CN111304285B

CN111304285B - Urinary metagenome sample library building and detecting method based on nanopore sequencing platform

Info

Publication number: CN111304285B
Application number: CN201911297880.2A
Authority: CN
Inventors: 杨帆; 吴苏生; 郭晓东; 赵成娜; 胡龙; 李杜衡; 涂浩波; 任用
Original assignee: Jiangsu Xiansheng Medical Diagnosis Co ltd; Nanjing Xiansheng Medical Testing Co ltd; Beijing Xiansheng Medical Examination Laboratory Co ltd
Current assignee: Jiangsu Xiansheng Medical Diagnosis Co ltd; Nanjing Xiansheng Medical Testing Co ltd; Beijing Xiansheng Medical Examination Laboratory Co ltd
Priority date: 2019-12-13
Filing date: 2019-12-13
Publication date: 2020-09-29
Anticipated expiration: 2039-12-13
Also published as: CN111304285A

Abstract

The invention relates to the technical field of biology, in particular to a urinary metagenome sample library building and detection method based on a nanopore sequencing platform. The urinary metagenome sample library construction method based on the nanopore sequencing platform is characterized in that a sample to be detected is subjected to host removal and genome extraction in sequence and then is marked, and the obtained marked template is subjected to PCR amplification to construct a library, wherein a reaction system for PCR amplification is subjected to temperature and time optimization, and contains 4% (v/v) of dimethyl sulfoxide, so that amplification preference and host sample detection are reduced, and the library construction method is long in sequence, high in concentration, strong in objectivity and high in accuracy, and simultaneously reduces sequencing difficulty and sequencing false negative.

Description

Urinary metagenome sample library building and detecting method based on nanopore sequencing platform

Technical Field

The invention relates to the technical field of biology, in particular to a urinary metagenome sample library building and detection method based on a nanopore sequencing platform.

Background

Infectious diseases, such as urinary-type infectious diseases, are one of the major threats to human health. The timely and accurate diagnosis of infection is a concern for patient prognosis. The existing detection and diagnosis methods mainly based on morphology and serology have the defects of long culture time, low sensitivity and the like. Since this century, with the rapid development and updating of nucleic acid sequencing technologies, pathogen analysis based on gene sequence analysis has been widely used in the field of infectious diseases. However, the current molecular diagnostic method for simultaneously detecting multiple pathogens in China is still lacking, generally, the method is the directional detection of single known pathogen, and the coverage range in clinical practice and infectious disease prevention and control is limited. For example, the conventional PCR amplification molecular diagnostic technique has many of the above-mentioned difficult-to-solve bottleneck problems, and is difficult to meet the requirements of clinical diagnosis and treatment.

The rapid development of sequencing technology has pushed the research of pathogenic genomics to a new stage in the last decade, and the whole genome information of viruses, bacteria, fungi and parasites is increasing; the development of genomics and bioinformatics, including metagenomics and corresponding sequencing technologies, has greatly expanded the means of research on complex microorganisms, including pathogens. The importance of the application of genomics in the prevention and control of infectious diseases is also increasing with the development of technologies.

Oxford Nanopore Technology (ONT) Nanopore single molecule sequencing Technology has the advantage of a three-generation sequencing long read length, which can be as high as 900 kb. The MinION product of the portable sequencer breaks through the limitation on time and space and perfectly conforms to the requirement of clinical detection by the volume of the U disk, flexibly controlled sequencing flux, the sequencing speed generated by real-time data and an algorithm for synchronizing and paralleling sequencing and data analysis. The metagenome analysis aiming at clinical infection is just an application field which is competent for nanopore sequencing and has unique advantages, and is particularly suitable for whole genome sequencing aiming at pathogens which are difficult to culture in vitro and can not be separated into pure strains.

However, the nanopore monomolecular sequencing technology has extremely high requirements on sample size, the library construction is particularly important, and the nanopore sequencing library construction method aiming at microorganisms is mainly an amplification method at present. The method needs a small amount of nucleic acid starting amount and is suitable for library construction of individual strains, however, the method has high single sample cost and low sequencing flux, and GC preference and a large amount of human nucleic acid occupation data volume can be shown if clinical samples rich in hosts (human beings), especially clinical samples containing various pathogenic bacteria, are faced, namely the problem to be solved is metagenome sequencing. The GC bias is that pathogenic bacteria with low GC content in a genome are preferentially amplified, pathogenic bacteria with high GC content are little or even not amplified, so that the amplification opportunities of each kind of bacteria in mixed pathogenic bacteria of a metagenome are different, the missing detection or the few detection of important pathogenic bacteria can be caused, and the accuracy of result output is influenced. Meanwhile, if the nucleic acid ratio of human is too high, the detection and data amount of real important pathogenic bacteria can be further influenced. This makes it difficult to effectively detect samples such as urinary infections by this method, and creates a bottleneck for downstream sequencing and analysis.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a method for establishing a database of urinary metagenome samples based on a nanopore sequencing platform, so as to relieve the problems that the human nucleic acid proportion in the existing metagenome is too high, the amplification opportunities of infectious bacteria are different, and the accuracy of result output is influenced.

The second purpose of the invention is to provide a urinary metagenome sample detection method based on a nanopore sequencing platform, provide a detection method with higher detection accuracy, and improve the false negative problem of missing detection or less detection.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a urinary metagenome sample library building method based on a nanopore sequencing platform comprises the steps of sequentially removing a host from a sample to be detected, extracting a genome, marking, and performing PCR amplification on an obtained template with a mark to build a library;

the reaction system of the PCR amplification contains 3-5% (v/v) of dimethyl sulfoxide;

the reaction procedure for the PCR amplification facilitates unwinding and extension of the high GC content template.

Further, the content of dimethyl sulfoxide was 4% (v/v).

Further, in the reaction procedure of PCR amplification, the pre-denaturation temperature is 96.5-97.5 ℃, the pre-denaturation time is 2-4min, the denaturation temperature is 96.5-97.5 ℃, the denaturation time is 15-25s, and the extension time is 5-7 min;

further, the pre-denaturation temperature is 97 ℃, the pre-denaturation time is 3min, the denaturation temperature is 97 ℃, the denaturation time is 20s, and the extension time is 6.5 min;

further, the reaction system for PCR amplification also comprises a labeled template, an amplification universal primer, DNA polymerase and a buffer solution;

preferably, the DNA polymerase is LongAmp Taq DNA polymerase;

preferably, the concentration of the labelled template is between 0.02 and 0.1 ng/. mu.l.

Further, the reaction procedure of the PCR amplification comprises:

preferably, the reaction procedure of the PCR amplification comprises:

further, the host removal sequentially comprises host DNA dissociation, host DNA degradation and host DNA removal.

Further, the free host DNA comprises a sample to be detected, and the sample is treated for 5-15min by 0.1-0.5 w/v% of saponin;

preferably, degrading the host DNA comprises dissociating the host DNA from the sample to be detected, and treating with 0.3-1% (v/v) HL-SAN for 10-20 min.

Further, the marking comprises incubation reaction of the sample to be detected and FRM, wherein the sample to be detected is subjected to host removal and genome extraction in sequence;

preferably, the procedure of the incubation reaction comprises: reacting at 29-31 deg.C for 0.5-1.5min, then at 79-81 deg.C for 0.5-1.5min, and storing at 11-13 deg.C;

preferably, the extent of the incubation reaction comprises: reacting at 30 deg.C for 1min, reacting at 80 deg.C for 1min, and storing at 12 deg.C.

A urinary metagenome sample detection method based on a nanopore sequencing platform comprises the library building method.

A kit for establishing a urinary metagenome sample based on a nanopore sequencing platform comprises the components in an RPB004 kit and further comprises DMSO with a final concentration of 4% (v/v).

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

1) according to the method, a database building system suitable for the urinary infection sample is obtained by optimizing the database building system, the objectivity is strong, the accuracy is high, and effective database building and sequencing of the urinary infection sample are guaranteed;

2) according to the invention, 4% (v/v) of dimethyl sulfoxide (DMSO) is added into the urinary sample library building system, so that the unwinding and extension of the GC content template of the high urinary sample can be obviously promoted, and the library building efficiency is improved.

3) The optimized amplification system of the urinary infection sample improves denaturation and pre-denaturation temperatures, prolongs denaturation and extension time, enables melting to be more sufficient, contributes to increase of product concentration, improves sequencing long fragment ratio, improves average length and reduces sequencing difficulty.

4) The database building method is simple and feasible for urinary samples, is practical, has higher accuracy of nanopore sequencing after database building, effectively solves the problem of false negative of missing measurement or less measurement, improves the detection rate, and is suitable for popularization.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is the statistics of the proportion result of each strain in the library in the comparative example of the present invention;

FIG. 2 is the statistics of the ratio of each strain in the library in example 1 of the present invention;

FIG. 3 is the statistics of the ratio of each strain in 2% DMSO pool building in example 2;

FIG. 4 is the statistics of the percentage of each strain in 4% DMSO pool building in example 2;

FIG. 5 is the statistics of the ratio of each strain in 5% DMSO pool establishment in example 2 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

A urinary metagenome sample library building method based on a nanopore sequencing platform sequentially carries out host removal and genome extraction on a sample to be detected, then labeling is carried out on the sample, and an obtained labeled template is subjected to PCR amplification to build a library, wherein a reaction system of the PCR amplification contains 3-5% (v/v) of dimethyl sulfoxide, and a reaction program of the PCR amplification promotes unwinding and extension of a template with high GC content.

The invention aims at the problems that when the existing nanopore sequencing library building method faces to species with large difference of nucleic acid GC content in a metagenome, strains with low GC content are easy to preferentially amplify, so that amplification preference is caused in detection, and the problem is more serious in a sample with low initial amount, so that the invention optimizes the existing amplification system and amplification program, and on one hand, dimethyl sulfoxide is added into a reaction system to reduce the amplification preference; on the other hand, the reaction program promotes the unwinding and the extension of the high GC template, and the library building accuracy, objectivity and efficiency are ensured by improving an amplification system for the high GC content template with higher denaturation temperature, denaturation time, extension time and the like.

It should be noted that, the term "host-removing" refers to a step of removing host DNA, for example, the host DNA may be dissociated, degraded, and finally removed; the marking refers to the marking modification of a sample to be detected after the host is removed and the genome is extracted by using substances such as FRM; the content of dimethyl sulfoxide in the reaction system for PCR amplification is typically, but not limited to, 3% (v/v), 4% (v/v) or 5% (v/v).

In a preferred embodiment, the content of dimethyl sulfoxide is 4% (v/v). Dimethyl sulfoxide (DMSO) is a sulfur-containing organic compound with high polarity, high boiling point and good thermal stability, and is known as an universal solvent. The addition of DMSO in the amplification system of the invention is beneficial to reducing the secondary structure of DNA, so that the template with high GC content is easy to completely denature, and the amplification process is easier to carry out. However, the activity of the amplification enzyme can be inhibited by excessively high content of dimethyl sulfoxide, even the template with high GC content in the gene is preferentially amplified, and the effect cannot be achieved by excessively low content of dimethyl sulfoxide.

In a preferred embodiment, the reaction sequence of PCR amplification has a pre-denaturation temperature of 96.5-97.5 deg.C, a pre-denaturation time of 2-4min, a denaturation temperature of 96.5-97.5 deg.C, a denaturation time of 15-25s, and an extension time of 5-7 min. The DNA chain with high GC content in the genome is harder to melt than the DNA chain with low GC content, and the required melting time is longer, so that the denaturation temperature and the pre-denaturation temperature are increased, the denaturation time is prolonged, the melting is more sufficient, and the increase of the product concentration can be further facilitated; meanwhile, the extension time is increased, so that the DNA amplification enzyme has more sufficient time to carry out new nucleic acid extension, the length-fragment ratio is further increased, and the average length is improved.

In a preferred embodiment, the reaction system for PCR amplification further comprises a labeled template, an amplification universal primer, a DNA polymerase and a buffer. The DNA polymerase is preferably LongAmp Taq DNA polymerase, and the concentration of the labeled template is preferably 0.02-0.1 ng/. mu.l. The universal amplification primer is a common amplification primer for library construction in the prior art.

In a preferred embodiment, the reaction system for PCR amplification is in 50. mu.l, including nucleic-free water 12. mu.l; 4 mul of template with mark; RLB (01-12A) 1. mu.l; LongAmp Taq 2 × master mix (NEB)25 μ l; 25% (v/v) dimethyl sulfoxide DMSO in an amount of 8. mu.l. Among them, RLB is Rapid Barcode Primers, total 12 molecular tags (purchased from ONT, contained in RPB004 kit).

In a preferred embodiment, the reaction sequence for PCR amplification comprises:

in a more preferred embodiment, the reaction sequence for PCR amplification comprises:

in a preferred embodiment, derhosting comprises sequentially episomal host DNA and degradative host DNA, followed by removal of the host DNA.

In a preferred embodiment, the free host DNA comprising the sample to be tested is treated with 0.1-0.5 w/v% saponin for 5-15 min.

In a preferred embodiment, degrading the host DNA comprises treating the sample to be detected with 0.3-1% (v/v) HL-SAN for 10-20min after dissociating the host DNA.

In a preferred embodiment, the labeling comprises incubating the sample to be detected, after sequentially dephosphating and genome extraction, with FRM (Fragmentation Mix, transposase, fragmenting DNA). The procedure for incubating the reaction preferably comprises: reacting at 29-31 deg.C for 0.5-1.5min, reacting at 79-81 deg.C for 0.5-1.5min, and storing at 11-13 deg.C. The extent of the incubation reaction further preferably comprises: reacting at 30 deg.C for 1min, reacting at 80 deg.C for 1min, and storing at 12 deg.C.

The urinary metagenome sample detection method based on the nanopore sequencing platform, which applies the library construction method, has higher detection accuracy, effectively solves the problem of false negative of missing detection or less detection, and improves the detection rate.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The detection microorganisms used in the present invention: purchasingMicrobial commenity standard (# D6306), which contains nucleic acids of 12 different GC contents of pathogenic bacteria, the composition and GC contents of which are shown in the following table:

the basic library building kit used in the invention is an RPB004 kit, and is purchased from ONT company.

Comparative example

The test is carried out on the basis of an RPB004 kit of ONT company, and all reagents including the library-establishing primer come from the kit. The specific library PCR amplification system is as follows, and this is used as a comparative example of the present invention.

The method comprises the following steps: a total of 4 gradients of the above standard and 1. mu.l FRM (contained in the RPB004 kit) were added to each sample in a 0.2ml PCR tube at 0.1ng/0.25ng/0.5ng/1ng to mimic the lower initial volume in practice.

The specific PCR amplification procedure is as follows:

after the PCR is finished, 1 mul is taken, the concentration of the PCR product is detected by using the Qubit, and the quality control standard is 4 ng/mul.

Adding 0.6 × beads, rotary incubating at room temperature for 5min, and centrifuging to remove supernatant. 200 μ l of freshly prepared 75% alcohol was washed 2 times with beads. Add 12. mu.l 10mM Tris-HCl (50mM NaCl) pH 8.0 eluent, spin incubate for 2min at room temperature, and flash release rack to clear. Carefully pipette 11. mu.l of the supernatant into a 1.5ml low adsorption centrifuge tube. Mu.l RAP was added to 10. mu.l of the supernatant, and the mixture was reacted at room temperature for 15min to prepare a machine.

According to the Oxford nanopore machining process: priming and loading the SpotON Flow Cell for operation. The obtained data are shown in the following table 1, the percentage means the percentage of the read length of a certain bacterium to the read length of all the bacteria after the bacteria are removed, and the statistical result is shown in the figure 1:

the data obtained by this comparative example are shown in the following table, with the results shown in table 1:

as can be seen from the results in the comparative example, when bacteria with different GC contents are co-amplified, the amplification of various bacteria is reduced along with the increase of GC contents, which is very unfavorable for metagenomic detection. The average library size of the comparative example was 1.78kb, making it more difficult to exploit the advantage of long read length for ONT sequencing.

Example 1 temperature and time isoparametric optimization of library pcr

Since the RPB004 reagent is only a basic library construction kit of ONT company, and the library construction effect is not obvious for the special sample of urinary system (such as the above comparative example), the application carries out multi-factor repeated optimization on the amplification system of the above comparative example in the development process, including pre-denaturation temperature, denaturation time, extension temperature and time, and the like, and finally determines an ideal amplification parameter system.

The method comprises the following steps: a total of 4 gradients of the above standard and 1. mu.l FRM (fraction of RPB004 kit) were added to each sample in a 0.2ml PCR tube at 0.1ng/0.25ng/0.5ng/1ng to mimic the lower initial volume in practice. Mixing and centrifuging gently, and reacting on a PCR instrument at 30 deg.C for 1min, 80 deg.C for 1min, and 12 deg.C for infinity.

A PCR mix system (50. mu.l) was formulated as follows:

Nuclease-free water	22μl
		Tagmented DNA	4μl
RLB(01-12A)	1μl
		LongAmp Taq 2X master mix(NEB)	25μl

the optimized PCR reaction conditions are as follows:

According to the Oxford nanopore machining process: priming and loading the SpotON Flow Cell for operation. The obtained data are shown in the following table, the percentage means the percentage of the number of the read-out strips of a certain bacterium to the number of all the read-out strips after the bacteria are taken off the machine, and the statistical results are shown in the table 2:

in example 1, it can be seen that the average length of the sequences in the off-line data is 2.22kb, which is higher than that in comparative example 25%, indicating that changing the amplification parameters can effectively extend the average length of the amplified product, and at the same time, can increase the concentration of the PCR product, which is beneficial for sequencing analysis, but the problem of GC bias is not significantly improved, as shown in FIG. 2.

Example 2 DMSO introduction and concentration optimization of library pcr

The method A comprises the following steps: a total of 4 gradients of the above standard and 1. mu.l FRM (purchased from ONT and included in the RPB004 kit) were added to each sample in a 0.2ml PCR tube at 0.1ng/0.25ng/0.5ng/1ng to mimic the lower initial volume in practice.

PCR mix systems (50. mu.l) were formulated as in the following Table, with a final DMSO concentration of 2% (v/v):

Nuclease-free water	18μl
		Tagmented DNA	4μl
RLB(01-12A)	1μl
		LongAmp Taq 2X master mix(NEB)	25μl
25% dimethyl sulfoxide DMSO	4μl

And (3) carrying out reaction by using a PCR instrument under the following conditions:

According to the Oxford nanopore machining process: priming and loading the SpotON Flow Cell for operation. The data obtained by sequencing analysis are shown in the following table 3, the percentage means the percentage of the read length of a certain bacterium to the read length of all the following bacteria, and the statistical result is shown in the following table 3:

it can be seen that a small amount of DMSO (2% v/v) slightly improved the phenomenon of preferential amplification of strains with a higher GC content in the metagenomic amplification of urine samples, but the effect was not significant, as detailed in fig. 3; at the same time, the average read length was 2.6kb, which is higher than that of comparative example 46%.

Second, method B is the same as method A except that 25% of dimethyl sulfoxide is used in an amount of 8. mu.l (final concentration: 4% v/v) in the PCR amplification reaction system.

The data obtained by sequencing analysis are shown in the following table, and the statistical results are shown in table 4:

as can be seen from the results in the table above, the amount of DMSO used reaches a final concentration of 4%, when bacteria with different GC contents are co-amplified, the sequencing amounts of various bacteria tend to be balanced (see FIG. 4), and fluctuate in a theoretical proportion range (12%), which is beneficial to metagenome detection, and meanwhile, the average sequencing read length is 2.18kb, and the comparison proportion length is improved by 22%.

The method is the same as the method A, except that the amount of 25% dimethyl sulfoxide in the reaction system for PCR amplification is 10. mu.l (final concentration is 5% v/v).

The data obtained by sequencing analysis are shown in the following table, and the statistical results are shown in table 5:

it can be seen that when the DMSO dosage is increased to reach a final concentration of 5%, the bacteria with high GC content are excessively amplified, so that the amplification of the bacteria with low GC content is inhibited, and the method is not suitable for the amplification of metagenome, the average read length of the off-line sequencing read length is 2.03kb, and the comparison ratio is improved by 14%, as shown in fig. 5.

In conclusion, DMSO with different concentrations has extremely obvious influence on the amplification result, and the excessive (5%) or high (2%) DMSO concentration has obvious effect difference on the establishment of the urinary sample, and the experiment determines that the DMSO concentration of 4% is most suitable for the establishment of the urinary sample.

Example 3 clinical sample testing

Microorganisms: clinical urinary samples collected by hospital clinical laboratory, each sample has clinical urine culture result. And (3) carrying out cold chain transportation on the sample at 4 ℃, centrifuging for 5min by fully and softly mixing 12300g in a biological safety cabinet, abandoning the supernatant, adding 1ml of LPBS for resuspension and precipitation, and transferring into a 2ml low-adsorption centrifuge tube.

1, removing a host:

a) taking the host sample needed to be removed in the last step, and centrifuging for 5min at 12300 g.

b) Carefully discard the supernatant (without touching the pellet, can leave 50. mu.l supernatant), resuspend the pellet in 250. mu.l PBS, vortex and mix well, if clumped, blow up and down with a gun and mix well.

c) Saponin was added to a final concentration of 0.22% (w/v), vortexed and vortexed on a vortexer for 10min at room temperature. Add 350. mu.l sterile water, vortex and mix well, add 12. mu.l 5M NaCl after 30s, shake and mix well immediately.

d) The supernatant was carefully removed by centrifugation at 10000g for 5min (without touching the pellet, 50. mu.l of supernatant could be left), 100. mu.l of PBS was resuspended in the pellet, and HL-SAN was added to a final concentration of 0.49% (v/v), incubated at 37 ℃ and 1300rpm for 15 min.

e) Resuspend the pellet with 800. mu.l PBS and vortex to mix.

2 MP FastPrep-245G wall breaking treatment

a) After host removal treatment, a clinical sample is directly blown and uniformly mixed by a pipette and then transferred into MP lysine Matrix Etube, and wall breaking treatment is carried out according to the requirements of MP FastPrep PROGRAM ManUALLY, wherein the SPEED is 6.0m/sec, the ADAPTER is quickPrep, the TIME is 40s, and the lysine Matrix is E.

b) And centrifuging the mixture for 10min by using an Eppendorf model 5424R centrifuge at 14000rpm, and transferring a supernatant to extract a genome in the next step.

3 Maxwell RSC automated nucleic acid extraction

a) The computer to which the Maxwell RSC extractor was connected was first turned on, and then the Maxwell RSC extractor instrument was turned on.

b) Among them, a program corresponding to "white Blood DNA (CATALOG NUMBER AS 1520)" was selected and extracted AS indicated.

c) The Qubit measures concentration.

4 library and superior sequencing analysis

The clinical experimental effect of the present invention was verified by performing comparative tests according to method 2 of example 2 and the method of comparative example, respectively. The specific results are as follows:

the results show that the method of the present invention is superior to the comparative examples, in both concentration of the amplification product and the average value of the product. The method is also obviously superior to the comparative example in the positive consistency rate of the detection result and the clinical culture: in the clinical test of the comparative example, No. 27 sample, No. 40 sample and No. 60 sample can not detect mixed infection, while No. 6 sample urine sample has low content of bacteria and cannot detect the proportion, so the detection rate is 9/13-100-69%; the invention can be detected completely and accurately, and the detection rate is 13/13-100 percent, which is obviously superior to the comparative example.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

1. A urinary metagenome sample library building method based on a nanopore sequencing platform is characterized in that a sample to be detected is sequentially subjected to host removal and genome extraction and then labeled, and an obtained labeled template is subjected to PCR amplification to build a library;

the reaction system of the PCR amplification contains 4% (v/v) of dimethyl sulfoxide;

the reaction procedure of the PCR amplification facilitates unwinding and extension of the high GC content template;

the PCR amplification reaction system also comprises a labeled template, an amplification universal primer, DNA polymerase and a buffer solution;

the DNA polymerase is LongAmp Taq DNA polymerase;

the concentration of the template with the mark is 0.002-0.02 ng/mul;

the reaction procedure of the PCR amplification comprises the following steps:

2. the library construction method of claim 1, wherein the derogating comprises in order episomal host DNA, degradative host DNA, and derogating host DNA.

3. The library-building method of claim 2, wherein the free host DNA comprises a sample to be tested treated with 0.1-0.5% (w/v) saponin for 5-15 min.

4. The library construction method according to claim 2, wherein the degraded host DNA comprises treating the sample to be detected with 0.3-1% (v/v) HL-SAN for 10-20min after dissociating the host DNA.

5. The library-building method of claim 1, wherein the labeling comprises incubating the sample to be tested with FRM after sequentially dephosgenating and extracting the genome.

6. The library construction method of claim 5, wherein the incubation reaction procedure comprises: reacting at 29-31 deg.C for 0.5-1.5min, reacting at 79-81 deg.C for 0.5-1.5min, and storing at 11-13 deg.C.

7. The library construction method of claim 6, wherein the incubation reaction procedure comprises: reacting at 30 deg.C for 1min, reacting at 80 deg.C for 1min, and storing at 12 deg.C.