阅读说明：本技术 一种用于检测呼吸道样本中病原体dna文库的构建方法及试剂盒 (Construction method and kit for detecting pathogen DNA library in respiratory tract sample ) 是由 蒋析文 梁志坤 马伟原 吴轶兰 于 2021-08-11 设计创作，主要内容包括：本申请属于生物技术领域,涉及一种用于检测呼吸道样本中病原体DNA文库的构建方法及试剂盒,包括通过一步反应对DNA样本以及与所述DNA样本相对应的内标进行片段化处理,以实现对所述DNA样本及所述内标进行末端修复、磷酸化及加A,获得第一产物；将所述第一产物依次进行接头连接和第一次纯化处理,获得第二产物；对所述第二产物依次进行PCR富集和第二次纯化处理,获得用于检测下呼吸道样本中病原体DNA文库。本申请的DNA文库构建过程简便快捷,能够适用于较低起始量及较差质量的DNA建库；并添加有内标,以便后续生信分析结果的解读。(The application belongs to the technical field of biology, and relates to a construction method and a kit for detecting a pathogen DNA library in a respiratory tract sample, which comprises the steps of fragmenting a DNA sample and an internal standard corresponding to the DNA sample through one-step reaction to realize terminal repair, phosphorylation and A addition of the DNA sample and the internal standard so as to obtain a first product; sequentially performing joint connection and first purification treatment on the first product to obtain a second product; and sequentially carrying out PCR enrichment and secondary purification treatment on the second product to obtain a DNA library for detecting pathogens in the lower respiratory tract sample. The DNA library construction process is simple, convenient and quick, and can be suitable for constructing DNA libraries with lower initial quantity and poorer quality; and an internal standard was added for subsequent interpretation of the assay results.)

1. A method for constructing a DNA library for detecting pathogens in a respiratory sample, comprising the steps of:

fragmenting a DNA sample and an internal standard corresponding to the DNA sample through a one-step reaction to realize end repair, phosphorylation and A addition of the DNA sample and the internal standard and obtain a first product;

sequentially performing joint connection and first purification treatment on the first product to obtain a second product;

and sequentially carrying out PCR enrichment and secondary purification treatment on the second product to obtain a DNA library for detecting pathogens in the respiratory tract sample.

2. The method of claim 1, wherein the fragmenting comprises: placing the DNA sample, the internal standard, the reaction solution B and the enzyme mixed solution C into a PCR tube,

wherein the reaction solution B comprises:

the final concentration of Tris-HCL was 250mM, pH 7.5;

MgCl₂to a final concentration of 50 mM;

the final concentration of NaCl was 250 mM;

the final concentration of DTT was 50 mM;

the final concentration of Triton X-100 is 0.75%;

the final concentration of ATP was 2.5 mM;

final concentration of dATP was 2.5 mM;

the final concentration of dGTP is 2.5 mM;

the final concentration of dCTP was 2.5 mM;

the final concentration of dTTP was 2.5 mM;

the enzyme mixed solution C is a mixed solution of Vvn endonuclease mutan, T7 endonuclease mutant and Taq DNA polymerase.

3. The method of claim 2, wherein the fragmenting process is performed under the following reaction conditions:

firstly, reacting for 10-30 minutes at 37 ℃; then, the reaction is carried out at 65 ℃ for 20 to 30 minutes.

4. The method for constructing a DNA library for detecting pathogens in respiratory tract samples according to claim 1, wherein the DNA fragmentation process comprises:

when the size of the inserted DNA fragment is 100 bp; firstly, reacting for 25-30 minutes at 37 ℃; then, reacting for 20-30 minutes at 65 ℃;

when the size of the inserted DNA fragment is 150 bp; firstly, reacting for 15-25 minutes at 37 ℃; then, reacting for 20-30 minutes at 65 ℃;

when the size of the inserted DNA fragment is 200-700 bp; firstly, reacting for 10-15 minutes at 37 ℃; then, the reaction is carried out at 65 ℃ for 20 to 30 minutes.

5. The method according to claim 1, wherein the step of fragmenting the DNA sample and the internal standard corresponding to the DNA sample by a one-step reaction to perform end repair, phosphorylation and adding a on the DNA sample and the internal standard to obtain the first product comprises:

the internal standardThe added mass being of the corresponding DNA sample

6. The method of claim 1, wherein the step of linker ligating the first product comprises:

adding the first product, the reaction solution D, the reaction solution E and the enzyme F into a PCR tube to carry out DNA fragment ligation reaction, so as to obtain a DNA fragment ligation sample;

wherein the content of the first and second substances,

the reaction solution D contains:

the final concentration of Tris-HCl was 75mM, pH 7.5;

MgCl₂to a final concentration of 15 mM;

the final concentration of DTT was 15 mM;

the final concentration of ATP was 2.75 mM;

the final concentration of PEG8000 was 20%;

the reaction solution E contained a linker at a concentration of 2 uM;

enzyme F is a ligase.

7. The method of claim 6, wherein the reaction conditions for linker ligation of the first product are as follows:

the reaction was carried out at 20 ℃ for 30 minutes.

8. The method of claim 6, wherein the first purification step comprises:

subpackaging the magnetic beads into low adsorption PCR tubes;

and adding the DNA fragment connection sample into a PCR tube, and purifying by using DNA magnetic beads to obtain a second product.

9. The method of claim 1, wherein the second PCR enrichment step comprises:

adding the second product, the reaction solution G and the reaction solution H into a PCR tube for centrifugal reaction to obtain a DNA amplification sample;

wherein:

the reaction solution G comprises a reaction buffer solution, four dNTPs and DNA polymerase in a one-step reaction system, wherein the four dNTPs are dATP, dGTP, dCTP and dTTP;

the reaction solution H is an amplification primer with the concentration of 15 mu M.

10. The method of claim 9, wherein the reaction conditions for PCR enrichment of the second product are as follows:

pre-denaturation treatment: reacting at 95 ℃ for 3 min;

and (3) denaturation treatment: reacting at 95 ℃ for 20 s;

annealing treatment: reacting at 58 ℃ for 20 s;

and (3) extension treatment: reacting at 72 ℃ for 20s, and continuing for 10-14 circulation flows;

and (3) complete extension treatment: the reaction is carried out for 5min at 72 ℃ and kept at 4 ℃ without changing the temperature.

11. The method of claim 9, wherein the second purification step comprises:

subpackaging the magnetic beads into low adsorption PCR tubes;

and adding the DNA amplification sample into a PCR tube, and purifying by magnetic beads to obtain the DNA library for detecting the pathogens in the respiratory tract sample.

12. A kit for detecting a DNA library of a pathogen in a sample of the respiratory tract comprising a module a, a module B and a module C;

the component A is a reagent or a reagent combination which is used for fragmenting a DNA sample and an internal standard corresponding to the DNA sample through one-step reaction so as to realize the end repair, phosphorylation and A addition of the DNA sample and the internal standard and obtain a first product;

the component B is a reagent or a reagent combination for sequentially carrying out joint connection and first purification treatment on the first product to obtain a second product;

and the component C is used for sequentially carrying out PCR enrichment and secondary purification treatment on the second product to obtain the kit for detecting the pathogen DNA library in the respiratory tract sample.

Technical Field

The application relates to the technical field of biology, in particular to a construction method and a kit for detecting a pathogen DNA library in a respiratory tract sample.

Background

The hospital infection rate of each department of China hospital is obviously different, and researches indicate that the hospital infection susceptible departments sequentially comprise surgery ICU, hematology department, emergency department, respiratory medicine department and neurology department. The ages of infected patients are V-shaped, and are mainly infants less than two years old and elderly patients over 60 years old. The immune system of infants is not mature, the elderly patients are seriously ill, the immune function is relatively low, invasive operations are more, and basic diseases are more accompanied, so the two groups are high risk groups infected in hospitals at present.

With the development of sequencing technology, secondary sequencing has generally become a clinical auxiliary diagnostic technical means, such as the prior whole exons, tumors, genetic disease sequencing and other technical means, in recent years, metagenomic sequencing has become a hotspot, and few effective library-building kits for metagenomic secondary sequencing are available in the market. The market lacks a kit suitable for library construction of human sputum, bronchoalveolar lavage fluid and other samples.

For the library construction step, most of the library construction reagent processes in the market are separately made for fragmentation and end repair, and mainly comprise the following steps: 1. carrying out ultrasonic fragmentation on the DNA; 2. magnetic bead purification is carried out on the fragmentation products; 3. performing end repair and A addition on the fragmentation purification product; 4. performing joint connection on the product A; 5. purifying the connection product by magnetic beads; 6. enriching the joint connection purified product; 7. and carrying out magnetic bead sorting or recovery on the enriched product. The steps comprise three steps of purification, the time consumption of the step of establishing the library is long, and the loss of the DNA sample is inevitably increased by multiple times of magnetic bead purification, so the requirements on the total amount and the quality of the DNA are high.

The existing kits for fragmentation, end repair and A-adding one-step reaction in the market at present amplify the template quantity to different degrees and cannot truly reflect the nucleic acid composition condition in a sample.

The host proportion difference of lower respiratory tract samples such as alveolar lavage fluid, sputum and the like is large, a large number of field planting bacteria exist, the existing reagent lacks an internal standard product to carry out normalization correction on the result, and the accuracy of later-stage data analysis can be influenced.

Therefore, the existing library building steps are complicated, the time consumption is long in multiple times of magnetic bead purification, the requirements on the total amount and quality of DNA are high, the steps of fragmentation, terminal repair and A addition cannot be integrated, or the steps can be integrally completed, and the template amount is amplified to a certain degree.

Disclosure of Invention

The embodiment of the application aims to provide a library construction method, the DNA library construction process is simple, convenient and quick, and the method is suitable for the DNA library construction process with low initial quantity and poor quality.

In order to solve the above technical problems, the embodiments of the present application provide a method for constructing a library, which adopts the following technical solutions:

a method for constructing a DNA library for detecting pathogens in a respiratory sample, comprising the steps of:

fragmenting a DNA sample and an internal standard corresponding to the DNA sample through a one-step reaction to realize end repair, phosphorylation and A addition of the DNA sample and the internal standard and obtain a first product;

sequentially performing joint connection and first purification treatment on the first product to obtain a second product;

and sequentially carrying out PCR enrichment and secondary purification treatment on the second product to obtain a DNA library for detecting pathogens in the respiratory tract sample.

Further, the fragmentation process comprises: placing the DNA sample, the internal standard, the reaction solution B and the enzyme mixed solution C into a PCR tube,

wherein the reaction solution B comprises:

the final concentration of Tris-HCL was 250mM, pH 7.5;

MgCl₂to a final concentration of 50 mM;

the final concentration of NaCl was 250 mM;

the final concentration of DTT was 50 mM;

the final concentration of Triton X-100 is 0.75%;

the final concentration of ATP was 2.5 mM;

final concentration of dATP was 2.5 mM;

the final concentration of dGTP is 2.5 mM;

the final concentration of dCTP was 2.5 mM;

the final concentration of dTTP was 2.5 mM;

the enzyme mixed solution C is a mixed solution of Vvn endonuclease mutan, T7 endonuclease mutant and Taq DNA polymerase.

Further, the reaction conditions of the fragmentation treatment are as follows:

firstly, reacting for 10-30 minutes at 37 ℃; then, the reaction is carried out at 65 ℃ for 20 to 30 minutes.

Further, the DNA fragmentation treatment comprises the following steps:

when the size of the inserted DNA fragment is 100 bp; firstly, reacting for 25-30 minutes at 37 ℃; then, reacting for 20-30 minutes at 65 ℃;

when the size of the inserted DNA fragment is 150 bp; firstly, reacting for 15-25 minutes at 37 ℃; then, reacting for 20-30 minutes at 65 ℃;

when the size of the inserted DNA fragment is 200-700 bp; firstly, reacting for 10-15 minutes at 37 ℃; then, the reaction is carried out at 65 ℃ for 20 to 30 minutes.

Further, the fragmenting, by a further reaction, a DNA sample and an internal standard corresponding to the DNA sample to achieve end repair, phosphorylation and a addition of a to the DNA sample and the internal standard, and the obtaining of the first product includes:

the mass added by the internal standard is that of the corresponding DNA sample

Further, the step of linker attaching the first product comprises:

adding the first product, the reaction solution D, the reaction solution E and the enzyme F into a PCR tube to carry out DNA fragment ligation reaction, so as to obtain a DNA fragment ligation sample;

wherein the content of the first and second substances,

the reaction solution D contains:

the final concentration of Tris-HCl was 75mM, pH 7.5;

MgCl₂to a final concentration of 15 mM;

the final concentration of DTT was 15 mM;

the final concentration of ATP was 2.75 mM;

the final concentration of PEG8000 was 20%;

the reaction solution E contained a linker at a concentration of 2 uM;

enzyme F is a ligase.

Further, the reaction conditions for linker connecting the first product are as follows:

the reaction was carried out at 20 ℃ for 30 minutes.

Further, the first purification treatment comprises:

subpackaging the magnetic beads into low adsorption PCR tubes;

and adding the DNA fragment connection sample into a PCR tube, and purifying by using DNA magnetic beads to obtain a second product.

Further, the second product PCR enrichment step comprises:

adding the second product, the reaction solution G and the reaction solution H into a PCR tube for centrifugal reaction to obtain a DNA amplification sample;

wherein:

the reaction solution G comprises a reaction buffer solution, four dNTPs and DNA polymerase in a one-step reaction system, wherein the four dNTPs are dATP, dGTP, dCTP and dTTP;

the reaction solution H is an amplification primer with the concentration of 15 mu M.

Further, the reaction conditions for PCR enrichment of the second product are as follows:

pre-denaturation treatment: reacting at 95 ℃ for 3 min;

and (3) denaturation treatment: reacting at 95 ℃ for 20 s;

annealing treatment: reacting at 58 ℃ for 20 s;

and (3) extension treatment: reacting at 72 ℃ for 20s, and continuing for 10-14 circulation flows;

and (3) complete extension treatment: the reaction is carried out for 5min at 72 ℃ and kept at 4 ℃ without changing the temperature.

Further, the second purification treatment step comprises:

subpackaging the magnetic beads into low adsorption PCR tubes;

and adding the DNA amplification sample into a PCR tube, and purifying by magnetic beads to obtain the DNA library for detecting the pathogens in the respiratory tract sample.

The application also protects a kit for detecting a pathogen DNA library in a respiratory sample, comprising a component a, a component B and a component C;

the component A is a reagent or a reagent combination which is used for fragmenting a DNA sample and an internal standard corresponding to the DNA sample through one-step reaction so as to realize the end repair, phosphorylation and A addition of the DNA sample and the internal standard and obtain a first product;

the component B is a reagent or a reagent combination for sequentially carrying out joint connection and first purification treatment on the first product to obtain a second product;

and the component C is used for sequentially carrying out PCR enrichment and secondary purification treatment on the second product to obtain the kit for detecting the pathogen DNA library in the respiratory tract sample.

The application also protects the use of any of the methods described above in sequencing. The sequencing is high throughput sequencing.

The application also protects the application of the kit in sequencing. The sequencing is high throughput sequencing.

Compared with the prior art, the embodiment of the application mainly has the following beneficial effects:

the method for constructing the DNA library for detecting the respiratory pathogens, provided by the invention, has the advantages of simple and rapid library construction process, less purification times and short consumed time. The integration of fragmentation, end repair, phosphorylation and A adding steps is realized, the requirements on the total amount and quality of DNA are low, and the method is suitable for constructing a DNA library with low initial amount and poor quality.

Drawings

In order to more clearly illustrate the solution of the present application, the drawings needed for describing the embodiments of the present application will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and that other drawings can be obtained by those skilled in the art without inventive effort.

FIG. 1 is a flow diagram of one embodiment of a method of library construction according to the present application;

Detailed Description

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 application belongs; the terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "including" and "having," and any variations thereof, in the description and claims of this application and the description of the above figures are intended to cover non-exclusive inclusions. The terms "first," "second," and the like in the description and claims of this application or in the above-described drawings are used for distinguishing between different objects and not for describing a particular order.

Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. It is explicitly and implicitly understood by one skilled in the art that the embodiments described herein can be combined with other embodiments.

The following examples are presented to facilitate a better understanding of the present application and are not intended to limit the present application. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

With continuing reference to FIG. 1, a flow diagram of one embodiment of a method of library construction according to the present application is shown. The library construction method comprises the following steps:

s1: fragmenting a DNA sample and an internal standard corresponding to the DNA sample through a one-step reaction to realize end repair, phosphorylation and A addition of the DNA sample and the internal standard and obtain a first product;

s2: sequentially performing joint connection and first purification treatment on the first product to obtain a second product;

s3: and sequentially carrying out PCR enrichment and secondary purification treatment on the second product to obtain a DNA library for detecting pathogens in the respiratory tract sample.

Firstly, fragmenting the DNA sample and an internal standard corresponding to the DNA sample through a one-step reaction to realize the fragmentation of the DNA sample and the internal standard, and simultaneously performing end repair, phosphorylation and A addition on the DNA sample and the internal standard to obtain the first product.

The method specifically comprises the following steps: reacting the DNA sample and the corresponding internal standard with a reaction solution B and an enzyme mixed solution C, wherein the reaction solution B comprises Tris-HCL (Tris-hydroxymethyl aminomethane hydrochloride), MgCl₂(magnesium chloride), NaCl (sodium chloride), DTT (dithiothreitol), Triton X-100 (polyethylene glycol octylphenyl ether), ATP (adenosine triphosphate), dATP (deoxyadenosine triphosphate), dGTP (guanine deoxynucleotide triphosphate), dCTP (deoxycytidine triphosphate) and dTTP (deoxythymidine triphosphate), wherein the final concentration of Tris-HCL is 250mM and the pH value is 7.5; MgCl₂To a final concentration of 50 mM; the final concentration of NaCl was 250 mM; the final concentration of DTT was 50 mM; the final concentration of Triton X-100 is 0.75%; the final concentration of ATP was 2.5 mM; final concentration of dATP was 2.5 mM; the final concentration of dGTP is 2.5 mM; the final concentration of dCTP was 2.5 mM; the final concentration of dTTP was 2.5 mM; the enzyme mixture C is a mixture of Vvn endonuclease mutant (Vibrio vulnificus nuclease), T7 endonuclease mutant (T7 endonuclease) and Taq DNA polymerase (DNA polymerase).

In this embodiment, many solutions are pre-configured to be stored as a high concentration mother liquor. In use, dilution from the mother liquor is generally required. The final concentration is the working concentration, i.e. the concentration at which the reaction solution B is actually used. Wherein mM means millimole per liter, e.g. 200mM ═ 0.200 mol/L. Wherein Vvn endonuclease mutant (Vibrio vulnificus nuclease) and T7 endonuclease mutant (T7 endonuclease) are both endonucleases.

1. Taking out the reaction solution B and the enzyme mixed solution C from a refrigerator at the temperature of-20 ℃. And thawing the reaction solution B at room temperature, reversing, uniformly mixing and centrifuging the reaction solution B for a short time for later use. And unfreezing the enzyme mixed solution C on ice, reversing and uniformly mixing, centrifuging the enzyme mixed solution C for a short time, and placing the enzyme mixed solution C on an ice box for later use. The following reaction systems were prepared in a PCR tube, as shown in table 1:

TABLE 1

Composition of	Volume (μ l)
		Add (Input) DNA sample + internal standard	35
Reaction solution B	10
		Enzyme mixture C	5
Volume Total (Total)	50

The internal standard is a proper pure compound added into a detection material in quantitative analysis, and the measured value is used for measuring whether the processes of library building and sequencing and a system are stable or not, so that the quality control and correction effects are achieved. The specific implementation mode is that the data of the sequencing on the computer is subjected to letter generation analysis statistics, and whether the detection ratio of the known genes contained in the internal standard is stable or not is judged to judge whether the processes and the systems of library construction and sequencing are stable or not. The present application adds an internal standard and sets the ratio of the internal standard addition, wherein the ratio of the internal standard addition is 1/1500 of the input nucleic acid (DNA sample), for example, 30ng of DNA sample and 0.02ng of internal standard addition. Quality control can be carried out on the steps of library building and sequencing based on the internal standard, and the result can be better corrected.

2. The PCR tube configured for the completed reaction system was gently vortexed and briefly centrifuged. The PCR tube was placed in a PCR instrument and the following reaction program was set up as shown in table 2:

TABLE 2

The fragmentation time was determined by the mass of the DNA sample added (Input) and the size of the target insert, as shown in Table 3:

TABLE 3

Expected insert size	Fragmentation time
		100bp	25-30min
150bp	15-25min
		200-700bp	10-15min

It should be noted that: the fragmentation time set above was obtained using high quality Hela cell gDNA as a template for the test. When high-quality Hela cell gDNA is used for library construction, different input amounts and the same reaction time are adopted, the distribution ranges of the fragmentation products are not greatly different, namely the distribution ranges are basically consistent, but the main peak positions may be slightly different. If the quality of the added (Input) DNA sample is not good or the fragmentation size is not in the expected range, the fragmentation time can be adjusted up and down in the range of 2-5 min.

II, performing joint connection and purification operation on the first product to obtain a second product, wherein the step of obtaining the second product comprises the following steps:

adding the first product, a reaction solution D, a reaction solution E and an enzyme F into a PCR tube to perform DNA fragment ligation reaction to obtain a DNA fragment ligation sample, wherein the reaction solution D comprises Tris-HCl and MgCl₂(magnesium chloride), DTT, ATP and PEG8000 (polyethylene glycol), wherein the final concentration of Tris-HCL is 75mM, and the pH value is 7.5; MgCl₂To a final concentration of 15 mM; the final concentration of DTT was 15 mM; the final concentration of ATP was 2.75 mM; the final concentration of PEG8000 was 20%; the reaction solution E contained linker X at a concentration of 2uM, and enzyme F was ligase.

1. The specific process of joint connection is as follows:

taking out the reaction solution D and the reaction solution E from a refrigerator at the temperature of-20 ℃, thawing at room temperature, mixing uniformly, and centrifuging for a short time for later use; taking out the enzyme F from a refrigerator at the temperature of-20 ℃, centrifuging for a short time, and putting the enzyme F on an ice box for later use.

The following components were added to the first product after fragmentation, end-point repair, phosphorylation, and addition of a in sequence, as shown in table 4:

TABLE 4

Slightly vortexing, mixing and centrifuging for a short time;

the PCR tube was placed in a PCR instrument and the following reaction program was set up:

2. the specific steps of the first purification of the ligation product are as follows:

after the step 1 is completed, taking out the DNA purified magnetic beads from a refrigerator at 2-8 ℃ 30min in advance, and standing to balance the temperature to room temperature; then, the DNA purified magnetic beads are fully and uniformly mixed by inversion or vortex oscillation, and 80 mu L of magnetic beads are subpackaged into 1.5mL of low adsorption centrifuge tubes; then, adding 80 μ L of the ligation product (i.e., the DNA fragment ligation sample) obtained in step 1 into the magnetic beads, and repeatedly and uniformly mixing by pipetting, wherein the following reminders are required: the sucker does not leave the liquid surface during mixing so as to prevent excessive bubbles; then, incubating for 5min at room temperature to enable the DNA to be combined on the magnetic beads; the sample was then placed on a magnetic stand and after the solution cleared (about 5min), the supernatant was carefully removed. The sample was then kept on the magnetic stand, the beads were rinsed by adding 200 μ L of freshly prepared 80% ethanol, incubated at room temperature for 30sec (sec), and the supernatant carefully removed.

Repeating the following steps once: keeping the sample on the magnetic stand all the time, add 200 μ L of freshly prepared 80% ethanol to rinse the beads, incubate for 30sec at room temperature, carefully remove the supernatant. The total number of rinses was two.

And (3) instantaneously centrifuging the sample for 5s, placing the sample on a magnetic frame, completely removing the residual supernatant by using a 10-mu-L pipette, keeping the sample on the magnetic frame all the time, and covering and drying the magnetic beads at room temperature for about 5-10min until the surfaces of the magnetic beads are not reflected. The sample was then removed from the magnetic stand and 22. mu.l of water (H) from which the nuclease had been removed was added₂O), gently sucking and beating the mixture by using a pipette, standing the mixture at room temperature for 2min, placing the mixture on a magnetic frame, carefully sucking 20 mu l of supernatant serving as a second product into a new PCR tube after the solution is clarified (approximately equal to 5 min).

Thirdly, the step of sequentially carrying out PCR enrichment and secondary purification treatment on the second product to obtain the DNA library for detecting the pathogens in the respiratory tract sample comprises the following steps:

adding the second product, a reaction solution G and a reaction solution H into a PCR tube for centrifugal reaction to obtain a DNA amplification sample, wherein the reaction solution G comprises a reaction buffer solution, 4 dNTPs (namely dATP, dGTP, dTTP, dCTP) and DNA polymerase in a one-step reaction system; the reaction solution H is an amplification primer with the concentration of 15 mu M. Wherein, the amplification primer is a specific base sequence, and the specific sequence of the amplification primer can be the sequence of the amplification primer commonly used in the field. The reaction buffer solution comprises magnesium ions, Tris-HCL (Tris hydrochloride), dimethyl sulfoxide, glycerol, Tween20/40 (Tween 20 or Tween 40), D- (+) trehalose dihydrate, tetramethylammonium chloride and DTT (dithiothreitol).

1. The specific steps of library amplification (i.e., PCR enrichment) are:

taking out the reaction liquid G and the reaction liquid H from a refrigerator at the temperature of-20 ℃, unfreezing the reaction liquid G on ice for 20min, unfreezing the reaction liquid H at room temperature for 20min, uniformly mixing, centrifuging for a short time for standby, and sequentially adding the following components into the PCT tube with the second product (namely the connecting product) in the step two 2:

composition of	Volume (μ l)
		Second product (ligation product)	20
Reaction solution G	25
		Reaction solution H	5
Total (Total)	50

Mixing by slight vortex, and centrifuging for a short time; the amplification was then run in a thermal cycler as follows:

it should be noted that: when the input amount of the nucleic acid (DNA sample) for library construction is less than 10ng, X is 14; when the input amount of the nucleic acid is 10-30 ng, X is 12; when the input amount of the nucleic acid is 30-50 ng, X is 10.

2. The specific steps of sorting (i.e. second purification) the amplified product fragments are as follows:

taking out the DNA purified magnetic beads from a refrigerator at 2-8 ℃ 30min in advance, and standing to balance the temperature to room temperature; then, the DNA purified magnetic beads are fully and uniformly mixed by reversing or vortex oscillation, and 85 mu L of the DNA purified magnetic beads are subpackaged into 1.5mL low adsorption centrifuge tubes; then 50 μ L of PCR product (i.e. DNA amplification sample) after PCR enrichment is supplemented with water to 100 μ L, and added into magnetic beads to be repeatedly blown, beaten and mixed uniformly, wherein, the following matters need to be noted: when in mixing, the suction head does not leave the liquid level, so as to prevent excessive bubbles;

incubating at room temperature for 5min to allow DNA to bind to the magnetic beads; then placing the sample on a magnetic frame, and taking the supernatant into a low-adsorption centrifuge tube containing 40 mu L of DNA purification magnetic beads after the solution is clarified (about 5 min); then, incubating for 5min at room temperature to enable the DNA to be combined on the magnetic beads; the sample was then placed on a magnetic stand and after the solution cleared (about 5min), the supernatant was carefully removed; then keeping the sample on the magnetic frame all the time, adding 200 mul of freshly prepared 80% ethanol to rinse the magnetic beads, incubating at room temperature for 30sec, and carefully removing the supernatant;

repeating the steps once: keeping the sample on the magnetic stand all the time, add 200 μ l of freshly prepared 80% ethanol to rinse the beads, incubate for 30sec at room temperature, carefully remove the supernatant. Rinsing twice in total;

instantly centrifuging the sample for 5sec, placing the sample on a magnetic frame, completely removing the residual supernatant by using a 10-microliter pipettor, keeping the sample on the magnetic frame all the time, and covering and drying the magnetic beads at room temperature for about 5-10min until the surfaces of the magnetic beads are not reflected; the sample was then removed from the magnetic rack, 22. mu.l of the enucleating enzyme H2O was added, gently pipetted and mixed, left to stand at room temperature for 2min and placed on the magnetic rack, and after the solution cleared (. apprxeq.5 min), 20. mu.l of the supernatant was carefully pipetted into a new 1.5ml EP tube.

This application has following advantage: the system is completed by DNA fragmentation, end repair, phosphorylation and A addition in one step, and the template amount is not amplified in the process. According to the QPCR result or the off-line data result, the amplification system with high specificity and high effective library content are obtained. And in the library building process, an internal standard is added into each sample for quality control in the steps of library building and sequencing, so that the effect of correcting the result is achieved. According to the method, magnetic bead purification is not needed after the tail end is repaired, so that the time for building the library is shortened and the cost is reduced. The method and the kit for constructing the DNA library for detecting the pathogens in the respiratory tract sample are suitable for detecting the pathogens in the respiratory tract sample.

It should be understood that, although the steps in the flowcharts of the figures are shown in order as indicated by the arrows, the steps are not necessarily performed in order as indicated by the arrows. The steps are not performed in the exact order shown and may be performed in other orders unless explicitly stated herein. Moreover, at least a portion of the steps in the flow chart of the figure may include multiple sub-steps or multiple stages, which are not necessarily performed at the same time, but may be performed at different times, which are not necessarily performed in sequence, but may be performed alternately or alternately with other steps or at least a portion of the sub-steps or stages of other steps.

It is to be understood that the above-described embodiments are merely illustrative of some, but not restrictive, of the broad invention, and that the appended drawings illustrate preferred embodiments of the invention and do not limit the scope of the invention. This application is capable of embodiments in many different forms and is provided for the purpose of enabling a thorough understanding of the disclosure of the application. Although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that the present application may be practiced without modification or with equivalents of some of the features described in the foregoing embodiments. All equivalent structures made by using the contents of the specification and the drawings of the present application are directly or indirectly applied to other related technical fields and are within the protection scope of the present application.