CN111020018B - Macrogenomics-based pathogenic microorganism detection method and kit - Google Patents
Macrogenomics-based pathogenic microorganism detection method and kit Download PDFInfo
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Abstract
The invention provides a pathogenic microorganism detection method and a kit based on metagenomics, wherein the method comprises the following steps: extracting nucleic acid; constructing a high-throughput sequencing library; quantifying the sequencing library constructed in the high-throughput sequencing library construction; diluting the quantitative sequencing library to 1-4pM, and performing high-throughput sequencing on a sequencer; sequencing data obtained in high-throughput sequencing was analyzed. According to the high-throughput sequencing library construction method and kit based on the metagenomics, provided by the invention, the DNA is fragmented by using the fragmenting enzyme, so that the loss of the DNA is smaller than that of a mechanical method, the fragmentation rate is higher, and the construction efficiency of the whole library is improved.
Description
The technical field is as follows:
the invention relates to the technical field of biology, in particular to a pathogenic microorganism detection method and a kit based on metagenomics.
Background art:
infectious diseases are a general term for diseases caused by pathogenic microorganisms (bacteria, viruses, fungi, parasites, etc.). According to statistics, infectious disease causes of death account for more than 25% of all causes of death, and are serious diseases seriously threatening human health in the world today. There are approximately 4000 million patients worldwide suffering from infectious diseases each year, and about one fourth of the patients in China, with the clinical diagnosis cause of disease being unknown accounting for nearly fifty percent (about 500 million). When infectious diseases of unknown cause occur, rapid and accurate identification of pathogens is critical for accurate treatment, effective monitoring, control of disease spread, and reduction of medical burden.
The metagenome technology based on high-throughput sequencing does not depend on traditional microorganism culture, directly performs high-throughput sequencing on nucleic acid in a clinical sample, can acquire all nucleic acid sequence information contained in the sample as far as possible, then performs comparison analysis with a database, judges the species information of microorganisms contained in the sample according to the sequence information obtained by comparison, and plays an important role in pathogen detection. High throughput sequencing for diagnosis of infectious diseases was first reported in 2014 in 1 patient with central nervous system infection. In addition, in respiratory tract infection, high-throughput sequencing is also used for various respiratory tract specimens including sputum, alveolar lavage fluid and the like, and shows good detection performance. Blood samples are also an important application field for high-throughput sequencing, and play a role in the pathogen detection of blood stream infection.
However, the existing technology for detecting pathogenic microorganisms based on a high-throughput sequencing technology generally adopts a traditional library construction method based on an Illumina sequencing platform, and comprises multiple steps of ultrasonic interruption, DNA fragment end repair, linker connection, PCR amplification and the like, so that the experimental operation is complex, and the price and the cost are high and the time is long due to the dependence on specific mechanical equipment such as a Covaris interruption instrument.
The invention is provided in view of the above.
The invention content is as follows:
the invention aims to provide a high-throughput sequencing library construction method based on metagenomics, a pathogenic microorganism detection method and a kit, so as to solve at least one technical problem in the prior art.
Specifically, in a first aspect of the present invention, a method for constructing a high-throughput sequencing library based on metagenomics is provided, the method comprising the following steps:
s101, fragmenting a DNA sequence by using a reaction system containing a fragmenting enzyme, and performing end repairing on the fragmented DNA fragment;
s102, connecting the joint sequences required by high-throughput sequencing to the two ends of the DNA fragment obtained in S101 by using a ligase reaction system, wherein the joint sequences are provided with specific label sequences;
s103, purifying the product in the S102 to obtain a high-throughput sequencing library.
By adopting the technical scheme, the DNA is fragmented by using the fragmenting enzyme, the loss of the DNA is smaller than that of a mechanical method, and meanwhile, the fragmentation rate is higher, so that the construction efficiency of the whole library is improved.
Preferably, in step S101, the reaction system comprises: according to the parts by volume, the weight percentage of the raw materials,
x parts of DNA sample, 1-2 parts of fragmentation Buffer solution, 2-4 parts of fragmentation enzyme and 2-15 parts of Buffer EB make-up system, wherein the concentration of the DNA sample is 5-30 ng/mul,
the reaction conditions are as follows:
preferably, the reaction system further comprises 0.5-1 part of an enhancer (Enhance).
Preferably, in the reaction system, the fragmentation Buffer is 1.5 parts, the fragmentation enzyme is 3 parts, the Enhance0.75 parts and Buffer EB makes up the system to 15 parts in parts by volume.
Preferably, in step S101, the fragmenting enzyme reaction system further includes polyethylene glycol 2000, and the content of polyethylene glycol 2000 is 0.1-0.3 parts by volume. More preferably, the reaction system of the fragmenting enzyme also comprises 0.02-0.04 parts of polyvinylpyrrolidone K30 (PVP-K30). Under the condition that polyethylene glycol exists in the system, a trace amount of PVP-K30 is added, so that the uniformity of the fragments can be further improved.
Preferably, in step S101, the fragmenting enzyme is DNA Fragmentase.
Preferably, in step S102, the reaction system comprises, in parts by volume:
10-20 parts of end repair product, 5-8 parts of connection Buffer solution, 2-5 parts of ligase, 4-6 parts of linker with tag sequence, 0.5-2 parts of Buffer EB, wherein the concentration of the ligase is 500-800U/mul,
the reaction conditions are as follows,
preferably, in step S102, the reaction system comprises, in parts by volume:
15 parts of a terminal repair product, 6 parts of a connection Buffer solution, 3 parts of ligase, 5 parts of a joint with a tag sequence and 1 parts of Buffer EB, wherein the concentration of the ligase is 600U/. mu.l.
Preferably, the ligation buffer comprises 300-350mM Tris-HCl, 30-60mM MgCl2, 3-6mM DTT, 3-6mM ATP. More preferably, the ligation buffer comprises 330mM Tris-HCl, 50mM MgCl2, 5mM DTT, 5mM ATP.
Preferably, in step S103, the product in step S102 is purified by magnetic beads.
Preferably, the step of purifying by magnetic beads comprises:
s201, sucking 10-30 mu L of EBBuffer and 30-50 mu L of DNA Clean Beads into 20-40 mu L of joint connection reaction products, and fully and uniformly mixing;
s202, incubating for 1-10min at room temperature;
s203, after centrifugation, separating the magnetic beads and the liquid under the action of an external magnetic field, and removing the supernatant after the solution is clarified;
s204, continuously adding 100-300 mu L of 70-90% ethanol solution to rinse the magnetic beads under the action of an external magnetic field, incubating at room temperature for 10-60S, and removing the supernatant;
s205, exposing the magnetic beads in air and drying for 5-10min,
s206, adding 10-30 mu L of EB into the magnetic beads for elution, fully and uniformly mixing, standing for 1-10min at room temperature, centrifuging, standing under an external magnetic field, and sucking 10-20 mu L of supernatant after the solution is clarified, thus obtaining the purified product.
Preferably, the step of S204 is repeated.
Preferably, the external magnetic field is a magnetic frame, and a test tube fixing part matched with the test tube is arranged on the magnetic frame.
Preferably, the diameter of the magnetic bead is0.5-1.5 μm. More preferably, the magnetic beads are selected from
DNA Clean Beads(VazymeN411)。
Preferably, in S204, the ethanol solution contains 0.02-0.05mol/L potassium chloride.
Further, in the second aspect of the present invention, there is provided a method for detecting a pathogenic microorganism based on metagenomics, comprising the steps of:
extracting nucleic acid;
constructing a high-throughput sequencing library by adopting the high-throughput sequencing library construction method;
sequencing library quantification: quantifying the sequencing library constructed in the high-throughput sequencing library construction step;
high-throughput sequencing: diluting the sequencing library quantified in the sequencing library quantifying step to 1-4pM, and performing high-throughput sequencing on a sequencer;
and (4) analyzing results: sequencing data obtained in high-throughput sequencing was analyzed.
Preferably, the analyzing method in the result analyzing step includes the steps of:
s401, splitting the sequencing data of the sample,
s402, calculating the proportion of the host sequence in the data,
s403, removing the host sequence,
s404, comparing the residual sequence with a pathogenic database to obtain the information of the pathogenic microorganism species.
Preferably, the resolution method is that the adapters connected with both ends of each library have a specific tag sequence, and after the data is downloaded, different reads are allocated to different libraries according to the tag sequence detected on each sequence.
Preferably, the tag sequence is 8nt in length.
Preferably, 20 libraries are loaded simultaneously during loading and sequencing.
Preferably, in S404, the method for comparing with the pathogen database is,
s501, generating a database hash index, splitting base sequences contained in the database based on the specific sequence length S, and storing the information of a genome position list where the base sequences are located by taking the base sequences as the hash index;
s502, sequence local comparison, namely splitting the query sequence into base segments with the same length, searching a corresponding sequence in a Hash index through Hash collision, and acquiring the position information of the segments in a genome;
s503, calculating the edit distance of the sequencing sequence at different positions of the reference sequence, and searching the optimal comparison result;
and S504, counting comparison results and outputting.
Preferably, in step S501, the base sequences included in the database are split by unit S, a hash index is constructed, and the genome position information is stored in the index. The typical sequence unit s is 20 bases in length. The reverse complement of the base sequence unit is also stored as an index in the index library.
Preferably, in the step S502, the sequence query method is to split the sequence into sequence units identical to those in step 1, and query the hash index constructed in step 1 by a hash collision method to obtain position information corresponding to the base sequence units.
Preferably, in step S503, the editing distance between the sequences and the genome is calculated according to the genome position information of the sequence unit split in step 2 in the index library. In order to avoid the influence caused by sequence insertion deletion, continuous 32 genome fragments of the genome are used as a statistical unit, and the editing distance in the statistical unit is used as the evaluation basis of the optimal comparison result. And calculating the edit distance between the query sequence and the reference sequence according to the number of sequence units matched by the same query sequence in the same reference sequence, and selecting the sequence with the lowest edit distance as the optimal comparison result of the query sequence in the reference database.
Preferably, in step S504, the distance between the sequencing sequence and the database reference sequence is calculated according to the sequence difference, if the optimal comparison result editing distance is m, the comparison result with the editing distance of m as the minimum and n as the maximum is searched according to a preset editing distance range n, and the result is output.
Further, in a third aspect of the present invention, there is provided a pathogenic microorganism detection kit comprising:
fragmenting enzyme, 80-85 μ L, preferably 83 μ L;
fragmenting enzyme buffer, 40-45 μ L, preferably 44 μ L;
linker, 24X, 4-8. mu.L, preferably 5. mu.L;
ligase, 80-85. mu.L, preferably 83. mu.L;
ligase buffer, 170 μ L.
In conclusion, the invention has the following beneficial effects:
1. according to the high-throughput sequencing library construction method based on the metagenomics, provided by the invention, the DNA is fragmented by using the fragmenting enzyme, the loss of the DNA is smaller than that of a mechanical method, the fragmentation rate is higher, and the construction efficiency of the whole library is improved.
2. According to the high-throughput sequencing library construction method based on the metagenomics, polyethylene glycol is added into a fragmentation enzyme reaction system, so that the small fragment proportion after DNA fragmentation can be effectively reduced, and most of fragments can be kept at 150-180 bp.
3. According to the high-throughput sequencing library construction method based on the metagenomics, a proper amount of potassium chloride is added in the magnetic bead purification process, so that the purification rate of DNA fragments is improved.
4. The pathogenic microorganism detection method based on the metagenomics provided by the invention obtains a more accurate pathogenic microorganism matching result through a comparison algorithm with a pathogenic database.
The specific implementation mode is as follows:
the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in this specification and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.
The present invention will be described in detail below by way of examples.
Sample example 1
This sample example was used to prepare DNA samples used in the following Experimental examples 1-2, and the specific procedures were as follows:
a nucleic acid extraction or purification reagent (Cat.1901, jin Ji 20190271, Tianjin Kingspoon medical science and technology Co., Ltd.) was used to extract the plasma samples of 1 clinical bloodstream infection patients, and the specific operation steps were as follows:
(1) taking 600. mu.l of plasma to a 3ml centrifuge tube, if less than 600. mu.l, adding buffer A to a final volume of 600. mu.l;
(2) adding 40 mul protease K solution, and uniformly mixing by vortex;
(3) add 600. mu.l of buffer B and 12. mu.l of Carrier RNA at a concentration of 1. mu.g/. mu.l, mix by gentle inversion, incubate at 70 ℃ for 15min, and shake the sample occasionally. Centrifuging briefly to remove liquid drops on the inner wall of the tube cover;
(4) after the temperature of the sample is reduced to room temperature, 600. mu.l of precooled absolute ethyl alcohol is added. Slightly inverting and mixing the sample, standing at room temperature for 5min, and centrifuging briefly to remove liquid drops on the inner wall of the tube cover;
(5) sucking 600 μ l of the solution obtained in the previous step into an adsorption column (the adsorption column is placed into a collecting pipe), centrifuging at 8000rpm for 30sec, discarding the waste liquid, and placing the adsorption column into a new 2ml centrifuge tube with a tube cover cut off;
(6) repeating the step 5;
(7) adding 500 μ l buffer solution C (checking whether absolute ethanol is added before use) into the adsorption column, centrifuging at 8000rpm for 30sec, discarding waste liquid, and placing into a new 2ml centrifuge tube with tube cover removed;
(8) adding 600 μ l of rinsing solution (checking whether absolute ethanol is added before use) into adsorption column, centrifuging at 8000rpm for 30sec, discarding waste liquid, and placing adsorption column into a new 2ml centrifuge tube with tube cover cut off;
(9) repeating the operation step 8;
(10) centrifuge at 12,000rpm (. about.13,400 Xg) for 2min and discard the waste. Sucking residual liquid in the adsorption column with 10 μ l gun head, uncovering the adsorption column, placing in a clean 1.5ml centrifuge tube, standing at room temperature for 2-5min until ethanol is fully volatilized;
(11) the purpose of the step is to remove the residual rinsing liquid in the adsorption column, and the residual ethanol in the rinsing liquid can influence the subsequent enzyme reaction (enzyme digestion, PCR and the like) experiment;
(12) suspending and dripping 50 mul of eluent into the middle position of the adsorption film, standing for 5min at room temperature, centrifuging for 2min at 12,000rpm (13,400 Xg), and collecting the solution into a centrifuge tube;
(13) the extracted nucleic acid DNA was quantified using the Qubit dsDNA HS assay Kit (Invitrogen Cat. Q32851) with the following quantification results:
experimental example 1
This example was conducted on a reaction system of a fragmenting enzyme, which is commercially available DNA Fragmentase.
Scheme a
DNA sample 1. mu.l, fragmentation Buffer 1. mu.l, fragmentation enzyme 2. mu.l, Enhance0.5. mu.l, Buffer EB made up the system to 15. mu.l,
scheme b
DNA sample 1. mu.l, fragmentation Buffer 2. mu.l, fragmentation enzyme 4. mu.l, Enhance 1. mu.l, Buffer EB made up the system to 15. mu.l, reaction conditions refer to protocol a.
Scheme c
DNA sample 1. mu.l, fragmentation Buffer 1.5. mu.l, fragmentation enzyme 3. mu.l, Enhance0.75. mu.l, Buffer EB made up the system to 15. mu.l, reaction conditions refer to scheme a.
Scheme d
DNA sample 1. mu.l, fragmentation Buffer 1.5. mu.l, fragmentation enzyme 5. mu.l, Enhance 1. mu.l, Buffer EB made up the system to 15. mu.l, reaction conditions refer to scheme a.
Scheme e
DNA sample 1. mu.l, fragmentation Buffer 1.5. mu.l, fragmentation enzyme 1. mu.l, Enhance 1. mu.l, Buffer EB made up the system to 15. mu.l, reaction conditions refer to scheme a.
Scheme f
Random breaks were performed using a sonicator. Wherein the ultrasonic power is 150W, the ultrasonic time is 2s every 8s, the total working time is 5min, and the ice bath is carried out.
Scheme g
Random breaks were performed using a sonicator. Wherein the ultrasonic power is 300W, the ultrasonic time is 5s every 5s, the total working time is 15min, and the ice bath is carried out.
Scheme h
Random breaks were performed using a sonicator. Wherein the ultrasonic power is 450W, the ultrasonic time is 3s at intervals of 7s, the total working time is 30min, and ice bath is carried out.
In schemes a-f, the length of the broken DNA fragments is detected, and the ratio of the DNA fragments with the length less than 100bp and the length more than 300bp is recorded as shown in Table 1:
TABLE 1
According to the results in Table 1, the fragmentation enzyme was used in an amount of 1-5. mu.l in a 15. mu.l system to achieve the fragmentation requirement, and the uniformity was significantly better than the random disruption protocol using a sonicator. Among them, the schemes a, b and c, namely the scheme that the using amount of the fragmentation enzyme is 2-4 mul, are more preferable by combining two indexes of the using amount and the uniformity of the enzyme.
Based on scheme b, the cleavage reaction time of the fragmenting enzyme was tested and the results are shown in Table 2:
TABLE 2
According to the results in Table 2, the enzyme cutting reaction time of the fragmenting enzyme can reach better fragmentation effect within 20-40min, wherein the reaction time is controlled within 35min, and the result is better.
Further, based on the scheme b, 0.2. mu.l of polyethylene glycol 2000 was added to the system, and the whole reaction time was tested, and the results are shown in Table 3:
TABLE 3
According to the results in table 3, the applicant unexpectedly found that, when the enzyme cutting time is longer after polyethylene glycol 2000 is added into the system, the small fragment ratio after the enzyme cutting reaction of the fragmentation enzyme is reduced, the small fragment increase caused by improper time control is reduced, and the uniformity of the fragmented DNA fragment is stronger.
Experimental example 2
As polyvinylpyrrolidone K30(PVP-K30) has a certain crosslinking property at a certain content as an auxiliary material of a pharmaceutical preparation, the applicant believes that the crosslinking property may positively promote the uniformity of the fragmentation reaction.
Therefore, based on the scheme b in the experimental example 1, 0.02mg of PVP-K30 was added to the system to perform the fragmentation reaction experiment, and the results are shown in Table 4:
TABLE 4
However, the results in Table 4 show that the uniformity after the fragmentation reaction is not improved but slightly decreased as compared with that in Table 2.
In subsequent experiments, applicants continued to perform fragmentation reaction experiments based on protocol b by adding 0.2. mu.l polyethylene glycol 2000 and 0.02mg PVP-K30 to the system, with the results shown in Table 5:
TABLE 5
According to the results in table 5, after polyethylene glycol 2000 and PVP-K30 were added simultaneously, although the number of large fragments varied insignificantly (P > 0.05), compared to table 3, the number of small fragments (less than 100bp) was significantly reduced (P < 0.01) except for one group of data in 10min (possibly due to too short reaction time), so that the effect of PVP-K30 added independently to the reaction system on the uniformity of fragmentation was not obvious overall, but when polyethylene glycol was present in the system, the addition of a small amount of PVP-K30 further improved the uniformity of fragments.
Experimental example 3
This example performs an experiment on the protocol for magnetic bead purification.
Sample 1: the product of scheme b in example 1 was taken as sample 1.
Sample 2: based on the scheme b in example 1, 0.2. mu.l of polyethylene glycol 2000 was added to the system, and the reaction product was sample 2.
Scheme a, a':
s1, sucking 10 mu L of EBBuffer and 30 mu L of DNA Clean Beads into 20 mu L of sample, and fully and uniformly mixing;
s2, incubating for 1min at room temperature;
s3, after centrifugation, separating the magnetic beads and the liquid under the action of an external magnetic field, and removing the supernatant after the solution is clarified;
s4, adding 100 mu L of 70% ethanol solution to rinse the magnetic beads under the action of the external magnetic field, incubating at room temperature for 10S, and removing the supernatant;
s5, exposing the magnetic beads to air for drying for 5min,
and S6, adding 10 mu L of EB into the magnetic beads for elution, fully and uniformly mixing, standing for 1min at room temperature, centrifuging, standing under an external magnetic field, and sucking 10 mu L of supernatant after the solution is clarified, thus obtaining the purified product.
Sample 1 was used as protocol A and sample 2 was used as protocol A'.
Scheme B, B':
s1, sucking 20 mu L of EBBuffer and 40 mu L of DNA Clean Beads into 30 mu L of sample, and fully and uniformly mixing;
s2, incubating for 5min at room temperature;
s3, after centrifugation, separating the magnetic beads and the liquid under the action of an external magnetic field, and removing the supernatant after the solution is clarified;
s4, under the action of the external magnetic field, adding 200 mu L of 80% ethanol solution to rinse the magnetic beads, incubating at room temperature for 30S, and removing the supernatant;
s5, exposing the magnetic beads to air for drying for 6min,
and S6, adding 20 mu L of EB into the magnetic beads for elution, fully and uniformly mixing, standing for 5min at room temperature, centrifuging, standing under an external magnetic field, and sucking 16 mu L of supernatant after the solution is clarified, thus obtaining the purified product.
Sample 1 was used as protocol B and sample 2 was used as protocol B'.
Scheme C, C':
s1, sucking 30 mu L of EBBuffer and 50 mu L of DNA Clean Beads into 40 mu L of sample, and fully and uniformly mixing;
s2, incubating for 10min at room temperature;
s3, after centrifugation, separating the magnetic beads and the liquid under the action of an external magnetic field, and removing the supernatant after the solution is clarified;
s4, adding 300 mu L of 90% ethanol solution to rinse the magnetic beads under the action of the external magnetic field, incubating at room temperature for 60S, and removing the supernatant;
s5, exposing the magnetic beads to air for drying for 10min,
and S6, adding 30 mu L of EB into the magnetic beads for elution, fully and uniformly mixing, standing for 10min at room temperature, centrifuging, standing under an external magnetic field, and sucking 20 mu L of supernatant after the solution is clarified, thus obtaining the purified product.
Sample 1 was used as protocol C and sample 2 was used as protocol C'.
On the basis of the scheme B, 0.03mol/L of sodium chloride, potassium chloride, magnesium chloride, potassium dihydrogen phosphate and dipotassium hydrogen phosphate are added in the step S4 to form the schemes D, E, F, G, H, D ', E ', F ', G ' and H '.
The purification rates of schemes A-I were examined and are reported in Table 6.
TABLE 6
| Group of | Purification ratio (%) |
| Scheme A | 55.2 |
| Scheme A' | 48.5 |
| Scheme B | 54.4 |
| Scheme B' | 48.0 |
| Scheme C | 56.7 |
| Scheme C' | 45.6 |
| Scheme D | 48.0 |
| Scheme D' | 45.2 |
| Scheme E | 48.3 |
| Scheme E' | 62.2 |
| Scheme F | 50.3 |
| Scheme F' | 42.0 |
| Scheme G | 44.3 |
| Scheme G' | 44.8 |
| Scheme H | 47.7 |
| Scheme H' | 44.6 |
As is clear from the results in Table 6, in the case where the sample group containing polyethylene glycol 2000 was purified without adding other components (schemes A-C, A '-C'), the purification rate was lower than that of the sample group not containing polyethylene glycol 2000 (p < 0.01), demonstrating that polyethylene glycol 2000 can reduce the fragmentation rate of DNA by the action of the fragmenting enzyme, but the presence of this component adversely affects the purification rate. According to the results of schemes D to H, when components such as sodium chloride, potassium chloride, magnesium chloride, potassium dihydrogen phosphate and dipotassium hydrogen phosphate were added to the sample, these components also adversely affected the purification rate, which was significantly reduced (p < 0.01) as compared with schemes A to C. In the sample group containing polyethylene glycol 2000, the applicant unexpectedly found that the purification rate of the potassium chloride group is obviously increased, not only higher than that of other sample groups (schemes D ', F', G ', H'), but also higher than that of schemes a-C, which indicates that the addition of potassium chloride and polyethylene glycol 2000 cooperate to play a synergistic role in the purification process of magnetic beads, so that the purification rate is obviously increased.
Sample example 2
This sample example was used to prepare DNA samples used in the following Experimental examples 1-2, and the specific procedures were as follows:
a nucleic acid extraction or purification reagent (Cat.1901, jin Ji 20190271, Tianjin Kingspoon medical science and technology Co., Ltd.) was used to extract the plasma samples of 1 clinical bloodstream infection patients, and the specific operation steps were as follows:
(1) taking 600. mu.l of plasma to a 3ml centrifuge tube, if less than 600. mu.l, adding buffer A to a final volume of 600. mu.l;
(2) adding 40 mul protease K solution, and uniformly mixing by vortex;
(3) add 600. mu.l of buffer B and 12. mu.l of Carrier RNA at a concentration of 1. mu.g/. mu.l, mix by gentle inversion, incubate at 70 ℃ for 15min, and shake the sample occasionally. Centrifuging briefly to remove liquid drops on the inner wall of the tube cover;
(4) after the temperature of the sample is reduced to room temperature, 600. mu.l of precooled absolute ethyl alcohol is added. Slightly inverting and mixing the sample, standing at room temperature for 5min, and centrifuging briefly to remove liquid drops on the inner wall of the tube cover;
(5) sucking 600 μ l of the solution obtained in the previous step into an adsorption column (the adsorption column is placed into a collecting pipe), centrifuging at 8000rpm for 30sec, discarding the waste liquid, and placing the adsorption column into a new 2ml centrifuge tube with a tube cover cut off;
(6) repeating the step 5;
(7) adding 500 μ l buffer solution C (checking whether absolute ethanol is added before use) into the adsorption column, centrifuging at 8000rpm for 30sec, discarding waste liquid, and placing into a new 2ml centrifuge tube with tube cover removed;
(8) adding 600 μ l of rinsing solution (checking whether absolute ethanol is added before use) into adsorption column, centrifuging at 8000rpm for 30sec, discarding waste liquid, and placing adsorption column into a new 2ml centrifuge tube with tube cover cut off;
(9) repeating the operation step 8;
(10) centrifuge at 12,000rpm (. about.13,400 Xg) for 2min and discard the waste. Sucking residual liquid in the adsorption column with 10 μ l gun head, uncovering the adsorption column, placing in a clean 1.5ml centrifuge tube, standing at room temperature for 2-5min until ethanol is fully volatilized;
(11) the purpose of the step is to remove the residual rinsing liquid in the adsorption column, and the residual ethanol in the rinsing liquid can influence the subsequent enzyme reaction (enzyme digestion, PCR and the like) experiment;
(12) suspending and dripping 50 mul of eluent into the middle position of the adsorption film, standing for 5min at room temperature, centrifuging for 2min at 12,000rpm (13,400 Xg), and collecting the solution into a centrifuge tube;
(13) the extracted nucleic acid DNA was quantified using the Qubit dsDNA HS assay Kit (Invitrogen Cat. Q32851) with the following quantification results:
| sample numbering | Concentration (ng/. mu.l) | Volume (μ l) | Yield (ng) |
| LYT | 0.608 | 50 | 12.16 |
Example 1
S101, fragmenting a DNA sequence by using a reaction system containing a fragmenting enzyme, and performing end repairing on the fragmented DNA fragment; the fragmenting enzyme is DNA Fragmentase.
Specifically, the fragmentation buffer was thawed and mixed by inversion, and the following reactions were prepared in sterile PCR tubes:
mix gently by pipetting (Do not shake) and centrifuge briefly to collect the reaction solution to the bottom of the tube. Temporarily placing the PCR tube on ice, setting the following program on a PCR instrument, pressing a pause key after the temperature of the PCR instrument is reduced to 4 ℃, placing the PCR tube into the PCR instrument, and then continuously running the program:
s102, connecting the joint sequences required by high-throughput sequencing to the two ends of the DNA fragment obtained in S101 by using a ligase reaction system, wherein the joint sequences are provided with specific label sequences;
specifically, the linkers (10 μ M) were diluted N-fold using EB as in the table below;
after thawing the ligation buffer, the mixture was inverted and mixed and placed on ice for use.
In the PCR tube of the end repair product obtained in the step S101, the following reaction was prepared:
the ligation buffer and ligase were premixed and stored at 4 ℃ for no more than 24h, and the linker was added during the reaction.
#X is the label number, one for each sample, Indexprime number.
Mix gently by pipetting (Do not shake) and centrifuge briefly to collect the reaction solution to the bottom of the tube. Placing the PCR tube in a PCR instrument, and carrying out the following reactions:
and S103, purifying the product in the S102 through magnetic beads to obtain a high-throughput sequencing library.
Specifically, the magnetic bead purification step is as follows:
s200, taking the magnetic Beads (DNA Clean Beads) out 30min in advance, carrying out vortex oscillation, mixing uniformly, balancing to room temperature, carrying out vortex oscillation and mixing uniformly after the DNA Clean Beads are balanced to the room temperature.
S201, sucking 10 mu L of EBBuffer and 30 mu L of DNA Clean Beads into 20 mu L of adaptor ligation reaction products, and performing vortex oscillation or gently blowing and beating 10 times by using a pipettor to fully mix the components.
S202, incubation for 1min at room temperature.
S203, the PCR tube is centrifuged briefly and placed in a magnetic rack to separate the magnetic beads and the liquid, and after the solution is clarified (about 5min), the supernatant is carefully removed (note: remove the supernatant as much as possible).
S204, keeping the PCR tube in a magnetic frame, adding 100 mu L of freshly prepared 70% ethanol to rinse the magnetic beads, carefully moving the PCR tube to the opposite side of the magnetic strip, incubating at room temperature for 10S, and carefully removing the supernatant until the magnetic beads are completely moved to the tube wall close to the magnetic strip. This step was repeated once for a total of two rinses.
S205, keeping the PCR tube in the magnetic frame all the time, and opening the cover to dry the magnetic beads in air for 5min until no ethanol remains.
S206, taking out the PCR tube from the magnetic frame, adding 10 mu L of EB for elution, carrying out vortex oscillation or gently blowing and beating by using a pipette to fully mix the mixture, standing the mixture at room temperature for 1min, centrifuging the PCR tube for a short time, placing the PCR tube on the magnetic frame for standing, carefully sucking 10 mu L of supernatant into a new PCR tube after the solution is clarified (about 5min), and cutting the supernatant without touching magnetic beads to obtain a purified product.
Example 2
S101, fragmenting a DNA sequence by using a reaction system containing a fragmenting enzyme, and performing end repairing on the fragmented DNA fragment; the fragmenting enzyme is DNA Fragmentase.
Specifically, the fragmentation buffer was thawed and mixed by inversion, and the following reactions were prepared in sterile PCR tubes:
mix gently by pipetting (Do not shake) and centrifuge briefly to collect the reaction solution to the bottom of the tube. Temporarily placing the PCR tube on ice, setting the following program on a PCR instrument, pressing a pause key after the temperature of the PCR instrument is reduced to 4 ℃, placing the PCR tube into the PCR instrument, and then continuously running the program:
s102, connecting the joint sequences required by high-throughput sequencing to the two ends of the DNA fragment obtained in S101 by using a ligase reaction system, wherein the joint sequences are provided with specific label sequences;
specifically, the linkers (10 μ M) were diluted N-fold using EB as in the table below;
after thawing the ligation buffer, the mixture was inverted and mixed and placed on ice for use.
In the PCR tube of the end repair product obtained in the step S101, the following reaction was prepared:
the ligation buffer and ligase were premixed and stored at 4 ℃ for no more than 24h, and the linker was added during the reaction.
#X is the label number, one for each sample, Indexprime number.
Mix gently by pipetting (Do not shake) and centrifuge briefly to collect the reaction solution to the bottom of the tube. Placing the PCR tube in a PCR instrument, and carrying out the following reactions:
and S103, purifying the product in the S102 through magnetic beads to obtain a high-throughput sequencing library.
Specifically, the magnetic bead purification step is as follows:
s200, taking the magnetic Beads (DNA Clean Beads) out 30min in advance, carrying out vortex oscillation, mixing uniformly, balancing to room temperature, carrying out vortex oscillation and mixing uniformly after the DNA Clean Beads are balanced to the room temperature.
S201, sucking 30 mu L of EBBuffer and 50 mu L of DNA Clean Beads into 40 mu L of adaptor ligation reaction products, and performing vortex oscillation or gently blowing and beating 10 times by using a pipettor to fully mix the components.
S202, incubation for 10min at room temperature.
S203, the PCR tube is centrifuged briefly and placed in a magnetic rack to separate the magnetic beads and the liquid, and after the solution is clarified (about 5min), the supernatant is carefully removed (note: remove the supernatant as much as possible).
S204, keeping the PCR tube in a magnetic frame, adding 300 mu L of freshly prepared 90% ethanol to rinse the magnetic beads, carefully moving the PCR tube to the opposite side of the magnetic strip, incubating at room temperature for 60S, and carefully removing the supernatant until the magnetic beads are completely moved to the tube wall close to the magnetic strip. This step was repeated once for a total of two rinses.
S205, keeping the PCR tube in the magnetic frame all the time, uncovering the magnetic beads and drying the magnetic beads in air for 10min until no ethanol remains.
S206, taking out the PCR tube from the magnetic frame, adding 30 mu L of EB for elution, carrying out vortex oscillation or gently blowing and beating by using a pipette, fully and uniformly mixing, standing for 10min at room temperature, carrying out short-time centrifugation on the PCR tube, placing on the magnetic frame for standing, carefully sucking 20 mu L of supernatant into a new PCR tube after the solution is clarified (about 5min), and cutting without touching magnetic beads to obtain a purified product.
Example 3
S101, fragmenting a DNA sequence by using a reaction system containing a fragmenting enzyme, and performing end repairing on the fragmented DNA fragment; the fragmenting enzyme is DNA Fragmentase.
Specifically, the fragmentation buffer was thawed and mixed by inversion, and the following reactions were prepared in sterile PCR tubes:
mix gently by pipetting (Do not shake) and centrifuge briefly to collect the reaction solution to the bottom of the tube. Temporarily placing the PCR tube on ice, setting the following program on a PCR instrument, pressing a pause key after the temperature of the PCR instrument is reduced to 4 ℃, placing the PCR tube into the PCR instrument, and then continuously running the program:
s102, connecting the joint sequences required by high-throughput sequencing to the two ends of the DNA fragment obtained in S101 by using a ligase reaction system, wherein the joint sequences are provided with specific label sequences;
specifically, the linkers (10 μ M) were diluted N-fold using EB as in the table below;
after thawing the ligation buffer, the mixture was inverted and mixed and placed on ice for use.
In the PCR tube of the end repair product obtained in the step S101, the following reaction was prepared:
the ligation buffer and ligase were premixed and stored at 4 ℃ for no more than 24h, and the linker was added during the reaction.
#X is the label number, one for each sample, Indexprime number.
Mix gently by pipetting (Do not shake) and centrifuge briefly to collect the reaction solution to the bottom of the tube. Placing the PCR tube in a PCR instrument, and carrying out the following reactions:
and S103, purifying the product in the S102 through magnetic beads to obtain a high-throughput sequencing library.
Specifically, the magnetic bead purification step is as follows:
s200, taking the magnetic Beads (DNA Clean Beads) out 30min in advance, carrying out vortex oscillation, mixing uniformly, balancing to room temperature, carrying out vortex oscillation and mixing uniformly after the DNA Clean Beads are balanced to the room temperature.
S201, sucking 20 mu L of EBBuffer and 40 mu L of DNA Clean Beads into 30 mu L of adaptor ligation reaction products, and performing vortex oscillation or gently blowing and beating 10 times by using a pipette to fully mix the components.
S202, incubation for 5min at room temperature.
S203, the PCR tube is centrifuged briefly and placed in a magnetic rack to separate the magnetic beads and the liquid, and after the solution is clarified (about 5min), the supernatant is carefully removed (note: remove the supernatant as much as possible).
S204, keeping the PCR tube in a magnetic frame, adding 200 mu L of freshly prepared 80% ethanol to rinse the magnetic beads, carefully moving the PCR tube to the opposite side of the magnetic strip, incubating at room temperature for 30S, and carefully removing the supernatant until the magnetic beads are completely moved to the tube wall close to the magnetic strip. This step was repeated once for a total of two rinses.
S205, keeping the PCR tube in the magnetic frame all the time, uncovering the magnetic beads and drying the magnetic beads in air for 5-10min until no ethanol remains.
S206, taking out the PCR tube from the magnetic frame, adding 19 mu L of EB for elution, carrying out vortex oscillation or gently blowing and beating by using a pipette, fully and uniformly mixing, standing for 5min at room temperature, carrying out short-time centrifugation on the PCR tube, placing on the magnetic frame for standing, carefully sucking 16 mu L of supernatant into a new PCR tube after the solution is clarified (about 5min), and cutting without touching magnetic beads to obtain a purified product.
Example 4
In step S101 of example 1, 0.1. mu.L of polyethylene glycol 2000 was added to the fragmenting enzyme reaction system.
Example 5
In step S101 of example 2, 0.3. mu.L of polyethylene glycol 2000 was added to the fragmenting enzyme reaction system.
Example 6
In step S101 of example 3, 0.2. mu.L of polyethylene glycol 2000 was added to the fragmenting enzyme reaction system.
Example 7
In step S204 of example 4, the ethanol solution contains 0.02mol/L potassium chloride.
Example 8
In step S204 of example 5, the ethanol solution contains 0.05mol/L potassium chloride.
Example 9
In step S204 of example 6, the ethanol solution contains 0.04mol/L potassium chloride.
Example 10
In step S101 of example 6, 0.02mg of PVP-K30 was added to the fragmenting enzyme reaction system.
Example 11
In step S101 of example 6, 0.04mg of PVP-K30 was added to the fragmenting enzyme reaction system.
Example 12
In step S101 of example 6, 0.03mg of PVP-K30 was added to the fragmenting enzyme reaction system.
Comparative example 1
In step S101 of example 2, DNA was fragmented by the ultrasonic method. Specifically, random interruptions were performed using a sonicator. Wherein the ultrasonic power is 300W, the ultrasonic time is 5s every 5s, the total working time is 15min, and the ice bath is carried out.
Comparative example 2
In step S101 of example 5, DNA was fragmented by the ultrasonic method. Specifically, random interruptions were performed using a sonicator. Wherein the ultrasonic power is 300W, the ultrasonic time is 5s every 5s, the total working time is 15min, and the ice bath is carried out.
Comparative example 3
In step S101 of example 8, DNA was fragmented by the ultrasonic method. Specifically, random interruptions were performed using a sonicator. Wherein the ultrasonic power is 300W, the ultrasonic time is 5s every 5s, the total working time is 15min, and the ice bath is carried out.
The purified products of examples 1 to 9 and comparative examples 1 to 3 were subjected to fragment length measurement, and the measurement results are shown in Table 7:
TABLE 7
According to the results in Table 7, it can be seen that, after DNA fragmentation is performed by using the fragmenting enzyme in the present invention, the proportion of library fragments in 150bp-200bp is higher than that in comparative examples 1-3, which is more favorable for the subsequent pathogen detection. In examples 4-9, i.e., the group using polyethylene glycol 2000, the proportion of 150bp-200bp in the library was higher than in examples 1-3, demonstrating the positive effect of polyethylene glycol 2000 in constructing the library. Examples 10-12 demonstrate the fact that the addition of PVP-K30 to a system with polyethylene glycol 2000 further improves the homogeneity of the DNA fragments after the reaction.
Examples 13 to 20
S301, extracting nucleic acid;
s302, constructing a high-throughput sequencing library as described above;
s303, quantifying the sequencing library constructed in the S302;
s304, diluting the quantitative sequencing library to 1-4pM, and performing high-throughput sequencing on a sequencer;
s305, the sequencing data obtained in S304 is analyzed.
S301, nucleic acid extraction, which comprises the following specific steps:
a nucleic acid extraction or purification reagent (Cat.1901, jin Ji 20190271, Tianjin Kingspoon medical science and technology Co., Ltd.) was used to extract the plasma samples of 1 clinical bloodstream infection patients, and the specific operation steps were as follows:
(1) taking 300. mu.l of plasma into a 1.5ml centrifuge tube, if the volume is less than 300. mu.l, adding the buffer solution A to a final volume of 300. mu.l;
(2) adding 20 mul protease K solution, and uniformly mixing by vortex;
(3) add 300. mu.l of buffer B and 6. mu.l of Carrier RNA at a concentration of 1. mu.g/. mu.l, mix by gentle inversion, incubate at 70 ℃ for 15min, and shake the sample occasionally. Centrifuging briefly to remove liquid drops on the inner wall of the tube cover;
(4) after the temperature of the sample is reduced to room temperature, 300 mul of precooled absolute ethyl alcohol is added. Slightly inverting and mixing the sample, standing at room temperature for 5min, and centrifuging briefly to remove liquid drops on the inner wall of the tube cover;
(5) sucking 600 μ l of the solution obtained in the previous step into an adsorption column (the adsorption column is placed into a collecting pipe), centrifuging at 8000rpm for 30sec, discarding the waste liquid, and placing the adsorption column into a new 2ml centrifuge tube with a tube cover cut off;
(6) repeating the step 5;
(7) adding 500 μ l buffer solution C (checking whether absolute ethanol is added before use) into the adsorption column, centrifuging at 8000rpm for 30sec, discarding waste liquid, and placing into a new 2ml centrifuge tube with tube cover removed;
(8) adding 600 μ l of rinsing solution (checking whether absolute ethanol is added before use) into adsorption column, centrifuging at 8000rpm for 30sec, discarding waste liquid, and placing adsorption column into a new 2ml centrifuge tube with tube cover cut off;
(9) repeating the operation step 8;
(10) centrifuge at 12,000rpm (. about.13,400 Xg) for 2min and discard the waste. Sucking residual liquid in the adsorption column with 10 μ l gun head, uncovering the adsorption column, placing in a clean 1.5ml centrifuge tube, standing at room temperature for 2-5min until ethanol is fully volatilized;
(11) note that: the purpose of the step is to remove the residual rinsing liquid in the adsorption column, and the residual ethanol in the rinsing liquid can influence the subsequent enzyme reaction (enzyme digestion, PCR and the like) experiment;
(12) suspending and dripping 50 mul of eluent into the middle position of the adsorption film, standing for 5min at room temperature, centrifuging for 2min at 12,000rpm (13,400 Xg), and collecting the solution into a centrifuge tube;
(13) the extracted nucleic acid DNA was quantified using the Qubit dsDNA HS assay Kit (Invitrogen Cat. Q32851), and the quantification results are shown in Table 8:
TABLE 8
| Group of | Sample numbering | Concentration (ng/. mu.l) | Volume (μ l) | Yield (ng) |
| Example 13 | ZYR | 0.538 | 20 | 10.76 |
| Example 14 | CKY | 0.408 | 20 | 8.16 |
| Example 15 | RJY | 0.478 | 20 | 9.56 |
| Example 16 | XJY | 0.486 | 20 | 9.72 |
| Example 17 | HQ | 0.456 | 20 | 9.12 |
| Example 18 | WRK | 0.508 | 20 | 10.16 |
| Example 19 | GM | 0.488 | 20 | 9.76 |
| Example 20 | ZLL | 0.486 | 20 | 9.72 |
S302, constructing a high throughput sequencing library using the method described in example 9;
s303, quantifying the sequencing library constructed in the S302, and specifically comprising the following steps:
the sequencing libraries were quantified using the QubitdsDNAHSassaykit (Invitrogen Cat. Q32851) and the VAHTS Library Quantification Kit for Illumina (VazyMesat. NQ101), the Quantification results are shown in Table 9:
TABLE 9
| Group of | Sample numbering | Library concentration (ng/. mu.l) | Total amount of library (ng) | qPCR results (nM) |
| Example 13 | ZYR | 0.428 | 8.56 | 0.479 |
| Example 14 | CKY | 0.35 | 7 | 0.181 |
| Example 15 | RJY | 0.45 | 9 | 0.284 |
| Example 16 | XJY | 0.386 | 7.72 | 0.445 |
| Example 17 | HQ | 0.348 | 6.96 | 0.339 |
| Example 18 | WRK | 0.434 | 8.68 | 0.483 |
| Example 19 | GM | 0.424 | 8.48 | 0.416 |
| Example 20 | ZLL | 0.374 | 7.48 | 0.387 |
S304, diluting the quantitative sequencing library to 1-4pM, and performing high-throughput sequencing on a sequencer, wherein the specific steps are as follows:
the sequencing libraries of examples 12-14 were diluted to 10pM after mixing in equal amounts, the sequencing libraries of examples 15-16 to 20pM after mixing in equal amounts, the sequencing libraries of examples 16-19 to 40pM after mixing in equal amounts, and high throughput sequencing was performed on an Illumina NextSeq550 sequencer using the NextSeq 500/550 high throughput v2 kit (75 cycles) (illuminacat. FC-404-.
S305, analyzing the sequencing data obtained in S304, and specifically comprising the following steps:
according to the specific molecular sequence added in the library construction process of each sample, the sequencing data belonging to each sample is split, the split data is analyzed by bioinformatics analysis software, the data quality, GC content, repeated sequence proportion and the like of the split data are firstly evaluated, meanwhile, a host database is compared, the host sequence proportion in the data is calculated, and the specific results are shown in Table 10:
watch 10
The sequence after host removal is compared with an autonomously developed pathogenic database containing 9705 types of microbial sequence information, and the pathogenic microbial species information contained in the sample is obtained by analysis, as shown in table 11:
TABLE 11
As can be seen from the results in tables 9-11, the pathogen detection using the method of the present invention has more accurate and reliable detection results compared to the prior art in terms of the number of matched pathogen reads and the gene coverage.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. A method for the detection of pathogenic microorganisms based on metagenomics, said method being a non-disease diagnostic method, said method comprising the steps of:
extracting nucleic acid;
constructing a high-throughput sequencing library;
sequencing library quantification: quantifying the sequencing library constructed in the high-throughput sequencing library construction step;
high-throughput sequencing: diluting the sequencing library quantified in the sequencing library quantifying step to 1-4pM, and performing high-throughput sequencing on a sequencer;
and (4) analyzing results: analyzing sequencing data obtained in high-throughput sequencing;
the method for constructing the high-throughput sequencing library comprises the following steps:
s101, fragmenting a DNA sequence by using a reaction system containing a fragmenting enzyme, and performing end repairing on the fragmented DNA fragment;
s102, connecting the joint sequences required by high-throughput sequencing to the two ends of the DNA fragment obtained in S101 by using a ligase reaction system, wherein the joint sequences are provided with specific label sequences;
s103, purifying the product in the S102 to obtain a high-throughput sequencing library;
the analysis method of the result analysis step includes the steps of:
s401, splitting the sequencing data of the sample,
s402, calculating the proportion of the host sequence in the data,
s403, removing the host sequence,
s404, comparing the residual sequence with a pathogen database, wherein the method comprises the following steps:
s501, generating a database hash index, splitting base sequences contained in the database based on the specific sequence length S, and storing the information of a genome position list where the base sequences are located by taking the base sequences as the hash index;
s502, sequence local comparison, namely splitting the query sequence into base segments with the same length, searching a corresponding sequence in a Hash index through Hash collision, and acquiring the position information of the segments in a genome;
s503, calculating the edit distance of the sequencing sequence at different positions of the reference sequence, and searching the optimal comparison result;
s504, counting and outputting comparison results, calculating the distance between the sequencing sequence and a database reference sequence according to sequence difference, if the optimal comparison result editing distance is m, searching a comparison result with the minimum editing distance m and the maximum editing distance n according to a preset editing distance range n, and outputting the result;
in step S101, the reaction system includes: according to the parts by volume, X parts of DNA sample, 1-2 parts of fragmentation Buffer solution, 2-4 parts of fragmentation enzyme and Buffer EB complement the system to 15 parts, wherein the concentration of the DNA sample is 5-30 ng/mu l, X is less than 12,
the reaction conditions are as follows:
the fragmentation enzyme reaction system also comprises polyethylene glycol 2000, wherein the content of the polyethylene glycol 2000 is 0.1-0.3 part by volume, and the fragmentation enzyme reaction system also comprises 0.02-0.04 part by volume of polyvinylpyrrolidone K30 (PVP-K30).
2. A method for detecting pathogenic microorganisms based on metagenomics according to claim 1, characterized in that: in the step S102, the reaction system includes, in parts by volume:
10-20 parts of end repair product, 5-8 parts of connection Buffer solution, 2-5 parts of ligase, 4-6 parts of linker with tag sequence, 0.5-2 parts of Buffer EB, wherein the concentration of the ligase is 500-800U/mu l,
the reaction conditions are as follows,
3. a method for the detection of pathogenic microorganisms based on metagenomics according to any one of claims 1-2, characterized in that: in step S103, purifying the product in step S102 with magnetic beads, where the step of purifying with magnetic beads includes:
s201, sucking 10-30 mu L of Buffer EB and 30-50 mu L of DNA Clean Beads into 20-40 mu L of joint connection reaction product, and fully and uniformly mixing;
s202, incubating for 1-10min at room temperature;
s203, after centrifugation, separating the magnetic beads and the liquid under the action of an external magnetic field, and removing the supernatant after the solution is clarified;
s204, continuously adding 100-300 mu L of 70-90% ethanol solution to rinse the magnetic beads under the action of an external magnetic field, incubating at room temperature for 10-60S, and removing the supernatant;
s205, exposing the magnetic beads in air and drying for 5-10 min;
s206, adding 10-30 mu L of EB into the magnetic beads for elution, fully and uniformly mixing, standing for 1-10min at room temperature, centrifuging, standing under an external magnetic field, and sucking 10-20 mu L of supernatant after the solution is clarified, thus obtaining the purified product.
4. A method for detecting pathogenic microorganisms based on metagenomics according to claim 3, characterized in that: in S204, the ethanol solution contains 0.02-0.05mol/L potassium chloride.
5. A method for detecting pathogenic microorganisms based on metagenomics according to claim 1, characterized in that: the method for splitting the sample sequencing data comprises the steps that a specific label sequence is arranged on a joint connected with two ends of each library, and after the data is downloaded, different reads are distributed to different libraries according to the label sequence measured on each sequence.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004009814A1 (en) * | 2002-07-23 | 2004-01-29 | Nuevolution A/S | Gene shuffling by template switching |
| WO2007046817A1 (en) * | 2005-10-19 | 2007-04-26 | E.I. Dupont De Nemours And Company | A mortierella alpina c16/18 fatty acid elongase |
| CN110381980A (en) * | 2017-01-06 | 2019-10-25 | 艾维迪提生物科学有限责任公司 | The method that nucleic acid-peptide composition and inducing exon are skipped |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2002221582A1 (en) * | 2000-12-19 | 2002-07-01 | Danmarks Jordbrugsforskning | A novel tissue specific plant promoter |
| US8530228B2 (en) * | 2010-12-28 | 2013-09-10 | Bexmart | Integrated versatile and systems preparation of specimens |
| CN103805697B (en) * | 2013-12-22 | 2016-05-04 | 上海翰宇生物科技有限公司 | A kind of method that can be used for the full genomic gene function of Rapid identification microorganism |
| CN106244578B (en) * | 2015-06-09 | 2021-11-23 | 生捷科技控股公司 | Method for sequencing nucleic acids |
| CN105255998A (en) * | 2015-06-18 | 2016-01-20 | 西南大学 | Method for fast searching pathogen specific detection target |
| CN107674870A (en) * | 2016-08-01 | 2018-02-09 | 武汉生命之美科技有限公司 | A kind of method of the target RNA sequence of rna binding protein in improved identification of cell sample |
| CN108998434B (en) * | 2017-06-07 | 2021-05-28 | 深圳华大生命科学研究院 | Fragmentation enzyme breaking buffer solution and method for improving fragmentation enzyme breaking efficiency |
| CN107217308A (en) * | 2017-06-21 | 2017-09-29 | 北京贝瑞和康生物技术股份有限公司 | A kind of sequencing library construction method and kit for being used to detect chromosome copies number variation |
| CN112375807B (en) * | 2017-12-30 | 2023-02-21 | 浙江安诺优达生物科技有限公司 | Method for randomly breaking DNA |
| CN108330546B (en) * | 2018-03-21 | 2019-02-01 | 东莞博奥木华基因科技有限公司 | A kind of library constructing method and reagent of simplification |
| CN109610008A (en) * | 2018-11-08 | 2019-04-12 | 广州华大基因医学检验所有限公司 | Cental system pathogenic infection detection library constructing method, detection method and kit based on high-flux sequence |
-
2019
- 2019-11-28 CN CN201911191572.1A patent/CN111020018B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004009814A1 (en) * | 2002-07-23 | 2004-01-29 | Nuevolution A/S | Gene shuffling by template switching |
| WO2007046817A1 (en) * | 2005-10-19 | 2007-04-26 | E.I. Dupont De Nemours And Company | A mortierella alpina c16/18 fatty acid elongase |
| CN110381980A (en) * | 2017-01-06 | 2019-10-25 | 艾维迪提生物科学有限责任公司 | The method that nucleic acid-peptide composition and inducing exon are skipped |
Non-Patent Citations (1)
| Title |
|---|
| Pima90-53细菌人工染色体（BAC）文库构建及抗黄萎病相关基因筛选;王文生;《中国优秀博硕士学位论文全文数据库 (硕士) 农业科技辑》;20160815(第8期);D046-11 * |
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