NaCl, 0.1-1% (w/w) triton, 0.2-5% (w/w) SDS; the second reagent comprises 1-500mM Tris, 1-200mM EDTA, 0.1-5M NaCl, 1-15% (w/w) PEG 2000.

Further, in the first reagent, the mass percentage of dithiothreitol is 1-5%, for example, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.

Further, in the first reagent, guanidinium isothiocyanate is present at a concentration of 1-5M, such as 1M, 1.5M, 2M, 2.5M, 3M, 3.5M, 4M, 4.5M, 5M, and the like.

Further, in the first reagent, the concentration of sodium acetate is 10 to 500mM, for example, 10mM, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, etc.

Further, in the first reagent, the concentration of Tris-HCl is 10-500mM, for example, 10mM, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, etc.

Further, in the first reagent, EDTA is present at a concentration of 5 to 200mM, for example, 5mM, 10mM, 50mM, 100mM, 150mM, 200mM, etc.

Further, in the first reagent, the concentration of NaCl is 0.2-5M, for example, 0.2M, 0.5M, 1M, 2M, 3M, 4M, 5M, etc.

Further, in the first reagent, the Triton is Triton X-100(Triton X-100), and the percentage by mass of Triton is 0.1 to 1%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, and the like.

Further, the percentage by mass of SDS in the first reagent is 0.2 to 5%, for example, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, etc.

Further, in the second reagent, Tris is at a concentration of 5 to 500mM, for example, 5mM, 10mM, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, etc.

Further, in the second reagent, EDTA is present at a concentration of 5 to 200mM, for example, 5mM, 10mM, 50mM, 100mM, 150mM, 200mM, etc.

Further, in the second reagent, the concentration of NaCl is 0.2-5M, for example, 0.2M, 0.5M, 1M, 2M, 3M, 4M, 5M, etc.

Further, in the second reagent, the mass percentage of PEG2000 is 2-15%, for example, 2%, 5%, 10%, 15%, and the like.

In some embodiments, the first reagent comprises 1-4% (w/w) Dithiothreitol (DTT), 2-5M Guanidinium Isothiocyanate (GITC), 50-200mM sodium acetate, 50-200mM Tris-HCl, 50-150mM EDTA, 0.2-5M NaCl, 0.1-1% (w/w) Triton X-100, 0.5-5% (w/w) SDS; the second reagent comprises 100-300mM Tris, 100-200mM EDTA, 1-3M NaCl and 5-15% (w/w) PEG 2000.

Further, the volume ratio of the first reagent to the second reagent is 1: 0.8-1.2; such as 1:0.8, 1:0.85, 1:1, 1:1.1, 1:1.2, etc.

In a second aspect of the present invention, a method for extracting a genome of a test sample is provided, which includes:

s1: adding 0.2-2 times of the first reagent of the invention into a sample to be detected, uniformly mixing, and incubating for 15-30min at 60-75 ℃ to obtain a first reaction solution;

s2: adding magnetic beads into the first reaction solution, incubating at room temperature, and performing vortex oscillation at least once during the incubation at room temperature to obtain a second reaction solution;

s3: carrying out first magnetic bead adsorption on the second reaction solution, discarding the solution, and then adding a second reagent of the invention, wherein the volume of the second reagent is 0.8-1.2 times of that of the first reagent; vibrating and uniformly mixing to obtain a third reaction solution;

s4: and carrying out second magnetic bead adsorption on the third reaction solution, discarding the solution, washing with an ethanol solution, and recovering to obtain magnetic beads combined with the sample genome.

According to the technical scheme of the invention, the prepared magnetic beads combined with the sample genome can be directly used for subsequent PCR amplification and the like; alternatively, DNA is recovered from the magnetic beads to which the genome of the sputum sample is bound, and a method for recovering DNA from the magnetic beads to which nucleic acid is bound is known to those skilled in the art.

Further, the sample to be detected can be selected from serum, plasma, tissue extract, swab lotion, urine, sputum and the like; sputum is preferred.

According to the technical scheme, the method can be suitable for extracting the genomes in various samples to be detected, and particularly, the existing defects of the sputum sample genome extraction are optimized and improved, so that the method is particularly suitable for extracting the sputum sample genome. The extraction method provided by the invention can realize the liquefaction of the sputum sample and the extraction of the genome of the sputum sample in the same reaction tube together, so that the nucleic acid extraction of the sputum sample becomes convenient and fast.

Further, in step S1, the incubation temperature may be selected from 60 ℃, 65 ℃, 70 ℃, 75 ℃, etc.; the incubation time may be selected from 15min, 20min, 25min, 30min, etc.

Further, in step S2, the magnetic beads are nano magnetic beads; the particle size of the nano magnetic beads is preferably 20-100 nm.

In some embodiments, the magnetic beads are superparamagnetic silica nanomagnetic beads.

Further, in step S2, the number of times of the vortex oscillation is 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more.

Further, in step S4, the concentration of the ethanol solution is 75-85%; e.g., 75%, 80%, 85%, etc.

Further, in step S4, the step of washing with an ethanol solution includes: and adding an ethanol solution into the magnetic beads, then performing magnetic bead adsorption and discarding the solution.

In some embodiments, the extraction method comprises the steps of:

(1) taking 0.3-1.5mL of sputum sample, adding 350-;

(2) adding 1-20 mu L of magnetic beads into the first reaction solution, incubating at room temperature for 3-5min, and performing vortex oscillation for at least one time to obtain a second reaction solution;

(3) transferring the reaction tube of the second reaction solution to a magnetic frame for adsorption for 1-5min, and pouring or sucking away the solution; then adding 300-;

(4) transferring the reaction tube of the third reaction solution to a magnetic frame for adsorption for 1-5min, and pouring or sucking away the solution; then adding an ethanol solution with the concentration of 80%, transferring the reaction tube to a magnetic frame for adsorption for 1-5min after vortex oscillation, and pouring or absorbing the solution;

(5) and (5) repeating the step (4) once or more than two times, and then collecting the magnetic beads by using a magnetic rod to obtain the magnetic beads combined with the sample genome.

In a third aspect of the present invention, there is provided a method for detecting a genome of a test sample, comprising: extracting a genome from a sample to be tested by the extraction method according to the second aspect of the present invention, and then detecting the extracted genome.

According to the technical scheme of the invention, the detection has non-disease diagnosis or treatment purposes.

Further, the detection includes immunoassay, RCP detection, and the like.

Compared with the prior art, the technical scheme of the invention has the following progress:

(1) the invention provides a reagent combination, which can realize the liquefaction and genome extraction of a sample to be detected, in particular a sputum sample, in the same reaction tube; and the reagent combination has fewer component types, the used reagent has low cost and low toxicity, the cost of nucleic acid extraction is saved, and the reagent combination has great advantages in the application in the fields of clinic, scientific research and the like.

(2) The invention also provides a corresponding extraction method, the steps of liquefying the sputum sample and extracting the genome by the magnetic bead extraction method can be completed in one reaction tube by using the extraction method, the step of extracting the genome from the sputum sample is greatly simplified, the whole process can be completed within one hour, the operation time is saved, the complexity of the operation is reduced, and the experimental error caused by the operation reason is reduced.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:

FIG. 1: the invention provides a flow chart of an extraction method of a sputum sample genome;

FIG. 2: the invention provides a sensitivity detection result of the extraction method of the sputum sample genome;

FIG. 3: the invention provides a repeatability detection result of the extraction method of the sputum sample genome;

FIG. 4: the comparison result of the sputum sample genome extraction method and the NaOH solution sputum extraction method provided by the invention is obtained;

FIG. 5: and comparing the inhibition rates of different sputum treatment methods.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Example 1

In this embodiment, nucleic acid extraction is performed on a plurality of bacteria liquid samples with standard concentrations, and the specific steps are as follows:

(1) respectively taking 300 mu L of mycobacterium tuberculosis (M.tuberculosis) bacterial liquid with the following standard concentration into a No. 1-4 centrifuge tube: 100cFU/mL, 1000cFU/mL, 5000cFU/mL, 10000 cFU/mL;

(2) respectively adding 500 μ L buffer A (2% DTT, 4M GITC, 100mM sodium acetate, 0.3% Triton X-100, 100mM Tris-HCI, 100mM EDTA, 2M NaCl, 3% SDS) into No. 1-4 centrifuge tubes, vortexing, shaking, mixing, and incubating at 70 deg.C for 20 min;

(3) respectively adding 15 mu L of magnetic beads (Magpure Particles N) into the No. 1-4 centrifuge tube treated in the step (2), standing at room temperature for 5min, and performing vortex oscillation for multiple times;

(4) transferring the centrifuge tube treated in the step (3) to a magnetic frame for adsorption for 3min, and pouring off the solution;

(5) adding 600 mu L of buffer B (200mM Tris, 150mM EDTA, 2M NaCl, 10% PEG 2000) into the centrifuge tube treated in the step (4), carrying out vortex oscillation, transferring the centrifuge tube to a magnetic frame for adsorption for 3min, and pouring off the solution;

(6) adding 80% ethanol solution into the centrifugal tube, performing vortex oscillation, transferring the centrifugal tube to a magnetic frame for adsorption for 3min, and discarding the solution;

(7) and (4) repeating the step (6) once, and then collecting the magnetic beads in the centrifugal tube by using a magnetic rod to obtain the magnetic beads combined with the sample genome.

And transferring the prepared magnetic beads combined with the sample genome into an amplification system, detecting nucleic acid in the sample by using an RT-PCR method in the amplification system, and identifying the nucleic acid extraction result by analyzing a fluorescent PCR amplification curve. As shown in FIG. 2, the curves from left to right are 10000cFU/mL, 5000cFU/mL, 1000cFU/mL and 100cFU/mL of the extracted bacterial liquid and the extracted negative sample. It can be seen that the sensitivity of the nucleic acid extraction method provided by the invention to mycobacterium tuberculosis can reach 100 cFU/mL.

Example 2

In this example, 20 samples of 100cFU/mL M.tuberculosis solution were subjected to nucleic acid extraction in the same manner as in example 1. The detection result is shown in fig. 3, the CT mean value of the 20 bacterial liquid sample amplifications is 35, and the CV value is 2.1%. It can be seen that the nucleic acid extraction method provided by the invention has good repeatability of nucleic acid extraction effect.

Example 3

This example compares the extraction method provided by the present invention with NaOH extraction. Since the clinical positive specimen of mycobacterium tuberculosis is infectious, this example employs a simulated clinical sputum positive specimen prepared by directly adding inactivated mycobacterium tuberculosis to clinical negative sputum.

The prepared clinical sputum positive simulation sample is evenly divided into six parts, each 300 mu L, and the extraction method and the NaOH sputum liquefaction method are respectively adopted for nucleic acid extraction and detection. The extraction method is carried out according to the steps of the embodiment 1, and the NaOH solution phlegm-resolving method is carried out according to the following steps:

adding a 4% NaOH solution with the volume twice that of the sputum sample of 300 mu L, standing for 30min, fully liquefying, centrifuging at 13000rpm for 5min, and discarding the supernatant; then washing with 1mL of physiological saline, centrifuging at 13000rpm for 5min, and removing 700 mu L of supernatant by suction; then adding 500 mu L of buffer A, mixing uniformly by vortex oscillation, and incubating for 20min at 70 ℃; the remaining steps are the same as steps (2) to (7) of example 1.

The products obtained by the two extraction methods are subjected to RT-PCR detection, the detection results are shown in figure 4, the nucleic acid detection results obtained by the method and the nucleic acid extraction results obtained by the NaOH liquefied sputum method are respectively shown from left to right, and the CT mean values are respectively 33.25 and 34.31. Therefore, the method provided by the invention can still achieve the same effect of extracting nucleic acid from the liquefied sputum sample after removing the pretreatment of liquefying the sputum sample by using NaOH.

Example 4

In this example, 50 clinical sputum positive samples prepared by directly adding inactivated mycobacterium tuberculosis to clinical negative sputum were subjected to genome extraction and detection, each sputum sample having a volume of 300 μ L, and nucleic acid extraction was performed on the 50 samples using the method of the present invention and the NaOH solution sputum method (the same procedure as in example 3).

The result is shown in figure 5, in 50 cases of simulated clinical sputum positive samples, 3 cases of NaOH solution sputum reduction method false negative samples have the inhibition rate of 6%; the method provided by the invention has no false negative and the inhibition rate is 0. The detection results show that the extraction method provided by the invention can be used for clinical samples and has excellent nucleic acid extraction efficiency when facing the clinical samples.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. A reagent combination comprising a first reagent and a second reagent; the first reagent comprises 1-5% dithiothreitol, 1-5M guanidinium isothiocyanate, 10-500mM sodium acetate, 5-500mM Tris-HCl, 1-200mM EDTA, 0.2-5M NaCl, 0.1-1% triton and 0.2-5% SDS; the second reagent comprises 1-500mM Tris, 1-200mM EDTA, 0.1-5M NaCl and 1-15% PEG 2000.

2. The reagent combination of claim 1, wherein the first reagent comprises 1-4% dithiothreitol, 2-5M guanidinium isothiocyanate, 50-200mM sodium acetate, 50-200mM Tris-HCl, 50-150mM EDTA, 0.2-5M NaCl, 0.1-1% triton, 0.5-5% SDS; the second reagent comprises 100-300mM Tris, 100-200mM EDTA, 1-3M NaCl, 5-15% PEG 2000.

3. The reagent combination of claim 1, wherein the volume ratio of the first reagent to the second reagent is 1:0.8 to 1.2.

4. A method for extracting a genome of a sample to be detected is characterized by comprising the following steps:

s1: adding 0.2-2 times of the first reagent of any one of claims 1-3 into a sample to be tested, mixing, and incubating at 60-75 deg.C for 15-30min to obtain a first reaction solution;

s2: adding magnetic beads into the first reaction solution, incubating at room temperature, and performing vortex oscillation for at least one time during the incubation at room temperature to obtain a second reaction solution;

s3: subjecting the second reaction solution to first magnetic bead adsorption, discarding the solution, and adding a second reagent according to any one of claims 1 to 3, the volume of the second reagent being 0.8 to 1.2 times that of the first reagent; vibrating and uniformly mixing to obtain a third reaction solution;

5. The method of claim 4, wherein the sample is selected from the group consisting of serum, plasma, tissue extract, swab wash, urine, and sputum.

6. The extraction method according to claim 4, wherein in step S2, the magnetic beads are nano magnetic beads; the particle size of the nano magnetic beads is preferably 20-100 nm.

7. The method of claim 4, wherein in step S2, the number of vortex oscillations is 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more.

8. The extraction method according to claim 4, wherein in the step S4, the concentration of the ethanol solution is 75-85%.

Preferably, in step S4, the step of washing with an ethanol solution includes: adding an ethanol solution to the magnetic beads, then carrying out magnetic bead adsorption and discarding the solution.

9. The extraction process according to any one of claims 4 to 8, characterized in that it comprises the following steps:

(1) taking 0.3-1.5mL of sputum sample, adding 350-;

(2) adding 1-20 mu L of magnetic beads into the first reaction solution, incubating at room temperature for 3-5min, and performing vortex oscillation at least once during the incubation period to obtain a second reaction solution;

(4) transferring the reaction tube of the third reaction solution to a magnetic frame for adsorption for 1-5min, and pouring or sucking away the solution; then adding an ethanol solution with the concentration of 80%, transferring the reaction tube to a magnetic frame for adsorption for 1-5min after vortex oscillation, and dumping or absorbing the solution;

(5) and (4) repeating the step (4) once or more than twice, and then collecting the magnetic beads by using a magnetic rod, namely preparing the magnetic beads combined with the sample genome.

10. A method for detecting a genome of a sample to be detected is characterized by comprising the following steps: extracting a genome from a test sample by the extraction method according to any one of claims 4 to 9, and then detecting the extracted genome.