CN111926394B - Metagenomics-based library construction method and detection kit - Google Patents

Abstract

The invention relates to a library building method and a detection kit based on metagenomics, and belongs to the technical field of infection pathogen diagnosis. The metagenomics-based library construction method includes the following steps: S1: extracting genomic nucleic acid in the sample to be tested; S2: in the enzyme digestion system, endonuclease is used for enzyme digestion and fragmentation, and end repair enzymes are used to lyse the DNA fragments. Perform end repair; S3: Add adapter and ligase to the solution obtained in step S2, and perform adapter ligation in a buffer system; S4: Use magnetic beads to purify the ligated product; S5: Amplify the purified product ; S6: Use magnetic beads to purify the amplified product to remove small fragments. The library construction method is suitable for laboratories that need to construct a large number of samples and nucleic acids of various sample types for metagenomic library construction. It not only improves the utilization rate of personnel and instruments, reduces the error rate, but also helps to ensure the success rate of library construction. Achieving the ultimate delivery target in 24 hours.

Description

Database building method and detection kit based on metagenomics

Technical Field

The invention relates to the technical field of infectious pathogen diagnosis, in particular to a database building method and a detection kit based on metagenomics.

Background

The metagenome (mNGS) technology has gradually become a widely adopted technology in many aspects of discovery and transformation research, has important application in pathogen diagnosis and analysis of urgent and difficult severe infectious diseases, and can perform unbiased sequencing on all nucleic acids in a sample, including nucleic acids of human sources and microorganisms.

In vitro infection diagnosis techniques can be used for the detection of clinical samples of all types of infection, such as sputum, alveolar lavage, pharyngeal swabs of respiratory tract infections; cerebrospinal fluid of central nervous system infections; blood from bloodstream infections, and other sample types, such as pleural effusion, tissues, paraffin sections, and the like. The quality of the extracted nucleic acids fluctuates greatly in different sample types due to the different contents of host cells and microorganisms. In order to meet the requirement of diversified sample library construction, the current commercial kits developed for macro-genome library construction all provide a large range of initial input amount of nucleic acid for library construction, and require users to properly dilute joints and properly adjust the number of PCR cycles for different input amounts. This is complicated for the medical examination laboratory that needs to carry out a large amount of samples and multiple sample types every day to carry out metagenome construction, consumes the flow of manpower and PCR appearance, needs medical laboratory to invest more manpower and material resources to ensure accuracy every day, and timely delivery work goes on smoothly.

Disclosure of Invention

Therefore, it is necessary to provide a standardized metagenomics-based library building method and a standardized metagenomics-based detection kit, which are suitable for laboratories that need to build a large number of samples and nucleic acids of various sample types, and can not only improve the utilization rate of personnel and instruments and reduce the error rate, but also be beneficial to ensuring the success rate of library building, and achieve the delivery goal of 24 hours.

A database building method based on metagenomics, comprising the steps of:

s1: extracting genome nucleic acid in a sample to be detected;

s2: in the restriction system, the genomic nucleic acid was present at 3. + -.2 ng: the usage amount of 20 plus or minus 5mU of endonuclease is used for carrying out enzyme slice segmentation on the endonuclease and carrying out end repair on the DNA fragment by using end repair enzyme;

s3: adding a linker and ligase into the solution obtained in the step S2, and performing linker connection in a buffer solution system;

s4: purifying the connected product by using magnetic beads, and removing incorrect connection, short joint fragments and residual joints;

s5: amplifying the purified product;

s6: and (3) purifying the amplified product by using magnetic beads, removing small fragments, and enabling the library peak shape to be single and concentrated.

In earlier studies, the inventor finds that in the metagenomic technology, the extracted nucleic acid quality greatly fluctuates due to the content difference of host cells and microorganisms in different sample types, and the inventor utilizes the sample advantages of self big data to summarize the extracted nucleic acid concentration of about 1000 samples selected from each sample type, finds that the extracted concentration of cerebrospinal fluid and blood samples is low and can be lower than the detection lower line of a nucleic acid concentration detector in some cases, and the extracted concentration of sputum and alveolar lavage fluid samples can be as high as 100 ng/mu L and the total amount is about 10-20 mu g, as shown in FIG. 1.

Aiming at the current situation, the invention researches and investigates samples of different types, prepares a metagenome library construction method suitable for the types of the used samples, unifies a detection method which needs to perform individual processing adjustment aiming at the characteristics of the samples of different types in the conventional technology into a standardized processing mode, improves the utilization rate of personnel and instruments, reduces the error rate and is beneficial to ensuring the success rate of library construction.

In one embodiment, in the step S1, the following method is used to extract the genomic nucleic acid from the test sample:

sample lysis: adding a sample to be tested into the lysate, uniformly mixing, incubating for 10-15min at 65-70 ℃ after centrifugation, centrifuging again, and cooling to room temperature;

DNA purification: transferring all the cracked lysate to an adsorption column, centrifuging to remove waste liquid, adding a buffer solution, centrifuging to remove protein bound on nucleic acid, adding the buffer solution, centrifuging to remove ions adsorbed on the adsorption column, and repeating the step for 1-3 times;

and (3) DNA drying: placing the adsorption column in a centrifuge tube, centrifuging for 3-5min at 10000-;

DNA dissolution: adding 40-70 μ L TB buffer solution or NF water (nanofiltration water) into the adsorption column, standing for 3-6min, centrifuging at 10000-.

In one embodiment, in the step S2, the concentration of the genomic nucleic acid solution obtained in the step S1 is detected, and then the feeding volume of the genomic nucleic acid is calculated according to the concentration of the genomic nucleic acid solution, so that the genomic nucleic acid in the enzyme digestion system is 3 ± 2 ng. By adjusting the volume of the nucleic acid solution, the total amount of the nucleic acid added to the reaction system is controlled, so that the subsequent process steps can be performed according to standardized operations.

In one example, in the step S2, the endonuclease is a non-restriction endonuclease, the end-repair enzyme is T4DNA polymerase or Klenow fragment enzyme, and the ratio of genomic nucleic acid: adding the end repairing enzyme in an amount of 1 plus or minus 0.2U; the conditions of the enzyme digestion fragmentation are as follows: incubating at 37 + -0.5 deg.C for 25 + -2 min; the conditions for end repair are as follows: incubating at 65 + -0.5 deg.C for 30 + -2 min; in the step S3, the genomic nucleic acid: adding a linker in an amount of 0.2 +/-0.1 ng of linker, wherein the ligase is T4DNA ligase, and the reaction conditions of linker ligation are as follows: keeping at 20 + -0.5 deg.C for 15 + -1 min.

In one embodiment, in the step S3, the buffer system further includes PEG8000 at a concentration of 30% to 35% by volume and hydroxypropyl methylcellulose at a concentration of 0.0001 ± 0.00002% by mass. The effect of the linker reaction can be improved by the combination of PEG8000 and hydroxypropyl methylcellulose (HPMC).

In one embodiment, in the step S4, the ligated product is purified using 0.6 × magnetic beads; in the step S6, the product after PCR amplification was purified using 0.9 × magnetic beads.

The magnetic beads refer to commercial magnetic beads (Beckmann), the system comprises magnetic beads, PEG, salt ions and the like, DNA can be adsorbed to the surface of the carboxyl modified polymer magnetic beads in the environment of PEG and salt ions with certain concentration, the process is reversible, and the DNA can be eluted under the condition of low salt. The screening of the magnetic beads on the size of the DNA fragments depends on PEG to a great extent, the concentration of PEG in a screening system is also high, and the screening effect on the DNA fragments is smaller. The 0.6 × PEG buffer magnetic bead refers to a magnetic bead buffer solution in which the PEG concentration in the working solution of the magnetic bead is 60% of the commercial product concentration, and similarly, the 0.9 × PEG buffer magnetic bead refers to a magnetic bead buffer solution in which the PEG concentration in the working solution of the magnetic bead is 90% of the commercial product concentration.

In one embodiment, the library construction method further comprises a step of S7, and the method for performing single-chain cyclization and nanosphere preparation on the product obtained from the library comprises the following steps:

calculating the input mass of each library according to the data quantity required to be generated by each library, and mixing the preset libraries; performing thermal denaturation on the mixed library and the short linker, and placing the denatured library on ice for later use; adding the denatured library into a single-chain cyclization reaction system for cyclization reaction; digesting the cyclized product by using an unclycled library, and purifying by using RNA magnetic beads; and (3) carrying out primer hybridization and rolling circle amplification on the purified product to obtain the nanosphere.

In one embodiment, the sample to be tested is cerebrospinal fluid, blood, sputum, alveolar lavage fluid, pleural effusion or urine.

The invention also discloses the metagenomic library obtained by the metagenomic library construction method.

The invention also discloses a pathogen detection method for non-diagnostic use, which comprises the database building method based on the metagenomics and also comprises the following steps:

sequencing: sequencing the constructed library;

and (3) letter generation analysis: removing the host sequence by bioinformatics analysis method to obtain the pathogenic gene information.

The invention also discloses a detection kit based on the metagenomics, which comprises:

enzyme digestion and end repair reagent: the enzyme preparation comprises an endonuclease and a terminal repair enzyme, wherein the working dosage of the endonuclease is 20 +/-5 mU, and the working dosage of the terminal repair enzyme is 1 +/-0.2U;

linker and linking reagent: the kit comprises a joint, ligase and a connection buffer solution, wherein the working dosage of the joint is 0.2 +/-0.1 ng, and the ligase is T4DNA ligase;

amplification reagents: comprises PCR amplification enzyme, an amplification buffer solution and an amplification primer;

and (3) purifying a reagent: including 0.6 x PEG buffer magnetic beads and 0.9 x PEG buffer magnetic beads.

In one embodiment, the ligation buffer comprises: PEG8000 with the volume percentage concentration of 30-35 percent and hydroxypropyl methylcellulose with the mass percentage concentration of 0.0001 +/-0.00002 percent.

Compared with the prior art, the invention has the following beneficial effects:

according to the method for establishing the library based on the metagenomics, a set of red genomics standardized library establishing method for different types of samples is established by testing and searching a large number of samples of different sample types, and through the method, the characteristics and the differences of the samples of different types can be ignored, and the library establishment and the on-machine test are performed in a standardized mode, so that the utilization rate of personnel and instruments is improved, the error rate is reduced, the success rate of library establishment is favorably ensured, and the goal of delivering the library within 24 hours is achieved.

According to the detection kit based on the metagenomics, the reagent is configured according to the database building method, so that the detection kit can be suitable for different types of samples, simplifies the using and operating steps, reduces the requirements on users, and has the advantages of high detection accuracy and convenience.

Drawings

FIG. 1 is a graph showing the concentration of nucleic acid extracted from different types of large samples according to the present disclosure;

FIG. 2 is a graph showing comparison between the results of the detection in example 2 and those of example 1;

wherein: "present invention" is the number of Reads in example 1 and "conventional method" is the number of Reads in example 2;

FIG. 3 is a graph showing the comparison of the concentration of the library in example 3 with that of example 1;

wherein: the "0.2" group was the example 1 library concentration, the remaining group was the example 3 library concentration;

FIG. 4 is a graph showing comparison between the results of example 3 and those of example 1;

wherein: the "0.2" group is the numbers of Reads in example 1, and the remaining groups are the numbers of Reads in example 2;

FIG. 5 is a graph showing comparison between the results of the detection in example 5 and those of example 1;

wherein: the "no HPMC added" group is the Reads number in example 5, and the "HPMC added" group is the Reads number in example 1;

FIG. 6 is a graph showing the comparison of the concentration of the library in example 6 with that of example 1;

wherein: the "PEG added" group was the example 6 library concentration and the "HPMC added" group was the example 1 library concentration.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The starting materials used in the following examples are all commercially available, unless otherwise specified.

Example 1

A method of pathogen detection comprising the steps of:

firstly, a sample source.

The experimental sample is a clinical positive sample M1-M18, and the types of pathogens determined by methods such as PCR, culture and the like are shown in the following table.

TABLE 1 pathogen species for each type of sample

And II, a method.

1. And (5) building a library.

S1: extracting all genome nucleic acid in a clinical sample to be detected by using a high-purity nucleic acid extraction method, wherein the specific method comprises the following steps:

s1.1, sample cracking:

adding a sample to be detected into the lysate, mixing uniformly, centrifuging, incubating for 15min at 70 ℃, centrifuging again, and cooling to room temperature.

S1.2, DNA purification:

transferring all the cracked lysate to an adsorption column, centrifuging to remove waste liquid, adding a buffer solution, centrifuging to remove protein bound on nucleic acid, adding the buffer solution, centrifuging to remove ions adsorbed on the adsorption column, and repeating the step for 1-3 times.

S1.3, DNA drying:

placing the adsorption column in a centrifuge tube, centrifuging at 12000r/min for 3min, air-drying, and air-drying for 3-5 min.

S1.4, DNA dissolution:

adding 70 μ L TB buffer solution into adsorption column, standing for 5min, centrifuging at 12000r/min for 2min, and collecting genome nucleic acid liquid.

S2: DNA fragmentation and end repair

In a fragmentation and end repair system, 3ng of genome nucleic acid, 20mU of non-restriction endonuclease and 1U T4DNA polymerase are added into each sample and mixed, the samples are firstly incubated at 37 ℃ for 15min for enzyme fragment fragmentation, and then incubated at 65 ℃ for 30min for end repair, and the reaction system components are shown in the table below.

TABLE 2 enzyme digestion fragmentation and end-repair reaction System

Note: y means that the total amount of nucleic acid DNA added was 3ng, and the addition volume was calculated from the concentration of nucleic acid obtained by extraction.

S3: joint connection

After the S2 reaction is finished, 0.2ng of linker and a T4 ligase system are added, and the mixture is incubated for 15min at 20 ℃ in a buffer solution system, wherein the components of the reaction system are shown in the following table:

TABLE 3 enzyme fragmentation and end-repair reaction System

Note: the PEG8000 is originally added into the buffer solution of the commercial linker reagent (the volume percentage concentration is 30-35%), and only HPMC is required to be additionally added during use, and the PEG8000 does not need to be added again.

S4: ligation product purification

The ligation product was purified with 0.6 × PEG buffer magnetic beads to remove incorrect ligations, linker short fragments and residual linker.

S5: library amplification

13 cycles of library amplification were performed using 10pmol of primers and other conventional amplification reagents according to the following PCR amplification procedure, and stored at 4 ℃.

TABLE 4 library amplification reaction System

Reaction system	The total amount is 50 μ L
		S2.3 step purified product	20μL
Primer and method for producing the same	10pmol
		PCR amplification reagent	25μL
Total	Supplement H2O to 50. mu.L

S6: library purification

And purifying the connection product by 0.9 XPEG buffer magnetic beads, and removing components of a PCR reaction system and small fragment nucleic acid to ensure that the library has single and concentrated peak patterns.

S7: single-chain cyclization and nanosphere preparation.

Calculating the input quality of each library according to the data quantity required to be generated by each library by conventional operation, and mixing the libraries required to be operated together; performing thermal denaturation on the mixed library and the short linker, wherein the reaction time is 98 ℃, and the mixed library and the short linker are immediately placed on ice after the thermal denaturation; adding the denatured library into a single-chain cyclization reaction system, and reacting for 15min at 37 ℃; the circularized product was digested with the non-circularized library and then purified using 1.625 × RNA magnetic beads; and (3) performing primer hybridization and rolling circle amplification on the purified product to obtain the nanospheres on the MGI platform.

2. And (5) sequencing.

Sequencing the constructed library. The specific concentration detection method is based on Invitrogen^TMThe 1X dsDNA HS Assay Kit instructions, fragment size was determined according to the Bioptic Qsep1 fragment analyzer protocol.

3. And (5) performing letter generation analysis.

Removing the host sequence by bioinformatics analysis method to obtain the pathogenic gene information.

Three, result in

The results of the measurements are shown in the following table.

TABLE 5 Key quality control and pathogen detection results

Note: "linker ratio" refers to the ratio of linker sequences in the assay results, and a lower linker ratio indicates a higher library quality.

Fourth, conclusion

The results show that the database is established according to the database establishing method based on the metagenomics for detection, the method is suitable for different types of clinical samples, and the detection result is accurate and reliable.

Example 2

A pathogen detection method screening is carried out as follows.

Firstly, a method is provided.

With reference to the process of example 1, the only difference is that:

1. in the step S2, the input amount of the genomic nucleic acid DNA is within the range of 100 pg-1. mu.g, and the usage amount of the endonuclease is still 20 mU;

2. in step S3, the amount of linker was added according to the amount of DNA added as follows:

TABLE 6 genomic nucleic acid input and linker usage

DNA input amount	Amount of joint used
		500ng–1μg	40±1ng
100ng–500ng	20±1ng
		25ng–100ng	10±1ng
5ng–25ng	2±0.5ng
		100pg–5ng	0.2±0.1ng

Note: the "amount of linker used" is an amount of linker used adjusted to match the amount of DNA nucleic acid to be added.

3. In step S5, the number of PCR amplification cycles was adjusted according to the amount of DNA put in the following table:

TABLE 7 genomic nucleic acid input and PCR cycle number

DNA input amount	Number of PCR cycles
		500ng–1μg	3
100ng–500ng	3±1
		25ng–100ng	5±2
5ng–25ng	10±2
		100pg–5ng	15±2

Note: the "number of PCR cycles" is adjusted according to the amount of DNA nucleic acid to be added.

And II, obtaining a result.

1. The results obtained according to the amounts of nucleic acid charged were as follows.

TABLE 8 genomic nucleic acid input and pathogen detection results

As shown in the above table and FIG. 2, the "conventional method" on the right side of FIG. 2 refers to the conventional library construction method (i.e., Reads detected in example 2) in which the amounts of other components in the digestion system and the linker system are adjusted according to the concentration and amount of the nucleic acid obtained by quantitative input of the endonuclease library construction nucleic acid, and the "present invention" on the left side refers to the library construction method in example 1 in which the amounts of other components in the digestion system and the linker system are adjusted, and the results of comparison of the Reads detected by the two methods show that the detection sensitivity of the conventional library construction method is lower than that of the present invention, for example, the detection results of the M6 and M14 samples by the conventional method are negative, and the detection Reads of the other samples all have a.

By analyzing key QC indexes, the deviation of the library peak value among samples in the conventional method has larger fluctuation compared with the method, and indirectly influences the uniformity of the Pooling on-machine library, thereby causing the nonuniformity of the data quantity among samples, wherein the data quantity is one of key factors influencing the sensitivity of metagenome. On the other hand, QC of the offline data shows that the joint in the offline data volume of the conventional method is higher than that of the invention, so that the efficiency of the data is directly reduced, and the sensitivity of the methodology is reduced.

Example 3

A pathogen detection method, performed with reference to the method of example 1, except that, in terms of DNA input: linker amounts were 3ng:0.07ng (group 1) or 3ng:0.52ng (group 2) or 3ng:1.36ng (group 3), and linker dosages were adjusted. The results obtained are as follows.

TABLE 9. different linker input amount library linker ratio (ng)

As shown in the above table and FIGS. 3-4, FIG. 3 is a comparison of library concentrations (ng/. mu.L) under different linker input conditions, and when the linker input amount is 0.07ng, the library concentration is significantly lower than that of example 1 in which the input amount is 0.2ng, wherein the library concentration of 3 samples is much lower than 1 ng/. mu.L, and the library concentration of 5 samples is around 1 ng/. mu.L, which cannot satisfy Pooling once for a plurality of samples or Pooling machine can be performed after the samples are concentrated, thus increasing the complexity of the operation.

When the input amount of the linker is increased to 0.52ng or 1.36ng, the concentration of the library is not obviously improved, but the proportion of the linker in the off-line data is gradually improved, although the sensitivity performance is not directly influenced, as shown in FIG. 4, and the ordinate of the graph of FIG. 4 is the number of detected pathogens Reads.

In order to increase the effective utilization rate of data, when the input amount of DNA is 3ng, the input amount of the linker is preferably 0.2 ng.

Example 4

A method of pathogen detection, carried out in accordance with the method of example 1, except that:

0.0001 +/-0.00002% of HPMC is not added into the connection system.

The results of the measurements are shown in the following table.

TABLE 10 Key quality control and pathogen detection results

The results are shown in the table and fig. 5, and fig. 5 shows that the pathogen detection performance of the test is not significantly affected by the connection system without adding HPMC, but it can be seen from the table that the yield of the library concentration in the embodiment is only 55%, especially for blood, cerebrospinal fluid and urine samples, the majority of the ex-warehouse concentration cannot reach 1ng/μ L, so that adding HPMC in the connection system has the advantage of enhancing the connection efficiency, and improves the conversion rate and the success rate of the library.

Example 5

A method of pathogen detection, carried out in accordance with the method of example 1, except that:

in addition to the PEG8000 originally added to the linker reagent buffer, 15% of PEG8000 was added to the linker system.

The results of the measurements are shown in the following table.

TABLE 11 Key quality control and pathogen detection results

Results as shown in the table above and fig. 6, fig. 6 is a comparison of the library concentration of this example with the library concentration of example 1, and the results show that adding 15% more PEG to the original system can help to increase the library concentration of blood, cerebrospinal fluid and urine samples, but the increase range is limited, probably because the PEG content in the original system is the more optimized dosage in the present connection system, the fine-tuning PEG content may achieve the optimization effect for a specific sample type, but it is difficult to achieve the uniform optimization effect for different sample types.

By comparing the above examples, a better linking efficiency can be achieved by adding 0.0001 ± 0.00002% HPMC to the linking system, probably due to the potential synergy between PEG molecules and HPMC molecules.

Example 6

A method of pathogen detection, carried out in accordance with the method of example 1, except that: the extraction concentration of the nucleic acid of the sample for library construction is lower than the lower detection limit.

Sample information is shown in the following table:

TABLE 12 samples with nucleic acid extraction concentrations below the detection bottom line

The detection results and key quality control indexes are shown in the following table:

TABLE 13 Key quality control and pathogen detection results

The results are shown in the table above, even for the sample with the nucleic acid concentration lower than the detection off-line, the qualified ex-warehouse concentration and the good detection performance can be obtained by using the scheme of the invention, which shows that the invention can meet the requirements of clinical samples, carry out unified standardized operation on the clinical samples, reduce the experimental complexity and the error rate, and improve the delivery efficiency within 24 hours.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (9)

1. A database building method based on metagenomics is characterized by comprising the following steps:

s1: extracting genome nucleic acid in a sample to be detected;

s2: in an enzyme digestion system, firstly detecting the concentration of the genomic nucleic acid solution obtained in the step S1, then calculating the feeding volume of the genomic nucleic acid according to the concentration of the genomic nucleic acid solution, and performing enzyme digestion according to the ratio of 3 +/-2 ng of the genomic nucleic acid: the usage amount of 20 plus or minus 5mU of endonuclease is used for carrying out enzyme slice segmentation on the endonuclease and carrying out end repair on the DNA fragment by using end repair enzyme; the endonuclease is a non-restriction endonuclease;

s3: adding a linker and ligase into the solution obtained in the step S2, and performing linker connection in a buffer solution system;

s4: purifying the connected product by using magnetic beads, and removing incorrect connection, short joint fragments and residual joints;

s5: amplifying the purified product;

s6: and (3) purifying the amplified product by using magnetic beads, removing small fragments, and enabling the library peak shape to be single and concentrated.

2. The metagenomics-based library building method of claim 1, wherein in the step S2, the end-repair enzyme is T4DNA polymerase or Klenow fragment enzyme, according to 3 ± 2ng genomic nucleic acid: adding the end repairing enzyme in an amount of 1 plus or minus 0.2U; the conditions of the enzyme digestion fragmentation are as follows: incubating at 37 + -0.5 deg.C for 25 + -2 min; the conditions for end repair are as follows: incubating at 65 + -0.5 deg.C for 30 + -2 min;

in the step S3, the genomic nucleic acid: adding a linker in an amount of 0.2 +/-0.1 ng of linker, wherein the ligase is T4DNA ligase, and the reaction conditions of linker ligation are as follows: keeping at 20 + -0.5 deg.C for 15 + -1 min.

3. The metagenomics-based library building method of claim 1, wherein in the step of S3, the buffer system further comprises PEG8000 in an amount of 30-35% by volume and hydroxypropylmethylcellulose in an amount of 0.0001 ± 0.00002% by mass.

4. The metagenomics-based library building method of any one of claims 1-3, further comprising a step of S7, wherein the library product is subjected to single-chain cyclization and nanosphere preparation, and the method comprises the following steps:

calculating the input mass of each library according to the data quantity required to be generated by each library, and mixing the preset libraries; performing thermal denaturation on the mixed library and the short linker, and placing the denatured library on ice for later use; adding the denatured library into a single-chain cyclization reaction system for cyclization reaction; digesting the cyclized product by using an unclycled library, and purifying by using RNA magnetic beads; and (3) carrying out primer hybridization and rolling circle amplification on the purified product to obtain the nanosphere.

5. The metagenomics-based database building method of claim 1, wherein the test sample is cerebrospinal fluid, blood, sputum, alveolar lavage fluid, pleural effusion or urine.

6. The metagenomic library obtained by the metagenomic-based library construction method according to any one of claims 1 to 5.

7. A method for pathogen detection for non-diagnostic use, comprising the method for metagenomics-based database construction according to any one of claims 1-5, further comprising the steps of:

sequencing: sequencing the constructed library;

and (3) letter generation analysis: removing the host sequence by bioinformatics analysis method to obtain the pathogenic gene information.

8. A metagenomics-based detection kit, comprising:

enzyme digestion and end repair reagent: the enzyme preparation comprises an endonuclease and a terminal repair enzyme, wherein the working dosage of the endonuclease is 20 +/-5 mU, and the working dosage of the terminal repair enzyme is 1 +/-0.2U;

linker and linking reagent: the kit comprises a joint, ligase and a connection buffer solution, wherein the working dosage of the joint is 0.2 +/-0.1 ng, and the ligase is T4DNA ligase; the ligation buffer comprises: PEG8000 with the volume percentage concentration of 30-35 percent and hydroxypropyl methylcellulose with the mass percentage concentration of 0.0001 +/-0.00002 percent;

amplification reagents: comprises PCR amplification enzyme, an amplification buffer solution and an amplification primer;

and (3) purifying a reagent: including PEG buffer magnetic beads.

9. The test kit of claim 8, wherein the purification reagents comprise 0.6 x PEG buffer magnetic beads and 0.9 x PEG buffer magnetic beads.

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