CN113637668A - Kit for simultaneously extracting pathogenic bacteria DNA of blood plasma and blood cells and application thereof - Google Patents
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Abstract
The invention relates to the technical field of molecular biology, in particular to a kit for simultaneously extracting DNA of pathogenic bacteria of blood plasma and blood cells and application thereof. Compared with the prior art, the method separates the plasma and the blood cells in the blood sample, firstly extracts the DNA of pathogenic bacteria in the blood cells, then combines the DNA with the plasma and purifies to obtain the DNA of all pathogenic bacteria in the blood sample, and provides a comprehensive and accurate pathogen detection result for clinical rapid detection; the obtained DNA has high purity, can be directly used for experiments such as PCR, enzyme digestion and the like, has simple and quick detection process, and can obtain the DNA of all bacteria in blood within 1 hour.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a kit for simultaneously extracting pathogenic bacteria DNA of blood plasma and blood cells and application thereof.
Background
With the continuous emergence of new pathogens and the abuse of antibiotics, pathogenic drug-resistant bacteria are continuously increased, the common infection pathogen spectrum is changed, and the novel infectious diseases and susceptible people are continuously increased. Conventional microbiological diagnostic techniques include: growth and isolation of microorganisms in culture, detection of pathogen-specific antibodies (serology) or antigens, and molecular identification of microbial nucleic acids (DNA or RNA) are typically performed by PCR. The disease condition deterioration caused by microbial infection is very rapid, so that how to rapidly, accurately and timely detect pathogenic microorganisms and accurately provide a pathogenic detection result for clinic in clinical medical diagnosis becomes a development trend of the detection technology at the present stage.
Metagenomic Next Generation Sequencing (mNGS) technology has been used clinically since its first use in diagnosing patients with central nervous system hookbody disease in 2014. When we performed the mNGS test on a clinical sample, we can obtain the gene sequence of human and the gene sequence of microorganism at the same time. Besides good detection performance in sputum, throat swab, alveolar lavage fluid, cerebrospinal fluid and other samples, the blood sample is also an important application field of the mNGS, and an effective solution is provided for solving the diagnosis problem of patients with clinical difficult infection. The current means for NGS detection in blood samples mainly aim at free nucleic acids (cfDNA) in plasma. However, it is well known that leukocytes phagocytose pathogenic microorganisms that invade the blood. Most of the bacteria are phagocytized by leukocytes and destroyed and dissolved by lysosomes, and some few of the bacteria are not destroyed and grow and propagate in the leukocytes and spread to other parts along with the leukocytes, and the bacteria are called intracellular parasitic bacteria, such as tubercle bacillus, typhoid bacillus, listeria monocytogenes and the like. Thus, since it is highly likely that misdiagnosis of a disease will be caused by detecting only plasma-free nucleic acids and omitting blood cell-parasitic bacteria, it is helpful to fully diagnose a disease only by fully extracting the DNA of all pathogenic bacteria in plasma and blood cells.
Disclosure of Invention
Aiming at the defects of the background technology, the invention provides the kit for simultaneously extracting the DNA of the pathogenic bacteria of the blood plasma and the blood cells and the application thereof, and solves the problem that only the free nucleic acid of the blood plasma is detected and the parasitic bacteria of the blood cells are ignored, thereby realizing the rapid and comprehensive detection of all the pathogens in the blood and providing an accurate pathogen detection result for clinic.
On one hand, the invention provides a kit for simultaneously extracting the DNA of pathogenic bacteria of blood plasma and blood cells, and the key points are that: comprises anticoagulant, erythrocyte lysate, leukocyte lysate, RNase A, protease K, bacterial cell lysate and protein precipitator.
Preferably, the anticoagulant comprises natural anticoagulant and Ca2+One of chelating agents.
Preferably, the protein precipitating agent comprises one of organic solvent, SDS, Tween 20, Span 85 and guanidine hydrochloride.
On the other hand, the invention provides the application of the kit for simultaneously extracting the DNA of the pathogenic bacteria of the blood plasma and the blood cells, and the key point is that the extraction method comprises the following steps:
s1, adding an anticoagulant into the blood sample, and performing centrifugal separation to obtain upper plasma and lower blood cells;
s2, adding erythrocyte lysate into the blood cells, mixing evenly until the erythrocytes are completely lysed, centrifuging and separating, and taking leukocyte sediment;
s3, adding the leukocyte lysate, RNase A and protease K into the leukocyte sediment, mixing uniformly until the leukocytes are completely lysed, centrifuging and separating to obtain sediment;
s4, adding the bacterial cell lysate and protease K into the precipitate obtained in the step S3, uniformly mixing until the bacterial cells are completely lysed, carrying out centrifugal separation, and taking supernatant;
s5, mixing the supernatant plasma in the S1 and the supernatant liquid obtained in the S4, adding a protein precipitator, uniformly mixing to precipitate the protein in the system, and performing centrifugal separation to obtain the supernatant liquid;
s6, transferring the supernatant obtained in the step S5 to a Clean column, filtering and removing waste liquid;
s7, adding rinsing liquid into the filtered Clean column, eluting impurities, removing waste liquid, adding the rinsing liquid again for elution, and circulating for 1-5 times;
s8, adding the eluent into the eluted Clean column, and eluting to obtain the DNA of the blood pathogenic bacteria.
Preferably, the mass ratio of the blood cells to the erythrocyte lysate in S2 is 1 (3-5).
Preferably, in the S3, the cracking temperature is 30-40 ℃, and the cracking time is 10-30 min.
Preferably, in the S4, the cracking temperature is 50-60 ℃, and the cracking time is 10-30 min.
Preferably, in the S6-S8, the Clean column is placed in a centrifuge for centrifugal treatment for acceleration of separation.
Preferably, the elution in S7 is circulated for 1-2 times.
Preferably, in S7, the Clean column after the last elution is centrifuged for 1 to 5min to remove the residual rinse solution.
Compared with the prior art, the invention has the following beneficial effects:
the method separates the plasma and the blood cells in the blood sample, firstly extracts the DNA of pathogenic bacteria in the blood cells, then combines the DNA with the plasma and purifies to obtain the DNA of all pathogenic bacteria in the blood sample, and provides a comprehensive and accurate pathogenic bacteria detection result for clinical rapidness, so that the method is suitable for multi-species blood, and is suitable for the DNA of whole blood pathogenic bacteria, including plasma free DNA and intracellular bacteria DNA; the obtained DNA has high purity, can be directly used for experiments such as PCR, enzyme digestion and the like, has simple and quick detection process, and can obtain the DNA of all bacteria in blood within 1 hour.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. Generally, terms used in connection with cellular and molecular biology, as well as chemistry, are well known and commonly used in the art. Certain experimental techniques, not specifically defined, are generally performed according to conventional methods already known in the art and as described in various comprehensive and more specific references cited and discussed in the present specification.
Reagent: the extraction-related reagents were purchased from Takara Shuzo (Dalian) Co., Ltd., and the PCR-related reagents were purchased from Takara Shuzo (Dalian) Co., Ltd.).
Example 1A method for simultaneously extracting pathogenic DNA from blood plasma and blood cells
(1) Separating plasma and blood cells: taking 2-3 ml of a blood sample, and adding an anticoagulant; centrifuging to separate plasma and blood cells; the upper plasma was aspirated into a 1.5ml ep tube for use;
(2) extracting DNA of intracellular pathogenic bacteria: adding erythrocyte lysate into the lower layer of blood cell sediment, wherein the mass ratio of the blood cells to the erythrocyte lysate is 1:4, uniformly mixing, centrifuging for 5min at the rotating speed of 3000rpm until the erythrocytes are completely lysed, and taking white sediment; adding leukocyte lysate 200 μ L, RNase A20 μ L and protease K20 μ L into the white precipitate, mixing, water-bathing at 37 deg.C for 10min, centrifuging, and removing supernatant; adding 100 μ L bacterial cell lysate and 10 μ L protease K into the precipitate, mixing, bathing at 56 deg.C for 10min, centrifuging at 12000rpm for 10min, and collecting supernatant.
(3) Combining and purifying pathogenic bacteria DNA: mixing the plasma with the supernatant obtained finally in the step (2), adding 200 mu L of protein precipitator for precipitating protein in a reaction system to promote DNA precipitation, then centrifuging for 10min at the rotating speed of 12000rpm, and taking the supernatant; placing the Clean column in a collecting pipe of a centrifugal device, transferring all supernatant liquor to the Clean column, and centrifuging for 1min at the rotating speed of 10000 rpm; taking out Clean column, pouring off waste liquid, putting Clean column back to collecting pipe, adding 400 μ L rinsing liquid, and centrifuging at 8000rpm for 1 min; adding the rinsing liquid again, eluting, centrifuging, taking out the Clean column, pouring off the waste liquid, putting the Clean column back to the collecting pipe again, centrifuging at 10000rpm for 1min, and removing the residual rinsing liquid; putting Clean column into new sterile 1.5ml EP tube, adding 50 μ L eluent, standing for 2min, centrifuging at 12000rpm for 1min, and collecting the tube liquid as blood pathogenic bacteria DNA.
Example 2A method for simultaneously extracting pathogenic DNA from blood plasma and blood cells
(1) Separating plasma and blood cells: selecting 2-3 ml of blood samples, and adding an anticoagulant; centrifuging to separate plasma and blood cells; the upper plasma was aspirated into a 1.5ml ep tube for use;
(2) extracting DNA of intracellular pathogenic bacteria: adding erythrocyte lysate into the lower layer of blood cell sediment, wherein the mass ratio of the blood cells to the erythrocyte lysate is 1:3, uniformly mixing, centrifuging for 5min at the rotating speed of 3000rpm until the erythrocytes are completely lysed, and taking white sediment; adding leukocyte lysate 200 μ L, RNase A20 μ L and protease K20 μ L into the white precipitate, mixing, water-bathing at 30 deg.C for 30min, centrifuging, and removing supernatant; adding 100 μ L bacterial cell lysate and 10 μ L protease K into the precipitate, mixing, bathing at 50 deg.C for 30min, centrifuging at 12000rpm for 10min, and collecting supernatant.
(3) Combining and purifying pathogenic bacteria DNA: mixing the plasma with the supernatant obtained finally in the step (2), adding 200 mu L of protein precipitator for precipitating protein in a reaction system to promote DNA precipitation, then centrifuging for 10min at the rotating speed of 12000rpm, and taking the supernatant; placing the Clean column in a collecting pipe of a centrifugal device, transferring all supernatant liquor to the Clean column, and centrifuging for 1min at the rotating speed of 10000 rpm; taking out Clean column, pouring off waste liquid, putting Clean column back to collecting pipe, adding 400 μ L rinsing liquid, and centrifuging at 8000rpm for 1 min; adding the rinsing liquid again, eluting, centrifuging, taking out the Clean column, pouring off the waste liquid, putting the Clean column back to the collecting pipe again, centrifuging at 10000rpm for 1min, and removing the residual rinsing liquid; putting Clean column into new sterile 1.5ml EP tube, adding 50 μ L eluent, standing for 2min, centrifuging at 12000rpm for 1min, and collecting the tube liquid as blood pathogenic bacteria DNA.
Example 3A method for simultaneously extracting pathogenic DNA from blood plasma and blood cells
(1) Separating plasma and blood cells: selecting 2-3 ml of blood samples, and adding an anticoagulant; centrifuging to separate plasma and blood cells; the upper plasma was aspirated into a 1.5ml ep tube for use;
(2) extracting DNA of intracellular pathogenic bacteria: adding erythrocyte lysate into the lower layer of blood cell sediment, wherein the mass ratio of the blood cells to the erythrocyte lysate is 1:5, uniformly mixing, centrifuging for 5min at the rotating speed of 3000rpm until the erythrocytes are completely lysed, and taking white sediment; adding leukocyte lysate 200 μ L, RNase A20 μ L and protease K20 μ L into the white precipitate, mixing, water-bathing at 40 deg.C for 20min, centrifuging, and removing supernatant; adding 100 μ L bacterial cell lysate and 10 μ L protease K into the precipitate, mixing, bathing at 60 deg.C for 20min, centrifuging at 12000rpm for 10min, and collecting supernatant.
(3) Combining and purifying pathogenic bacteria DNA: mixing the plasma with the supernatant obtained finally in the step (2), adding 200 mu L of protein precipitator for precipitating protein in a reaction system to promote DNA precipitation, then centrifuging for 10min at the rotating speed of 12000rpm, and taking the supernatant; placing the Clean column in a collecting pipe of a centrifugal device, transferring all supernatant liquor to the Clean column, and centrifuging for 1min at the rotating speed of 10000 rpm; taking out Clean column, pouring off waste liquid, putting Clean column back to collecting pipe, adding 400 μ L rinsing liquid, and centrifuging at 8000rpm for 1 min; circularly eluting for 2 times, taking out Clean column, pouring off waste liquid, putting Clean column back to the collecting pipe, centrifuging at 10000rpm for 1min, and removing residual rinsing liquid; putting Clean column into new sterile 1.5ml EP tube, adding 50 μ L eluent, standing for 2min, centrifuging at 12000rpm for 1min, and collecting the tube liquid as blood pathogenic bacteria DNA.
Selecting typhoid bacillus blood samples, wherein each sample is divided into two groups, one group is used for extracting blood pathogenic bacteria DNA by adopting the method of the embodiment 1, the other group is used for extracting pathogenic bacteria DNA in blood plasma by adopting the existing method, and then DNA library building, sequencing and biological information analysis are carried out:
(1) library construction: carrying out ultrasonic breaking on DNA, placing a fragmentation product into a PCR tube, adding reagents according to table 1, placing the PCR tube into a PCR instrument, carrying out DNA damage repair, filling in DNA tail ends, carrying out phosphorylation at a 5 'end and adding an A tail at a 3' end, and carrying out a reaction procedure: the hot cover is opened at 105 ℃ and reacts for 20 minutes at 30 ℃, 15 minutes at 65 ℃ and kept at low temperature of 4 ℃; the reagents are shown in table 1:
TABLE 1
(2) Adding a joint, and purifying magnetic beads: adding corresponding reagents into the system after the reaction is finished according to the following table 2, uniformly mixing by vortex, carrying out microcentrifugation, and placing the PCR tube into a PCR instrument for reaction, wherein the reaction procedure comprises the following steps: the hot cover is opened at 105 ℃, the reaction is carried out for 15 minutes at 20 ℃, and the temperature is kept at 4 ℃; purifying the product by using 60 μ l of magnetic beads according to the magnetic bead purification procedure;
TABLE 2
(3) PCR amplification, magnetic bead purification: adding corresponding reagents into the system after the reaction is finished according to the following table 3, uniformly mixing by vortex, carrying out microcentrifugation, and carrying out the reaction on a PCR instrument according to the set program of the following table 4;
TABLE 3
TABLE 4
After the reaction is finished, purifying the PCR amplification product by using 30 mul of magnetic beads according to the magnetic bead purification process.
(1) NGS sequencing: sequencing was accomplished using the Illumina platform, library results are shown in table 5, and pathogen indicator detection results are shown in table 6:
TABLE 5
TABLE 6
From the above table, it can be known that the existing method can only extract cfDNA in blood plasma by extracting pathogenic bacteria DNA in blood samples, but the method of the present invention can simultaneously extract and detect pathogenic bacteria DNA in blood plasma and blood cells, the DNA extraction concentration is far greater than that of the existing method, the pathogenic bacteria detection effect is obviously better than that of the existing method, and the present invention is helpful for comprehensive disease diagnosis.
Finally, it should be noted that the above-mentioned description is only a preferred embodiment of the present invention, and those skilled in the art can make various similar representations without departing from the spirit and scope of the present invention.
Claims (10)
1. An extraction method for simultaneously extracting pathogenic bacteria DNA of blood plasma and blood cells is characterized by comprising the following steps:
s1, adding an anticoagulant into the blood sample, and performing centrifugal separation to obtain upper plasma and lower blood cells;
s2, adding erythrocyte lysate into the blood cells, mixing evenly until the erythrocytes are completely lysed, centrifuging and separating, and taking leukocyte sediment;
s3, adding the leukocyte lysate, RNase A and protease K into the leukocyte sediment, mixing uniformly until the leukocytes are completely lysed, centrifuging and separating to obtain sediment;
s4, adding the bacterial cell lysate and protease K into the precipitate obtained in the step S3, uniformly mixing until the bacterial cells are completely lysed, carrying out centrifugal separation, and taking supernatant;
s5, mixing the supernatant plasma in the S1 and the supernatant liquid obtained in the S4, adding a protein precipitator, uniformly mixing to precipitate the protein in the system, and performing centrifugal separation to obtain the supernatant liquid;
s6, transferring the supernatant obtained in the step S5 to a Clean column, filtering and removing waste liquid; s7, adding rinsing liquid into the filtered Clean column, eluting impurities, removing waste liquid, adding the rinsing liquid again for elution, and circulating for 1-5 times;
s8, adding the eluent into the eluted Clean column, and eluting to obtain the DNA of the blood pathogenic bacteria.
2. The method for simultaneously extracting the DNA of pathogenic bacteria of blood plasma and blood cells as claimed in claim 1, wherein: the volume ratio of the blood cells to the erythrocyte lysate in the S2 is 1 (3-5).
3. The method for simultaneously extracting the DNA of pathogenic bacteria of blood plasma and blood cells as claimed in claim 1, wherein: in the S3, the cracking temperature is 30-40 ℃, and the cracking time is 10-30 min.
4. The method for simultaneously extracting the DNA of pathogenic bacteria of blood plasma and blood cells as claimed in claim 1, wherein: in the S4, the cracking temperature is 50-60 ℃, and the cracking time is 10-30 min.
5. The method for simultaneously extracting the DNA of pathogenic bacteria of blood plasma and blood cells as claimed in claim 1, wherein: in the S6-S8, the Clean column is placed in a centrifugal device for centrifugal treatment for accelerating separation.
6. The method for simultaneously extracting the DNA of pathogenic bacteria of blood plasma and blood cells as claimed in claim 1, wherein: and the elution is circulated for 1-2 times in the S7.
7. The method for simultaneously extracting the DNA of pathogenic bacteria of blood plasma and blood cells as claimed in claim 1, wherein: in the step S7, the Clean column after the last elution is centrifuged for 1-5min, and the residual rinsing liquid is removed.
8. A kit for simultaneously extracting and detecting pathogenic bacteria DNA of blood plasma and blood cells is characterized in that: comprising the anticoagulant, erythrocyte lysate, leukocyte lysate, RNase A, protease K, bacterial cell lysate, protein precipitant used in the extraction method according to any one of claims 1 to 7.
9. The kit for simultaneously extracting and detecting the DNA of the pathogenic bacteria of the blood plasma and the blood cells according to claim 8, which is characterized in that: the anticoagulant comprises natural anticoagulant and Ca2+One of chelating agents.
10. The kit for simultaneously extracting and detecting the DNA of the pathogenic bacteria of the blood plasma and the blood cells according to claim 8, which is characterized in that: the protein precipitant comprises one of organic solvent, SDS, Tween 20, Span 85 and guanidine hydrochloride.
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| CN114350749A (en) * | 2022-01-04 | 2022-04-15 | 长沙金域医学检验实验室有限公司 | Free nucleic acid library building method based on metagenome sequencing |
| CN116590283A (en) * | 2023-07-17 | 2023-08-15 | 北京微岩医疗器械有限公司 | Cell binding enhancer and method for removing human free nucleic acid |
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| CN114350749A (en) * | 2022-01-04 | 2022-04-15 | 长沙金域医学检验实验室有限公司 | Free nucleic acid library building method based on metagenome sequencing |
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| CN116590283A (en) * | 2023-07-17 | 2023-08-15 | 北京微岩医疗器械有限公司 | Cell binding enhancer and method for removing human free nucleic acid |
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Application publication date: 20211112 |