CN116334071A - Nucleic acid extraction kit for detecting pathogenic microorganisms and extraction method thereof - Google Patents
Nucleic acid extraction kit for detecting pathogenic microorganisms and extraction method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a nucleic acid extraction kit for detecting pathogenic microorganisms and an extraction method thereof. The kit comprises: lysate, binding solution, rinsing solution I, rinsing solution II, eluent, stopping solution, magnetic beads, lysate beads and complex enzyme. The invention adopts a mechanical method and an enzymolysis method, optimizes grinding and incubation conditions, shortens the cracking time while improving the DNA yield of gram positive bacteria and fungi, and reduces the fragment loss caused by nucleic acid breakage and subsequent library establishment. Compared with the column extraction method, the magnetic bead method reduces the loss of nucleic acid in the silica gel column purification process, and is beneficial to realizing the extraction automation and standardization.
Description
Technical Field
The invention belongs to the technical field of nucleic acid extraction, and particularly relates to a nucleic acid extraction kit for detecting pathogenic microorganisms and an extraction method thereof.
Background
The pathogenic microorganisms infected with human beings comprise bacteria, fungi, viruses, protozoa, mycoplasma, chlamydia and the like, and the metagenomic sequencing provides a novel method for detecting the composition of the microorganisms without separation culture. The method can realize high-flux and large-scale rapid detection of the pathogenic microbiome. However, pathogenic samples derived from clinical practice, such as alveolar lavage fluid, cerebrospinal fluid, ascites fluid, etc., tend to have low concentrations of microorganisms, with some pathogenic microorganisms being present in very small amounts. In addition, fungi and gram positive bacteria in microorganisms are structurally hard and difficult to lyse. In order to effectively analyze pathogenic microbiota of clinical samples, the extraction method of DNA is particularly important, and the quality of metagenomic sequencing and classification identification is directly related.
Currently, in microbial nucleic acid extraction and purification, mechanical lysis methods, such as bead beating, are considered to be lysed gold standard vertebrae. Since beading is effective in lysing a variety of microorganisms, this method has been widely used for the lysis of gram-positive and negative bacteria. However, most of the DNA obtained after bead beating is sheared into very small fragments, and the DNA obtained after fragment screening has very limited yield and tends to lose specific fragments of some pathogenic microorganisms. In addition, the bead beating method is biased towards a microorganism which is easier to lyse, and DNA fragments which are shorter and easier to shear. Enzymatic hydrolysis methods show relatively consistent lysis efficiency on gram-positive and gram-negative bacteria with minimal shearing, but require longer lysis treatment times. The method is mainly aimed at bacterial lysis, and the nucleic acid yield of fungi is low. Therefore, it has been a urgent problem to extract a sufficient amount of DNA from clinical samples, to increase DNA yields of gram positive bacteria and fungi, and to uniformly reflect the microbial composition of the samples.
Disclosure of Invention
The invention aims to provide a kit capable of improving the DNA yield of gram-positive bacteria and fungi.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided a kit for nucleic acid extraction of a pathogenic microorganism comprising: lysate, binding solution, rinsing solution I, rinsing solution II, eluent, stopping solution, magnetic beads, lysate beads and complex enzyme; the complex enzyme package: lywallase, chitinase, lysostaphin, lysozyme; wherein the enzyme activity ratio among muramidase, chitinase, lysostaphin and lysozyme is as follows: (5-10): (10-30): (25-35): (25-35).
In some embodiments of the invention, the lywallase is 5-10U/mL, chitinase is 10-30U/mL, lysostaphin is 25-35U/mL, and lysozyme is 25-35U/mL.
The lysate can denature proteins, inhibit degradation of DNA by nuclease, precipitate proteins, and provide a buffer system for released DNA to stabilize the DNA. The binding solution can denature proteins, resulting in disruption of cellular structures, while inhibiting nuclease activity. The rinsing liquid I and the rinsing liquid II are mainly used for rinsing DNA and removing residual salts. The eluate dissolves the DNA and provides an environment in which the DNA is stably preserved. The stop solution can denature the protein and stop the enzymatic reaction. The magnetic beads are mainly enriched in nucleic acids by groups on the surface of the magnetic beads. The lysis beads are used for breaking cell walls which are difficult to lyse through mechanical action, so that the lysis efficiency is improved. Complex enzymes are mainly synergistic in cleavage, reducing nucleic acid cleavage caused by excessive mechanical disruption.
In some embodiments of the invention, the lysate comprises: tris-HCl 0.2-0.5M, EDTA 40-50mM, naCl 0.2-0.3M, proClin, 0.01-0.05%.
In some embodiments of the invention, the binding fluid comprises: 3-5M guanidine hydrochloride, 5-10% Triton X-100 and 5-10% Tween.
In some embodiments of the invention, the tween may be tween 20, tween 40, tween 60, tween 80.
In some embodiments of the invention, the rinse liquid I comprises: 3-5M guanidine hydrochloride and 50-60% ethanol.
In some embodiments of the invention, the rinse liquid II comprises: tris-HCl 20-30mM and ethanol 70-80%.
In some embodiments of the invention, the eluent comprises: tris 10-20mM, EDTA 0.2-0.4mM, proClin300 0.01-0.05%.
In some embodiments of the invention, the stop solution comprises: proteinase K10-20mg/mL and SDS 0.1-0.2%.
In some embodiments of the invention, the magnetic beads are hydroxyl magnetic beads.
In some embodiments of the invention, the cleavage beads comprise at least one of garnet, zirconia, silica, glass beads.
In some embodiments of the invention, the zirconia beads have a particle size of 0.5 to 1.5mm.
In a second aspect of the invention there is provided the use of a kit according to the first aspect of the invention for extracting nucleic acids.
In some embodiments of the invention, the nucleic acid is DNA or RNA.
In some embodiments of the invention, the nucleic acid is a nucleic acid of a pathogenic microorganism.
In some embodiments of the invention, the pathogenic microorganism comprises: bacteria, fungi, viruses, protozoa, mycoplasma, chlamydia.
In a third aspect of the present invention, there is provided a method of extracting nucleic acid, the extraction method comprising the steps of:
s1: adding a lysate and lysis beads into a sample, grinding, adding lysozyme for reaction, and then adding a protease inhibitor to terminate the enzymatic reaction;
s2: adding nucleic acid binding solution into the supernatant, and eluting the magnetic beads with magnetic bead enrichment and eluent to obtain nucleic acid.
In some embodiments of the invention, the magnetic beads are further rinsed after enrichment, specifically comprising the steps of: the first rinsing and the second rinsing are carried out using rinsing liquid I and rinsing liquid II, respectively.
In some embodiments of the invention, the milling conditions are 59-65Hz milling for 10-15min or normal temperature 2000-3000rpm shaking for 10-15min; too long a time may cause nucleic acid cleavage.
In some embodiments of the invention, the reaction conditions of the lysozyme are: 36-38 ℃ for 10-15 min.
In some embodiments of the invention, the sample comprises: alveolar lavage fluid, cerebrospinal fluid, pus, ascites, sputum, blood, vaginal secretions, faeces, tissue, cells.
The beneficial effects of the invention are as follows:
the invention provides a kit for extracting nucleic acid of pathogenic microorganism, comprising: lysate, binding solution, rinsing solution I, rinsing solution II, eluent, stopping solution, magnetic beads, lysate beads and complex enzyme; the complex enzyme package: 5-10U/mL of muramidase, 10-30U/mL of chitinase, 25-35U/mL of lysostaphin, and 25-35U/mL of lysozyme. The DNA yield of gram-positive bacteria and fungi is improved, the cracking time is shortened, and the fragment loss caused by nucleic acid breakage and subsequent library establishment is reduced. Compared with the column extraction method, the magnetic bead method reduces the loss of nucleic acid in the silica gel column purification process, and is beneficial to realizing the extraction automation and standardization.
Drawings
FIG. 1 shows the colony formation (M: initial ratio of standard) of standard samples treated in experimental groups 1, 2, 3 and 4.
Fig. 2 amounts of pathogenic bacteria detected in clinical samples treated in experimental groups 1, 2, 3, and 4.
FIG. 3 shows the colony formation (M: initial ratio of standard) of standard samples treated in experimental groups 5, 6, 7 and 8.
Fig. 4 amounts of pathogenic bacteria detected in clinical samples treated in experimental groups 5, 6, 7, 8.
FIG. 5 shows the colony formation (M: initial ratio of standard) of standard samples treated in experimental groups 9, 10 and 11.
Fig. 6 amounts of pathogenic bacteria detected in clinical samples treated in experimental groups 9, 10, and 11.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Lywallase was purchased from the next holy creature, cat.no.10403ES81; lysozyme was purchased from the next holy organism, cat.no.10402ES08; chitinase was purchased from Sigma, cat.no. c6137; lysostaphin was purchased from Sigma, cat.no.L4402.
The cleavage beads were MP Lysing Matrix A (MP, cat.no. 116910050-CF).
EXAMPLE 1 Effect of different wall breaking methods on bacterial and fungal purification
1. Reagent preparation
1.1 lysate
Weighing 121g of Tris, and dissolving the Tris into ultrapure water to prepare mother solution of 1M for use; weighing 292g of EDTA, and dissolving with ultrapure water to prepare mother solution of 1M for use; weighing 11.7g of NaCl, weighing 200mL 1M Tris,40mL 1M EDTA,120 mu L of ProClin300, adding ultrapure water for dissolution, adjusting the pH to 8, and fixing the volume to 1L.
1.2 binding solution
478g of guanidine hydrochloride was weighed, 50mL of Triton X-100, 100mL of Tween 20 was weighed, dissolved in ultrapure water, pH was adjusted to 6, and the volume was set to 1L.
1.3 rinsing liquid I
287g of guanidine hydrochloride is weighed, ultra-pure water is added for dissolution, the pH is adjusted to 4.5, the volume is fixed to 430mL, and 570mL of ethanol is added for uniform mixing.
1.4 rinsing liquid II
20mL of 1M Tris is measured, ultrapure water is added for dissolution, the pH is adjusted to 7, the volume is fixed to 200mL, and 800mL of ethanol is added for uniform mixing.
1.5 eluent
10mL of 1M Tris, 200. Mu.L of 1M EDTA, 120. Mu.L of ProClin300 were measured, dissolved in ultrapure water, pH was adjusted to 8, and the volume was adjusted to 1L.
1.6 stop solution
The stop solution is proteinase K20 mg/mL and SDS 0.1%. 100g of SDS was dissolved in ultrapure water, and the volume was adjusted to 1L for use. Proteinase K was diluted to 20mg/mL for use.
1.7 magnetic beads
The magnetic beads are hydroxyl magnetic beads (50 mg/mL, particle size 1 mm) and the dosage is 1 mg/reaction.
1.8 cleavage beads
1.9 lysozyme mixtures
The amount of lysozyme mixture is 5U/mL of muramidase, 10U/mL of chitinase, 30U/mL of lysostaphin and 30U/mL of lysozyme.
2. Sample preparation
The experiment was evaluated using standard and clinical samples, each standard was mixed according to the proportions described in table 1.
TABLE 1
| Sequence number | Name of the name | Latin name | Gram staining | Proportion (%) | Bacterial liquid Content (CFU) |
| 1 | Salmonella typhimurium | Salmonella enterica | G- | 15 | 1.5×10 4 |
| 2 | Escherichia coli | Escherichia coli | G- | 15 | 1.5×10 4 |
| 3 | Shigella dysenteriae | Shigella dysenteriae | G- | 15 | 1.5×10 4 |
| 4 | Staphylococcus aureus | Staphylococcus aureus | G+ | 15 | 1.5×10 4 |
| 5 | Staphylococcus epidermidis | Staphylococcus epidermidis | G+ | 15 | 1.5×10 4 |
| 6 | Candida albicans | Monilia albicans | Fungi | 5 | 0.5×10 4 |
| 7 | Novel cryptococcus | Cryptococcus neoformans | Fungi | 5 | 0.5×10 4 |
| 8 | Adenovirus 3 | Adenovirus Ⅲ type | Virus (virus) | 15 | 1.5×10 4 |
Clinical samples were taken from 10 cases of alveolar lavage solutions of patients with different severe infections (Shenzhen Hua people Hospital).
3. Experimental grouping
Control group: DNA extraction was performed using QIAamp DNA Micro Kit (56304, qiagen).
Experiment group 1: extracting with self-prepared reagent.
Experiment group 2: self-assembling reagent and mechanical method.
Experiment group 3: extracting by self-matching reagent and enzymolysis method.
Experiment group 4: extracting by self-matching reagent, mechanical method and enzymolysis method.
3 standard samples and 10 clinical samples were taken for each experimental group.
4. Experimental procedure
(1) Centrifuging the standard bacterial liquid at 12000rpm for 5min, removing supernatant, and extracting with precipitate;
(2) The alveolar lavage fluid sample was centrifuged at 12000rpm for 5min at 1mL, and the supernatant was removed and the pellet was left for extraction.
4.1 control reagent extraction
(1) Nucleic acid extraction was performed using QIAamp DNA Micro Kit (56304, qiagen). The procedure was followed as described, eluting with 50. Mu.L of eluent.
4.2 self-assembling reagent extraction
(1) Adding 500 mu L of lysate into the sample, and vortex shaking for 15s;
(2) mu.L of protease K and 100. Mu.L of SDS were added and mixed.
(3) Transferring the supernatant to a new centrifuge tube, adding an equal volume of the binding solution, and uniformly mixing. Incubate at 70℃for 10min.
(4) 80. Mu.L of hydroxyl magnetic beads are added, vortexed and mixed well and incubated for 5min at room temperature. The EP tube was placed on a magnetic rack and the liquid in the tube was discarded after the solution was clear.
(5) Adding 500 mu L of rinsing liquid I, mixing, placing on a magnetic rack, and discarding the liquid after the magnetic beads are completely adsorbed.
(6) 500 mu L of rinse liquid II is added into the EP tube, the mixture is uniformly mixed by vortex, the mixture is placed on a magnetic rack, and after the magnetic beads are completely adsorbed, the liquid is discarded.
(7) Opening the tube cover, and airing at room temperature for about 5-10min.
(8) 50. Mu.L of eluent is added and mixed well, and incubated for 5min at room temperature.
(9) After incubation, the EP tube was placed on a magnetic rack and after the beads were completely adsorbed, the DNA product was transferred into a new EP tube.
4.3 self-matching reagent+mechanical extraction
After the completion of step (1), the solution was added to a holding tube containing the split beads, and the tube was placed in a mill, and milled at 59Hz for 10min. And then carrying out the subsequent self-matching reagent extraction step.
4.4 self-matching reagent+enzymatic extraction
After step (1) is completed, adding lysozyme mixture into the solution to make the muramidase be 5U/mL, chitinase 10U/mL, lysostaphin 30U/mL and lysozyme 30U/mL. Incubate at 37℃for 10min. And then carrying out the subsequent self-matching reagent extraction step.
4.5 self-matching reagent, mechanical method and enzymolysis method for extraction
After the completion of step (1), the solution was added to a holding tube containing the split beads, and the tube was placed in a mill, and milled at 59Hz for 10min. The lysozyme mixture was added to the solution to give a lywallase of 5U/mL, chitinase 10U/mL, lysostaphin 30U/mL, lysozyme 30U/mL and incubated at 37℃for 10min. And then carrying out the subsequent self-matching reagent extraction step.
4.6 detection
(1) The extracted nucleic acid was measured for DNA concentration and purity by an ultraviolet spectrophotometer.
(2) The qPCR method is adopted to detect 10 microorganisms and internal reference content in the standard sample. The primer sequences used are shown in table 2 below:
TABLE 2
The reaction system is as follows
Green qPCR Mix (P2092, GDSBio) instructions. The qPCR instrument detects the result of Ct value, and the delta Ct method is used for calculating the relative expression quantity.
(3) Metagenomic sequencing is used to detect pathogenic microorganisms in clinical samples.
5. Experimental results
(1) The concentration and purity of the extracted nucleic acid are shown in Table 3.
TABLE 3 Table 3
| Sample of | Purity of nucleic acid (A260/A280) | Average nucleic acid concentration (ng/. Mu.L) | Average total nucleic acid (. Mu.g) |
| B-control group | 1.8 | 55.3 | 2.765 |
| B-Experimental group 1 | 1.9 | 45.4 | 2.27 |
| B-Experimental group 2 | 1.9 | 64.2 | 3.21 |
| B-Experimental group 3 | 2.0 | 58.6 | 2.93 |
| B-Experimental group 4 | 2.0 | 70.5 | 3.525 |
| L-control group | 1.9 | 23.4 | 1.17 |
| L-test group 1 | 1.8 | 19.3 | 0.965 |
| L-test group 2 | 1.9 | 28.6 | 1.43 |
| L-test group 3 | 2.0 | 26.8 | 1.34 |
| L-test group 4 | 2.0 | 39.5 | 1.975 |
Note that: b is standard sample extraction, and L is clinical sample extraction. Average nucleic acid yield (average total nucleic acid) =average nucleic acid concentration×50 (eluent volume)/1000.
The result shows that the method adopts a mechanical method and an enzymolysis method to crack the sample on the basis of the existing microorganism DNA extraction method, and can obviously improve the yield of the sample DNA.
(2) The detection results of the standard sample flora are shown in figure 1.
The DNA yield of gram positive bacteria and fungi can be improved by adopting a mechanical method and an enzymolysis method to crack the sample on the basis of the existing microorganism DNA extraction method, and the whole flora is formed to be closer to the initial mixing proportion of each component in the standard sample.
(3) The detection result of the pathogenic bacteria of the clinical sample is shown in figure 2.
On the basis of the existing microorganism DNA extraction method, a mechanical method is combined with an enzymolysis method to crack a sample, so that the detection rate of pathogenic microorganisms, especially gram positive bacteria and fungi, is generally improved.
EXAMPLE 2 Effect of different treatment conditions on bacterial and fungal purification
1. And (3) preparation of a reagent: as described above.
2. Sample preparation: as described above.
3. Experimental grouping
Control group: DNA extraction was performed using QIAamp DNA Micro Kit (56304, qiagen).
The experimental group was set up on the basis of experimental group 4 in example 1, mainly investigating the effect of two factors, milling conditions and incubation conditions, on purification:
experimental group 5: grinding at 59Hz for 5min, incubating at 37℃for 5min, and the other conditions were as described above.
Experiment group 6: grinding at 59Hz for 10min, and incubating at 37℃for 10min, with the other conditions being as described above.
Experiment group 7: grinding at 59Hz for 20min, incubating at 37℃for 10min, and the other conditions were as described above.
Experiment group 8: grinding at 59Hz for 10min, and incubating at 37℃for 20min, with the other conditions being as described above.
3 standard samples and 10 clinical samples were taken for each experimental group.
4. Experimental procedure
(1) Centrifuging the standard bacterial liquid at 12000rpm for 5min, removing supernatant, and extracting with precipitate;
(2) The alveolar lavage fluid sample was centrifuged at 12000rpm for 5min at 1mL, and the supernatant was removed and the pellet was left for extraction.
4.1 control reagent extraction
(1) Nucleic acid extraction was performed using QIAamp DNA Micro Kit (56304, qiagen). The procedure was followed as described, eluting with 50. Mu.L of eluent.
4.2 self-assembling reagent extraction
(1) Adding 500 mu L of lysate into the sample, and vortex shaking for 15s;
(2) The solution was added to a holding tube containing the split beads and placed in a mill and milled as described in the experimental group above. A lysozyme mixture was added to the solution to give a lywallase of 5U/mL, chitinase 10U/mL, lysostaphin 30U/mL, and lysozyme 30U/mL, and incubations were performed as described in the above experimental groups.
(3) mu.L of protease K and 100. Mu.L of SDS were added and mixed.
(4) Transferring the supernatant to a new centrifuge tube, adding an equal volume of the binding solution, and uniformly mixing. Incubate at 70℃for 10min.
(5) 80. Mu.L of hydroxyl magnetic beads are added, vortexed and mixed well and incubated for 5min at room temperature. The EP tube was placed on a magnetic rack and the liquid in the tube was discarded after the solution was clear.
(6) Adding 500 mu L of rinsing liquid I, mixing, placing on a magnetic rack, and discarding the liquid after the magnetic beads are completely adsorbed.
(7) 500 mu L of rinse liquid II is added into the EP tube, the mixture is uniformly mixed by vortex, the mixture is placed on a magnetic rack, and after the magnetic beads are completely adsorbed, the liquid is discarded.
(8) Opening the tube cover, and airing at room temperature for about 5-10min.
(9) 50. Mu.L of eluent is added and mixed well, and incubated for 5min at room temperature.
(10) After incubation, the EP tube was placed on a magnetic rack and after the beads were completely adsorbed, the DNA product was transferred into a new EP tube.
4.3, detection: as described above
5. Experimental results
(1) The concentration and purity of the extracted nucleic acid are shown in Table 4.
TABLE 4 Table 4
| Sample of | Purity of nucleic acid (A260/A280) | Average nucleic acid concentration (ng/. Mu.L) | Average total nucleic acid (. Mu.g) |
| B-control group | 1.9 | 64.2 | 3.21 |
| B-Experimental group 5 | 2.0 | 78.3 | 3.915 |
| B-Experimental group 6 | 1.9 | 82.5 | 4.125 |
| B-Experimental group 7 | 2.0 | 88.7 | 4.435 |
| B-experiment group 8 | 2.0 | 84.2 | 4.21 |
| L-control group | 1.8 | 18.6 | 0.93 |
| L-test group 5 | 1.9 | 22.7 | 1.135 |
| L-test group 6 | 1.9 | 29.4 | 1.47 |
| L-experiment group 7 | 1.8 | 32.5 | 1.625 |
| L-test group 8 | 1.8 | 34.2 | 1.71 |
Note that: b is standard sample extraction, and L is clinical sample extraction. Average nucleic acid yield (average total nucleic acid) =average nucleic acid concentration×50 (eluent volume)/1000.
It is demonstrated that further increases in milling and incubation times can increase the yield of sample DNA.
(2) The detection results of the standard sample flora composition are shown in figure 3.
Comparing different treatment conditions, finding that adding the cracking beads, grinding for 10min at 59Hz, and adding enzyme, incubating for 10min at 37 ℃ to form an integral flora, wherein the initial mixing proportion of each component in the sample is closer to that of the standard sample; reducing the grinding and incubation time, increasing the proportion of gram-negative bacteria, and reducing the proportion of gram-positive bacteria, fungi and viruses; increasing grinding time on the basis of grinding at 59Hz for 10min, reducing the proportion of gram-negative bacteria and viruses, and increasing the proportion of gram-positive bacteria and fungi; the incubation time was increased on the basis of incubation at 37℃for 10min, with an increase in the proportion of gram-positive bacteria.
(3) The detection result of the pathogenic bacteria of the clinical sample is shown in figure 4.
Comparing different treatment conditions, grinding for 10min at 59Hz with lysis beads, incubating for 10min at 37 ℃ with enzyme, wherein the detection amount of gram-negative bacteria and viruses is highest, grinding for 10-20min at 59Hz with gram-positive bacteria and fungi, incubating for 10-20min at 37 ℃ with less difference, but incubating for 5min at 37 ℃ higher than that of control reagent and grinding for 5min at 59 Hz.
EXAMPLE 3 Effect of Complex enzymes at different concentrations on bacterial and fungal purification
1. And (3) preparation of a reagent: as described above.
2. Sample preparation: as described above.
3. Experimental grouping
Control group: DNA extraction was performed using QIAamp DNA Micro Kit (56304, qiagen).
Experimental group: self-assembled reagent extraction+lysis beads 59Hz milling for 10min, and incubation of complex enzyme at 37deg.C for 10min. The concentrations of the complex enzymes used for each group were as follows.
Experiment group 9: lywallase 2.5U/mL, chitinase 5U/mL, lysostaphin 15U/mL, lysozyme 15U/mL.
Experimental group 10: lywallase 5U/mL, chitinase 10U/mL, lysostaphin 30U/mL, lysozyme 30U/mL.
Experiment group 11: lywallase 10U/mL, chitinase 20U/mL, lysostaphin 60U/mL, lysozyme 60U/mL.
3 standard samples and 10 clinical samples were taken for each experimental group.
4. Experimental procedure
(1) Centrifuging the standard bacterial liquid at 12000rpm for 5min, removing supernatant, and extracting with precipitate;
(2) The alveolar lavage fluid sample was centrifuged at 12000rpm for 5min at 1mL, and the supernatant was removed and the pellet was left for extraction.
4.1 control reagent extraction
(1) Nucleic acid extraction was performed using QIAamp DNA Micro Kit (56304, qiagen). The procedure was followed as described, eluting with 50. Mu.L of eluent.
4.2 self-assembling reagent extraction
(1) Adding 500 mu L of lysate into the sample, and vortex shaking for 15s;
(2) The solution was added to a holding tube containing the split beads and placed in a mill and milled as described in the experimental group above. The lysozyme mixture was added to the solution in the amounts described above and incubated as described above.
(3) mu.L of protease K and 100. Mu.L of SDS were added and mixed.
(4) Transferring the supernatant to a new centrifuge tube, adding an equal volume of the binding solution, and uniformly mixing. Incubate at 70℃for 10min.
(5) 80. Mu.L of hydroxyl magnetic beads are added, vortexed and mixed well and incubated for 5min at room temperature. The EP tube was placed on a magnetic rack and the liquid in the tube was discarded after the solution was clear.
(6) Adding 500 mu L of rinsing liquid I, mixing, placing on a magnetic rack, and discarding the liquid after the magnetic beads are completely adsorbed.
(7) 500 mu L of rinse liquid II is added into the EP tube, the mixture is uniformly mixed by vortex, the mixture is placed on a magnetic rack, and after the magnetic beads are completely adsorbed, the liquid is discarded.
(8) Opening the tube cover, and airing at room temperature for about 5-10min.
(9) 50. Mu.L of eluent is added and mixed well, and incubated for 5min at room temperature.
(10) After incubation, the EP tube was placed on a magnetic rack and after the beads were completely adsorbed, the DNA product was transferred into a new EP tube.
4.3, detection: as described above
5. Experimental results
(1) The concentration and purity of the extracted nucleic acid are shown in Table 5.
TABLE 5
| Sample of | Purity of nucleic acid (A260/A280) | Average nucleic acid concentration (ng/. Mu.L) | Average total nucleic acid (. Mu.g) |
| B-control group | 1.9 | 48.2 | 2.41 |
| B-experiment group 9 | 2.0 | 56.6 | 2.83 |
| B- |
1.9 | 68.5 | 3.425 |
| B-Experimental group 11 | 2.0 | 72.1 | 3.605 |
| L-control group | 1.8 | 15.7 | 0.785 |
| L-test group 9 | 1.9 | 19.6 | 0.98 |
| L- |
1.9 | 28.4 | 1.42 |
| L-test group 11 | 1.8 | 32.7 | 1.635 |
Note that: b is standard sample extraction, and L is clinical sample extraction. Average nucleic acid yield (average total nucleic acid) =average nucleic acid concentration×50 (eluent volume)/1000.
The concentration of the complex enzyme is increased on the basis of the existing microorganism DNA extraction method, so that the yield of sample DNA can be improved.
(2) The detection results of the standard sample flora composition are shown in figure 5.
Comparing the different treatment conditions, the concentrations of complex enzymes used in experimental groups 2 and 3 were found to be such that the overall flora constituted an initial mix ratio of the components in the sample closer to that of the standard.
(3) The detection result of the pathogenic bacteria of the clinical sample is shown in figure 6.
Compared with different treatment conditions, the pathogenic bacteria detection amount of the experimental group 11 & gt and the pathogenic bacteria detection amount of the experimental group 10 & gt and the experimental group 9 are compared, but when the complex enzyme concentration of the experimental group 10 is adopted for extraction, the pathogenic bacteria detection amount of the experimental group is obviously superior to that of the experimental group 9, the complex enzyme concentration is continuously improved, the pathogenic bacteria detection amount improving effect is not obvious, and the experimental group 10 is adopted as the optimal extraction concentration due to the consideration of cost.
The present invention has been described in detail in the above embodiments, but the present invention is not limited to the above examples, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A kit for pathogenic microorganism nucleic acid extraction comprising: lysate, binding solution, rinse solution, eluent, stop solution, magnetic beads, lysate beads, and complex enzyme; the complex enzyme comprises: lywallase, chitinase, lysostaphin, lysozyme;
wherein the enzyme activity ratio among muramidase, chitinase, lysostaphin and lysozyme is as follows: (5-10): (10-30): (25-35): (25-35).
2. The kit of claim 1, wherein the lysate comprises: tris-HCl 0.2-0.5M, EDTA 40-50mM, EDTA-2Na 40-50mM, naCl 0.2-0.3M, KCl 0.15-0.2M, proClin 0.01-0.05%; preferably, the binding solution comprises: 3-5M guanidine hydrochloride, 5-10% Triton X-100 and 5-10% Tween.
3. The kit of claim 1, wherein the rinse solution comprises a rinse solution I and a rinse solution II, the rinse solution I comprising: 3-5M guanidine hydrochloride, 50-60% ethanol and 40-50% isopropanol; preferably, the rinse liquid II comprises: tris-HCl 20-30mM and ethanol 70-80%.
4. The kit of claim 1, wherein the eluent comprises: tris 10-20mM, EDTA 0.2-0.4mM, proClin300 0.01-0.05%; preferably, the stop solution comprises: proteinase K10-20mg/mL and SDS 0.1-0.2%.
5. The kit of claim 1, wherein the magnetic beads are hydroxyl magnetic beads; the cleavage beads comprise at least one of garnet, zirconia beads, silica beads, glass beads.
6. Use of a kit according to any one of claims 1 to 5 for extracting nucleic acids.
7. The use according to claim 6, wherein the nucleic acid is DNA or RNA; preferably, the nucleic acid is a nucleic acid of a pathogenic microorganism; preferably, the pathogenic microorganism comprises: at least one of bacteria, fungi, viruses, protozoa, mycoplasma, and chlamydia.
8. A method for extracting nucleic acid, comprising the steps of:
s1: adding a lysate and cleavage beads into a sample, grinding, adding complex enzyme for reaction, and then adding protease inhibitor to terminate enzymatic reaction;
s2: adding the supernatant into the binding solution, and eluting the magnetic beads by using magnetic bead enrichment and eluent to obtain the nucleic acid.
9. The method according to claim 8, wherein the grinding conditions are normal temperature, 59-65Hz grinding for 10-15min or normal temperature, 2500-3000rpm shaking for 10-15min; preferably, the complex enzyme reaction conditions are: 36-38 ℃ for 10-15 min.
10. The method of claim 8, wherein the sample comprises: alveolar lavage fluid, cerebrospinal fluid, pus, ascites, sputum, blood, vaginal secretions, faeces, tissue, cells.
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| CN116926065A (en) * | 2023-09-12 | 2023-10-24 | 天根生化科技（北京）有限公司 | Nucleic acid extraction kit suitable for detecting pathogenic microorganisms and host residues and extraction method thereof |
| CN116926065B (en) * | 2023-09-12 | 2023-12-29 | 天根生化科技（北京）有限公司 | Nucleic acid extraction kit suitable for detecting pathogenic microorganisms and host residues and extraction method thereof |
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