CN113832146A - Method for extracting pathogenic microorganism nucleic acid from formalin-fixed paraffin-embedded sample - Google Patents
Method for extracting pathogenic microorganism nucleic acid from formalin-fixed paraffin-embedded sample Download PDFInfo
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- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 34
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 34
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 34
- 244000000010 microbial pathogen Species 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000005119 centrifugation Methods 0.000 claims abstract description 12
- 239000000203 mixture Substances 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims abstract description 10
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000008096 xylene Substances 0.000 claims abstract description 8
- 239000012149 elution buffer Substances 0.000 claims abstract description 6
- 238000007664 blowing Methods 0.000 claims abstract description 5
- 238000010009 beating Methods 0.000 claims abstract description 4
- 238000002156 mixing Methods 0.000 claims abstract description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 4
- 239000012498 ultrapure water Substances 0.000 claims abstract description 4
- 239000012456 homogeneous solution Substances 0.000 claims abstract description 3
- 238000011534 incubation Methods 0.000 claims description 4
- 238000003260 vortexing Methods 0.000 claims description 3
- 238000001514 detection method Methods 0.000 abstract description 9
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 239000011324 bead Substances 0.000 description 6
- 239000006166 lysate Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 108010067770 Endopeptidase K Proteins 0.000 description 2
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
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- 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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Abstract
The invention provides a method for extracting pathogenic microorganism nucleic acid from a formalin-fixed paraffin-embedded sample, which comprises the following steps: adding xylene into a centrifuge tube filled with a formalin-fixed paraffin-embedded sample, and centrifuging; removing xylene in the centrifugal tube, and adding absolute ethyl alcohol for centrifugation; removing absolute ethyl alcohol in the centrifuge tube, and incubating until the absolute ethyl alcohol is completely evaporated; adding ultrapure water into a centrifuge tube, blowing, beating and uniformly mixing until the mixture is homogeneous; clarifying the homogeneous solution, swirling, adding an elution buffer solution, centrifuging, and purifying to obtain pathogenic microorganism nucleic acid; the results of the macro genes of the pathogenic microorganism nucleic acid show that the macro genes are below 10 percent, the macro gene high-throughput detection proves that the method is really effective, and the capability of removing human-derived nucleic acid is extremely strong, so that the method for extracting the pathogenic microorganism nucleic acid from the formalin-fixed paraffin-embedded sample can remove the human-derived background to the greatest extent, and the aim of extracting only the pathogenic microorganism nucleic acid is fulfilled.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for extracting pathogenic microorganism nucleic acid from a formalin-fixed paraffin-embedded sample.
Background
Nucleic acids are biological macromolecular compounds formed by polymerizing many nucleotides, and are classified into deoxyribonucleic acid (abbreviated as DNA) and ribonucleic acid (abbreviated as RNA) according to their chemical compositions. DNA is the primary material basis for storing, replicating, and transmitting genetic information. RNA plays an important role in protein synthesis, and thus nucleic acid plays a crucial role in a series of important life phenomena such as growth, heredity, mutation and the like. The research of nucleic acid can essentially reveal the structure and composition of microorganisms, even mysterious veil of pathogenesis, and can effectively research how to prevent and treat diseases generated by the microorganisms, thus having important significance for the research of nucleic acid.
Aiming at the current clinical requirements, metagenomic detection is required to be carried out on some retrospective specimens. As is well known, paraffin specimens are the first choice for retrospective research, so that how to find a method for extracting only pathogenic microorganism nucleic acid in paraffin specimens aiming at clinical pain spots is urgent. In addition, the extraction of the pathogenic microorganism nucleic acid is based on fresh samples (blood, alveolar lavage fluid, pus and the like), and no method for separately extracting the pathogenic microorganism nucleic acid from paraffin wax samples is available in the market.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for extracting pathogenic microorganism nucleic acid from a formalin-fixed paraffin-embedded sample.
In order to achieve the above purpose, the solution of the invention is as follows:
a method for extracting pathogenic microorganism nucleic acid from a formalin-fixed paraffin-embedded sample comprises the following steps:
(1) adding xylene into a centrifuge tube filled with a formalin-fixed paraffin-embedded sample, and centrifuging;
(2) removing xylene in the centrifuge tube, and adding absolute ethyl alcohol for centrifugation;
(3) removing the absolute ethyl alcohol in the centrifuge tube, and incubating until the absolute ethyl alcohol is completely evaporated;
(4) adding ultrapure water into the centrifuge tube, and blowing, beating and uniformly mixing until the mixture is homogeneous;
(5) and (5) clarifying the homogeneous solution obtained in the step (4), then vortexing, adding an elution buffer solution, centrifuging, and purifying to obtain the pathogenic microorganism nucleic acid.
Preferably, in the step (1), the rotation speed of centrifugation can be 12000-15000rpm, preferably 13000 rpm; the time for centrifugation may be 1-4min, preferably 2 min.
Preferably, in the step (2), the rotation speed of centrifugation can be 12000-15000rpm, preferably 13000 rpm; the time for centrifugation may be 1-4min, preferably 2 min.
Preferably, in step (3), the incubation temperature may be 50-60 ℃, preferably 56 ℃; the incubation time may be 1-10min, preferably 5 min.
Preferably, in step (5), the elution buffer may be 90 to 100. mu.L, preferably 90. mu.L.
Preferably, in the step (5), the rotation speed of the vortex can be 1200-1800rpm, preferably 1500 rpm; the time for vortexing may be 1-4min, preferably 3 min.
Due to the adoption of the scheme, the invention has the beneficial effects that:
the result of the macro gene of the pathogenic microorganism nucleic acid shows that the content of the macro gene is below 10 percent, the macro gene high-throughput detection proves that the method is really effective, and the capability of removing human-derived nucleic acid is extremely strong, so that the method can remove the human-derived background to the maximum extent and achieve the aim of extracting only the pathogenic microorganism nucleic acid.
Detailed Description
The invention provides a method for extracting pathogenic microorganism nucleic acid from a formalin-fixed paraffin-embedded sample.
The technical content of the present invention will be further described with reference to examples. The following examples are illustrative and not intended to be limiting, and are not intended to limit the scope of the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The method for extracting pathogenic microorganism nucleic acid from the formalin-fixed paraffin-embedded sample comprises the following steps:
(1) 1mL of xylene was added to the centrifuge tube containing the wax coil (i.e., formalin-fixed paraffin-embedded sample), and the mixture was centrifuged at 13000rpm for 2 min.
(2) After removing xylene from the centrifuge tube, 1mL of absolute ethanol was added and the mixture was centrifuged at 13000rpm for 2 min.
(3) The absolute ethyl alcohol in the centrifuge tube is discarded, and the centrifuge tube is incubated at 56 ℃ for 5min until the absolute ethyl alcohol is completely evaporated.
(4) And adding 200 mu L of ultrapure water into the centrifuge tube, and blowing and uniformly mixing by using a pipette until the mixture is homogeneous.
(5) And 400. mu.L of the clarified solution was added to the bead mill tube.
(6) And (3) adding 200 mu L of the tissue homogenate sample obtained in the step (4) into the bead grinding tube containing 400 mu L of the clear liquid obtained in the step (5).
(7) The mixture was shaken at 1500rpm for 15min using a vortexer with a microcentrifuge tube adapter.
(8) After 3min centrifugation at 15000rpm, 300. mu.L of supernatant (clarified lysate) was transferred to a new centrifuge tube and 10. mu.L of proteinase K was added to the centrifuge tube.
(9) And blowing and beating for several times by a liquid transfer machine to uniformly mix the clarified lysate with the proteinase K. Incubate at room temperature for 2 min.
(10) 720. mu.L of the lysate/conjugate/magnetic bead mixture (350. mu.L of lysate + 350. mu.L of conjugate + 20. mu.L of magnetic beads) was added to each tube, vortexed at 1500rpm for 3min, and centrifuged instantaneously for 10 s.
(11) The tube was placed on a magnetic frame and incubated at room temperature for 3 min.
(12) The tube was kept on a magnetic rack and the supernatant carefully aspirated and discarded.
(13) The tubes were removed from the rack, 500. mu.L of Wash 1 was added to each tube, vortexed at 1500rpm for 1min, and centrifuged instantaneously for 10 s.
(14) The centrifuge tube was placed on a magnetic frame and incubated at room temperature for 1 min.
(15) The tube was kept on a magnetic rack and the supernatant carefully aspirated and discarded.
(16) The tubes were removed from the rack, 500. mu.L of Wash solution 2 was added to each tube, vortexed at 1500rpm for 1min, and centrifuged instantaneously for 10 s.
(17) The centrifuge tube was placed on a magnetic frame and incubated at room temperature for 1 min.
(18) The tube was kept on a magnetic rack and the supernatant carefully aspirated and discarded.
(19) And keeping the centrifugal tube on the magnetic frame, opening the tube cover, and drying the magnetic beads at room temperature for 5 min.
(20) Then, the residual washing solution 2 was aspirated off using a 20. mu.L pipette.
(21) The tubes were removed from the magnetic stand, 90. mu.L of elution buffer was added to each tube, vortexed at 1500rpm for 3min, and centrifuged instantaneously for 10 s.
(22) The centrifuge tube was placed on a magnetic frame and incubated at room temperature for 3 min.
(23) And keeping the centrifuge tube on a magnetic frame, and transferring the purified nucleic acid into a clean centrifuge tube or a clean plate which is well marked.
Wherein, in the step (5), the clear solution is carried by the kit.
In step (10), the binding solution is a kit, and the magnetic beads are purchased from Saimer Feishel technologies.
The pathogenic microorganism nucleic acid is subjected to the following metagenome detection, and the detection results are shown in tables 1 and 2:
TABLE 1
And (4) detection conclusion:
the detection sequence at this time: mycobacterium tuberculosis 1 strip.
TABLE 2
And (4) detection conclusion:
the detection sequence at this time: mycobacterium tuberculosis 1 strip.
As can be seen from tables 1 and 2, the results of the macro genes are all below 10%, that is, the results of the macro gene sequencing show that the method can remove the human background to the maximum extent and achieve the purpose of extracting only the nucleic acid of the pathogenic microorganism.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.
Claims (6)
1. A method for extracting pathogenic microorganism nucleic acid from a formalin-fixed paraffin-embedded sample is characterized by comprising the following steps: which comprises the following steps:
(1) adding xylene into a centrifuge tube filled with a formalin-fixed paraffin-embedded sample, and centrifuging;
(2) removing xylene in the centrifuge tube, and adding absolute ethyl alcohol for centrifugation;
(3) removing the absolute ethyl alcohol in the centrifuge tube, and incubating until the absolute ethyl alcohol is completely evaporated;
(4) adding ultrapure water into the centrifuge tube, and blowing, beating and uniformly mixing until the mixture is homogeneous;
(5) and (5) clarifying the homogeneous solution obtained in the step (4), then vortexing, adding an elution buffer solution, centrifuging, and purifying to obtain the pathogenic microorganism nucleic acid.
2. The method of claim 1 for extracting nucleic acids from pathogenic microorganisms from formalin-fixed paraffin-embedded specimens, comprising: in the step (1), the rotation speed of the centrifugation is 12000-15000rpm, and the time of the centrifugation is 1-4 min.
3. The method of claim 1 for extracting nucleic acids from pathogenic microorganisms from formalin-fixed paraffin-embedded specimens, comprising: in the step (2), the rotation speed of the centrifugation is 12000-15000rpm, and the time of the centrifugation is 1-4 min.
4. The method of claim 1 for extracting nucleic acids from pathogenic microorganisms from formalin-fixed paraffin-embedded specimens, comprising: in the step (3), the incubation temperature is 50-60 ℃, and the incubation time is 1-10 min.
5. The method of claim 1 for extracting nucleic acids from pathogenic microorganisms from formalin-fixed paraffin-embedded specimens, comprising: in the step (5), the elution buffer solution is 90-100 mu L.
6. The method of claim 1 for extracting nucleic acids from pathogenic microorganisms from formalin-fixed paraffin-embedded specimens, comprising: in the step (5), the rotation speed of the vortex is 1200-1800rpm, and the time of the vortex is 1-4 min.
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