CN113046414A - Kit for human fecal pathogenic microorganism genome library construction and sequencing method - Google Patents

Abstract

The invention discloses a kit for establishing a human fecal pathogenic microorganism genome library and a sequencing method. The invention relates to a nucleic acid extraction kit for establishing a pathogenic microorganism genome library in human excrement, which comprises the following components: nucleic acid release agent lysine A, Enhancer lysine Enhancer, inhibitor IRT, Binding Buffer, washing solution 1 and washing solution 2. The invention optimizes the nucleic acid extraction link, and the extracted DNA has high purity and high yield, so as to ensure the normal operation of downstream tests even under the condition of limited sample size. Meanwhile, the PCR amplification conditions are optimized, the initial quantity of DNA library construction is reduced, the library construction success rate is improved, the PCR amplification bias is reduced, the library construction period is shortened, and the pain points that the existing library construction method has high requirement on the initial quantity of the template, the bias is easy to occur, the library construction period is long and the like are overcome.

Description

Kit for human fecal pathogenic microorganism genome library construction and sequencing method

Technical Field

The invention belongs to the field of DNA extraction and library construction, and particularly relates to a kit and a sequencing method for constructing a human fecal pathogenic microorganism genome library.

Background

The traditional clinical model for diagnosing infectious diseases of patients is to make differential diagnosis by doctors and then perform a series of tests to try to find out the cause of the disease. Routine detection of pathogens in clinical samples ranges from identification of microorganisms growing in culture (e.g., by biochemical phenotypic detection or matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry), detection of organism-specific biomarkers (e.g., antigen by latex agglutination assays or enzyme-linked immunosorbent assays (ELISA) antibody assays or nucleic acid assays using PCR on individual reagents).

Although molecular diagnostic assays provide a reasonably cost effective and rapid method for diagnosing the most common infections (< 2h turn-around time in general). However, almost all conventional microbiological tests currently in use can only detect one or a limited number of pathogens at a time, or require successful culture of microorganisms from clinical specimens.

Fortunately, a new etiologic diagnostic technology, mNGS (metagenomic second-generation sequencing), a method of sequencing all nucleic acids in a clinical sample and then comparing with the genome sequence of each species to find possible pathogens, would be expected to solve this problem. The mNGS can not only greatly shorten the sample turnover time, but also provide accurate and favorable diagnosis and treatment basis for clinic in time.

Currently, as one of the most important molecular biological analysis methods, mNGS not only provides important data for basic biological studies such as genetic information disclosure and gene expression regulation, but also plays an important role in application studies such as gene diagnosis and gene therapy. Since 2005, high throughput sequencing technologies were successively born and developed, as labeled by 454 technology by Roche, Solexa technology by Illumina, and SOLiD technology by ABI. Sequencing is achieved by the mNGS, typically by the following three steps: 1) obtaining nucleic acid molecules containing a region to be determined by a PCR amplification technology; 2) connecting different linker molecules to two ends of a nucleic acid molecule containing a target region to be determined respectively to obtain a determination library; 3) the universal primer amplifies the ligation product to the amount required for sequencing.

However, the amount of nucleic acid extracted from some precious samples is very limited, and the nucleic acid amount of the samples cannot meet the requirement of the on-machine process after the traditional host and other steps are usually carried out, so that the PCR cycle number is increased and the amplification bias is aggravated through PCR amplification, and thus, a serious problem occurs in the identification of bacteria with high GC content, and the abundance ratio of the strains is seriously unbalanced. Meanwhile, the problem that the period of partial library building kit is too long and the like exists.

In view of this, there is still a need in the prior art for a new rapid method suitable for micro-clinical library construction to simplify the experimental steps and improve the library construction efficiency.

Disclosure of Invention

The invention provides a kit and a sequencing method for a pathogenic microorganism gene library in human excrement, which optimize the nucleic acid extraction link, have high purity and high yield of extracted DNA and ensure that downstream tests can be normally carried out even under the condition of limited sample quantity. Meanwhile, the PCR amplification conditions are optimized, the initial quantity of DNA library construction is reduced, the library construction success rate is improved, the PCR amplification bias is reduced, the library construction period is shortened, and the pain points that the existing library construction method has high requirement on the initial quantity of the template, the bias is easy to occur, the library construction period is long and the like are overcome.

The first object of the present invention is to provide a nucleic acid extraction kit for the genomic library of pathogenic microorganisms in human feces, which comprises: a nucleic acid releasing agent Lysis A, a reinforcing agent Lysis Enhancer, a de-inhibitor IRT, a Binding solution Binding Buffer, a washing solution 1 and a washing solution 2;

the nucleic acid releasing agent lysine A comprises 20 mM-40 mM EDTA, 40 mM-80 mM Tris-HCl, 2% -5% PVP-40 (mass fraction), a solvent DEPC water and a pH value of 9-10;

the Enhancer lysine Enhancer comprises 12-15% of SDS (mass fraction), 4-8% of PVP-40 (mass fraction) and DEPC water as a solvent.

Preferably, the nucleic acid releasing agent LysisA comprises 20mM EDTA, 50mM Tri-HCl, 2% PVP-40 (mass fraction) and DEPC water as a solvent, the Enhancer Lysis Enhancer comprises 15% SDS (mass fraction) and 5% PVP-40 (mass fraction) and DEPC water as a solvent.

Preferably, the de-inhibitor IRT comprises: 120mM NH₄Al(SO₄)₂.12H₂O, DEPC water is used as a solvent, and the pH value is 3.0-4.0;

preferably, the Binding solution Binding Buffer comprises: 3M guanidinium isothiocyanate, 16.7% isopropanol (V/V), 2% Triton X100(V/V), 10mM EDTA, 5mM Tris-HCl in DEPC water at pH 6.0-7.0;

the washing solution 1 comprises: 20% ethanol (V/V), 20% isopropanol (V/V), 70mM sodium chloride, 20mM Tris-HCl, DEPC water as solvent, and pH 6.0-7.0;

the washing liquid 2 comprises: 70% ethanol (V/V), 20mM Tris-HCl, DEPC water as solvent, pH 6.0-7.0.

The second purpose of the invention is to provide a kit for fragmenting treatment and end-modifying part of a pathogenic microorganism genome library in human excrement, which comprises AC buffers and AC Enzymes;

the AC Buffer comprises: 500mM Tris-HCl, 200mM KCl, 100mM MgCl2, 1mM DTT, 10mM ATP, 10mM dATP, 2mM dTTP, 2mM dGTP, 2mM dCTP, 0.1% Triton X-100(v/v), and the solvent is DEPC water;

the AC Enzymes comprise: 1.5U/ul T4 Polynucleotide Kinase (Enzymatics, Y9040L), 1U/ul T4 DNA Polymerase (Enzymatics, P7080L), 0.5U/ul Klenow Fragment (Enzymatics, P7060L), 1U/ul Taq-B DNA Polymerase (Enzymatics, P7250L), 4mM Tris-HCl, 5mM KCl, 0.8mM DTT, 0.2mM EDTA, 22% (mass fraction) Glycerol, 0.5% Triton X-100(v/v), and DEPC water as a solvent.

The third purpose of the invention is to provide a kit for connecting and enriching joints of a pathogenic microorganism gene building library in human excrement, which comprises Ligation XP and AD Buffer;

the Ligation XP comprises: 14% Glycerol (v/v), 20% PEG-6000 (mass fraction), 25% 1,2-Propanediol (v/v), 40mM Tris-HCl, 10mM MgCl2, 10mM MATP, 10mM DTT, 80U/ul Rapid 4 DNAIGAse (EnzyValley), and DEPC water as a solvent;

the AD Buffer comprises: 10mM Tris-HCl, 0.5mM EDTA, 50mM NaCl, and DEPC water as solvent.

The fourth purpose of the invention is to provide a nucleic acid extraction method of a pathogenic microorganism genome library in human excrement, which comprises the following steps:

nucleic acid release: transferring the excrement sample to a bead grinding tube for grinding beads, adding a nucleic acid releasing agent LysisA and an Enhancer Lysis Enhancer into the sample, performing vortex scattering on the sample, incubating, and putting the grinding tube into a bead grinding instrument for bead grinding;

inhibitor removal: transferring all the supernatant into a centrifuge tube, adding inhibitor-removed IRT into the supernatant, uniformly mixing by vortex, and centrifuging;

column adsorption, namely transferring the supernatant into a centrifuge tube, adding Binding solution Binding Buffer into the supernatant, reversing and uniformly mixing to obtain mixed solution, putting a purification column into a collection tube, transferring the mixed solution into a column, centrifuging to discard effluent, and putting the column back into the collection tube;

desalting: adding washing solution 1 into the column, centrifuging, pouring off the filtrate, putting the column back into the collecting pipe, adding washing solution 2 into the column, centrifuging, repeating the operation of washing solution 2 for several times, pouring off the filtrate, putting the column back into the collecting pipe, and centrifuging;

nucleic acid elution: the column was placed in a centrifuge tube, sterilized water was added to the center of the membrane of the column, and the column was left at room temperature and then centrifuged to obtain nucleic acid.

The fifth object of the present invention is to provide a method for banking genomes of pathogenic microorganisms in human excrement, which comprises the following steps:

dna fragmentation:

preparing a breaking system in 50ul breaking pipes, and selecting corresponding breaking time according to different breaking systems:

B. end repair (A-Tailed Conversion):

preparing a terminal repair reaction system in a 0.2mL PCR tube:

Fragment ds_DNA	25μL
		AC Buffer	3μL
AC Enzymes	2μL
		Total Volume	30μL

blowing and beating for at least 10 times or carrying out vortex oscillation and uniform mixing, and carrying out short-time centrifugation to collect liquid on the pipe/hole wall;

the reaction system was placed on a PCR instrument with the hot lid set at 80 ℃ and the following procedure was run:

·20℃ 30min；

·65℃ 30min；

·4℃ Forever

C. connecting joints:

5min before the ligation reaction, the Adapter working solution was prepared according to the following table and placed on ice for use:

DNA input	Dilution
		50ng-500ng	No dilution
5ng-50ng	10-fold

the following reagents were added directly to the end-repair reaction system:

A-Tailing cDNA	30μL
		Ligation XP	20μL
Adapter(for illumina)	5μL
		Total Volume	55μL

placing the reaction system on a PCR instrument, setting the temperature of a hot cover as Off, and incubating for 20min at 20 ℃;

D. fragment selection and purification

Insert Size	～150bp	150-250bp	250-350bp	350-450bp	450-550bp	>550bp
							1st Beads Vol.(μL)	50	40	30	25	20	15
2nd Beads Vol.(μL)	25	20	15	10	10	10

Taking the Fapon DNA clean Beads out, standing to room temperature, and uniformly mixing in a vortex manner for later use;

to a 1.5mL tube or octal tube was added 30. mu.L ddH₂Adding O +30 mu LFapon DNAclear Beads into 55 mu L of the reaction product in the previous step, uniformly mixing by vortex, collecting the liquid on the tube wall instantly, and standing for 5min at room temperature;

placing a 1.5mL tube or an eight-connected tube on a magnetic frame until the solution in the tube is clarified;

carefully sucking the supernatant into a new 1.5mL EP tube or an eight-linked tube, adding 15 μ L Fapon DNA clean Beads, mixing well, and standing at room temperature for 5 min;

placing the EP tube or the eight-connected tube on a magnetic frame, and removing supernatant after the solution in the tube is clarified;

adding 200 mu L of freshly prepared 80% ethanol into an EP tube or an eight-connection tube, rotating a 1.5mL tube for one circle, standing the eight-connection tube for 30s, and then removing a supernatant;

repeating the steps, and removing the supernatant as much as possible;

placing a 1.5mL tube or an eight-connection tube on a magnetic frame, standing at room temperature for 10min until the magnetic beads are dried or opening the tube and placing the tube on a 45 ℃ metal bath until the surfaces of the magnetic beads are free of water;

removing the centrifugal tube from the magnetic frame, adding 22 mu L of ddH2O for resuspension of magnetic beads, uniformly mixing by vortex or blowing, collecting the liquid on the tube wall instantly, and standing at room temperature for 2 min;

placing the EP tube or the eight-connected tube on a magnetic frame, transferring 20 mu L of supernatant into a new PCR tube after the solution in the tube is clarified, and carrying out the next amplification;

C. library amplification:

preparing a library amplification reaction system on an ice box:

setting up a library amplification reaction program:

the cycle numbers are referenced as follows:

Total DNA input	Recommended PCR cycles	Total DNA input	Recommended PCR cycles
				1ug	5	50ng	10-11
500ng	6-8	10ng	12-13
				100ng	8-9	5ng	13-15

D. and (3) purifying an amplification product:

vortex Fapon DNA clean Beads, resuspend them;

transferring the PCR product to a new 1.5mL tube or an eight-connected tube, adding 0.8X 40 mu L Fapon DNA clean Beads, mixing uniformly by vortex, collecting the tube wall liquid instantly, and standing at room temperature for 5 min;

placing 1.5mL tube or eight-connected tube on a magnetic frame, standing for 2min, and standing until the solution in the tube is clear;

carefully sucking and discarding supernatant liquid, adding 200 μ L of newly prepared 80% ethanol into the tube, rotating the tube on a magnetic frame, and discarding supernatant after the solution is clarified;

repeating the steps, and removing the supernatant as much as possible;

placing a 1.5mL tube or an eight-connection tube on a magnetic frame, standing at room temperature for 10min until the magnetic beads are dried or opening the tube and placing the tube on a 45 ℃ metal bath until the surfaces of the magnetic beads are free of water;

adding 28 μ L ddH2O into the EP tube or the eight-link tube, blowing, beating, mixing, standing for 2min, and placing the EP tube on a magnetic frame until the liquid becomes clear;

and sucking 25 mu L of supernatant into a new 1.5mL EP tube, adhering a library label on the tube wall, using Qsep400 to check the quality to be qualified, and storing the product in a special library storage position at the temperature of-20 ℃.

The invention optimizes the nucleic acid extraction link, and the extracted DNA has high purity and high yield, so as to ensure the normal operation of downstream tests even under the condition of limited sample size. Meanwhile, the PCR amplification conditions are optimized, the initial quantity of DNA library construction is reduced, the library construction success rate is improved, the PCR amplification bias is reduced, the library construction period is shortened, and the pain points that the existing library construction method has high requirement on the initial quantity of the template, the bias is easy to occur, the library construction period is long and the like are overcome.

Description of the drawings:

FIGS. 1 and 2 are pre-machine Qsep400 quality checks of the library;

FIG. 3 is the results of sequencing data generating analysis.

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof. It should be understood that these examples are only for the purpose of the present invention and are not intended to limit the scope of the present invention. Unless defined or indicated otherwise, the scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Example 1:

firstly, preparing a reagent of a nucleic acid extraction part of the kit:

nucleic acid releasing agent Lysis a: 20mM EDTA, 50mM Tri-HCl, 2% PVP-40 (mass fraction), and DEPC water as a solvent.

Enhancer lysine Enhancer: 15% SDS (mass fraction), 5% PVP-40 (mass fraction), and DEPC water as solvent.

De-inhibitor IRT: 120mM NH₄Al(SO₄)₂.12H₂O, DEPC water is used as a solvent, and the pH value is 3.0-4.0;

binding solution Binding Buffer: 3M guanidinium isothiocyanate, 16.7% isopropanol (V/V), 2% Triton X100(V/V), 10mM EDTA, 5mM Tris-HCl in DEPC water at pH 7.0;

washing solution 1: 20% ethanol (V/V), 20% isopropanol (V/V), 70mM sodium chloride, 20mM Tris-HCl, DEPC water as solvent, pH 6.4;

washing solution 2: 70% ethanol (V/V), 20mM Tris-HCl, DEPC water as solvent, and pH 6.4;

the preparation method comprises the following steps: adding the above components according to their content, mixing, adjusting pH, and filtering with 0.22um filter membrane (raw technology, F513134-0001) for sterilization;

secondly, the extraction method of the nucleic acid extraction part of the kit comprises the following steps:

nucleic acid release: transfer 2-3 soybean-sized fecal samples to a 2ml bead mill tube containing milling beads. Immediately adding 800ul of nucleic acid releasing agent Lysis A and 100ul of enhancing agent Lysis Enhancer into the sample, vortexing at the maximum for 1min to fully disperse the sample, and incubating at 65 ℃ for 20 min. The grinding tube was put into a bead mill for bead milling at a strength of 30m/s for 15s, and repeated 3 times.

Inhibitor removal: transferring all the supernatant into a 1.5ml centrifuge tube, adding 100ul of inhibitor-removed IRT into the supernatant, mixing uniformly by vortex, standing at 4 ℃ for 5min, and centrifuging for 1min at 12,000x g;

column passing adsorption: transferring the whole supernatant (about 1ml) to a 2ml centrifuge tube, adding 1ml Binding Buffer to the supernatant, mixing by inversion, and placing the purification column (crude, B615025-1000) in a 2ml collection tube. Transfer 700. mu.l of the mixture to the column. Centrifuging for 30-60 s at 12,000x g. Pouring out the effluent, and putting the column back into the collecting pipe;

desalting: the filtrate was decanted and the column was returned to the collection tube. Add 500uL of Wash 1 to the column, centrifuge for 1min at 8,000Xg, discard the filtrate and load the column back into the collection tube. Add 500uL of Wash 2 to the column. Centrifuge at 8,000Xg for 1min, repeat washing 2 times, discard filtrate, pack column back into collection tube. Centrifuging at 8,000Xg for 3 min;

nucleic acid elution: the column was placed in a 1.5ml centrifuge tube. Adding 30-50 μ l of sterilized water preheated to 70 deg.C to the center of the membrane of the column, and standing at room temperature for 3 min. Centrifuge for 1min at 12,000x g.

Thirdly, a competitive product kit: magen fecal DNA extraction kit (cat # D3141) and assist in san-saint fecal DNA extraction kit (cat # 18820ES50)

The extraction steps are as follows:

nucleic acid release: transfer 2-3 soybean-size stool samples to a 2ml bead mill tube containing milling beads. Immediately adding 600 mu LBufferaTL/PVP-10 into a sample, putting a grinding tube into a bead grinding instrument for bead grinding, wherein the strength is 30m/s, the time is 15s, repeating for 3 times, and incubating for 10min at 65 ℃; 13,000x g for 10min, transfer 500. mu.l of the supernatant to a 2ml centrifuge tube, and add 20. mu.l of protease K and 500. mu.l of Buffer AL to the supernatant. Mix by inversion and digest at 70 ℃ for 10 min. Cooled to room temperature, 500. mu.l of absolute ethanol was added to the mixture, and vortexed and mixed for 15 seconds.

Column passing adsorption: HiPure DNA Mini Column II was loaded into a 2ml collection tube. Transfer 700. mu.l of the mixture to the column. Centrifuging for 30-60 s at 12,000x g. And pouring out the effluent, putting the column back into the collecting pipe, and centrifuging the residual mixed solution according to the steps until the transfer is finished.

Desalting: 500 μ L of Buffer GW1 diluted with ethanol was added to the column. Standing for 2min, and centrifuging for 30-60 s at 12,000x g. Pouring out the effluent, and putting the column back into the collecting pipe; 650 μ L of Buffer GW2 diluted with ethanol was added to the column. Standing for 2min, and centrifuging for 30-60 s at 12,000x g. Pouring out the effluent, and putting the column back into the collecting pipe; centrifuge for 2min at 12,000x g to dry the column.

Nucleic acid elution: the column was placed in a 1.5ml centrifuge tube. Adding 30-50 μ l of sterilized water preheated to 70 deg.C to the center of the membrane of the column, and standing at room temperature for 3 min. Centrifuge for 1min at 12,000x g.

Detection of the concentration and OD value of the DNA

The DNA extracted by the kit and the competitive product kit is subjected to concentration and OD detection by using Nandrop 300

Sample numbering	A	B	C	D	E	F
							Concentration (ng/ul)	451.708	474.141	234.592	254.354	207.06	251.115
A260/A280	2.09	2.18	1.72	1.68	1.858	1.944
							A260/A230	1.06	1.21	1.59	1.72	1.741	1.219

Note: a and B are the results of the extraction portion of the kit of the invention, C and D are the results of the competitive product extraction kit 1 (magenta, D3141), E and F are the competitive product extraction kit 2 (assist in san, 18820ES50)

From the results, the nucleic acid concentration of the same volume of the specimen obtained by the extraction method of the present invention using the kit of the present invention is higher than that of the competitive reagent kit, and the purity is also superior to that of the competitive reagent kit.

Fourthly, preparing a kit library building part as follows:

AC Buffer: 500mM Tris-HCl, 200mM KCl, 100mM MgCl2, 1mM DTT, 10mM ATP, 10mM dATP, 2mM dTTP, 2mM dGTP, 2mM dCTP, 0.1% Triton X-100(v/v), and the solvent is DEPC water;

AC Enzymes: 1.5U/ul T4 Polynucleotide Kinase (Enzymatics, Y9040L), 1U/ul T4 DNA Polymerase (Enzymatics, P7080L), 0.5U/ul Klenow Fragment (Enzymatics, P7060L), 1U/ul Taq-B DNA Polymerase (Enzymatics, P7250L), 4mM Tris-HCl, 5mM KCl, 0.8mM DTT, 0.2mM EDTA, 22% Glycerol (mass fraction), 0.5% Triton X-100(v/v), and DEPC water as solvent;

ligation XP: 14% Glycerol (v/v), 20% (mass fraction) PEG-6000, 25% 1,2-Propanediol (v/v), 40mM Tris-HCl, 10mM MgCl₂10mM ATP, 10mM DTT, 80U/ul Rapid T4 DNA Ligase (EnzyValley) and DEPC water as a solvent;

AD Buffer: 10mM Tris-HCl, 0.5mM EDTA, 50mM NaCl, and DEPC water as solvent;

the preparation method comprises the following steps: adding the above components according to their content, mixing, and filtering with 0.22um filter membrane (raw technology, F513134-0001) for sterilization;

the kit nucleic acid library building part library building method comprises the following steps:

isometric mixing of front-end extracted nucleic acid products A and B to obtain a sample 1, isometric mixing of front-end extracted products C and D to obtain a sample 2, isometric mixing of front-end extracted nucleic acid products E and F to obtain a sample 3, and entering three samples into a downstream experiment;

dna fragmentation (DNAFragment):

preparing a breaking system in 50ul breaking pipes, and selecting corresponding breaking time according to different breaking systems:

note:

Pico sonication device(B01080010)

B. end repair (A-Tailed Conversion):

preparing a terminal repair reaction system in a 0.2mL PCR tube:

Fragment ds_DNA	25μL
		AC Buffer	3μL
AC Enzymes	2μL
		Total Volume	30μL

blowing (at least 10 times) or vortex, shaking, mixing, and centrifuging for a short time to collect liquid on the tube/hole wall;

the reaction system was placed on a PCR instrument with the hot lid set at 80 ℃ and the following procedure was run:

·20℃ 30min；

·65℃ 30min；

·4℃ Forever

C. connector Ligation (Adapter Ligation):

5min before preparation of the ligation reaction, Adapter working solution (Adapter, illumina platform sequence, AD Buffer for dilution) was prepared according to the following table and placed on ice for use:

DNA input	Dilution
		50ng-500ng	No dilution
5ng-50ng	10-fold

the following reagents were added directly to the end-repair reaction system:

A-Tailing cDNA	30μL
		Ligation XP	20μL
Adapter(for illumina)	5μL
		Total Volume	55μL

placing the reaction system on a PCR instrument, setting the temperature of a hot cover as Off, and incubating for 20min at 20 ℃;

D. fragment selection and purification (Post-Ligation Clean-up)

Insert Size	～150bp	150-250bp	250-350bp	350-450bp	450-550bp	>550bp
							1st Beads Vol.(μL)	50	40	30	25	20	15
2nd Beads Vol.(μL)	25	20	15	10	10	10

Note: the following purification procedure is exemplified by 300bp

Taking the Fapon DNA clean Beads out, standing to room temperature, and uniformly mixing in a vortex manner for later use;

to a 1.5mL tube or octal tube was added 30. mu.L ddH₂Adding O +30 mu LFapon DNAclear Beads into 55 mu L of the reaction product in the previous step, uniformly mixing by vortex, collecting the liquid on the tube wall instantly, and standing for 5min at room temperature;

place 1.5mL tube or octal tube on magnetic rack until the solution in the tube is clear.

Carefully pipette the supernatant into a new 1.5mL EP tube or an octal tube (note: do not discard the supernatant), add 15. mu.L of Fapon DNA clean Beads, mix well, and stand at room temperature for 5 min;

note that: the supernatant is reserved in the operation, and the magnetic beads on the tube wall are not required to be contacted in the transferring process;

placing the EP tube or the eight-connected tube on a magnetic frame, and removing supernatant after the solution in the tube is clarified;

note that: this step should retain the beads.

Adding 200 mu L of freshly prepared 80% ethanol into an EP tube or an eight-connection tube, rotating a 1.5mL tube for one circle, standing the eight-connection tube for 30s, and then removing a supernatant;

repeating the steps, and removing the supernatant as much as possible;

placing a 1.5mL tube or an eight-connection tube on a magnetic frame, standing at room temperature for 10min until the magnetic beads are dried or opening the tube and placing the tube on a 45 ℃ metal bath until the surfaces of the magnetic beads are free of water;

removing the centrifugal tube from the magnetic frame, adding 22 mu L of ddH2O for resuspension of magnetic beads, uniformly mixing by vortex or blowing, collecting the liquid on the tube wall instantly, and standing at room temperature for 2 min;

the EP tube or the octal tube is placed on a magnetic rack, and after the solution in the tube is clarified, 20. mu.L of the supernatant is transferred to a new PCR tube for the next amplification.

Library amplification (library amplification):

preparing a library amplification reaction system on an ice box:

setting up a library amplification reaction program:

the cycle numbers are referenced as follows:

Total DNA input	Recommended PCR cycles	Total DNA input	Recommended PCR cycles
				1ug	5	50ng	10-11
500ng	6-8	10ng	12-13
				100ng	8-9	5ng	13-15

and (3) purifying an amplification product:

vortex Fapon DNA clean Beads, resuspend them;

transferring the PCR product to a new 1.5mL tube or an eight-linked tube, adding 0.8X (40. mu.L) Fapon DNA clean Beads, mixing uniformly by vortex, collecting the tube wall liquid instantly, and standing at room temperature for 5 min;

placing 1.5mL tube or eight-connected tube on a magnetic frame, standing for 2min, and standing until the solution in the tube is clear;

carefully sucking and discarding supernatant liquid, adding 200 μ L of newly prepared 80% ethanol into the tube, rotating the tube on a magnetic frame, and discarding supernatant after the solution is clarified;

repeating the steps, and removing the supernatant as much as possible;

and (3) placing a 1.5mL tube or an eight-connected tube on a magnetic frame, standing at room temperature for 10min until the magnetic beads are dried or opening the tube and placing the tube on a 45 ℃ metal bath until the surfaces of the magnetic beads have no water.

Adding 28 μ L ddH2O into the EP tube or the eight-link tube, blowing, beating, mixing, standing for 2min, and placing the EP tube on a magnetic frame until the liquid becomes clear;

and sucking 25 mu L of supernatant into a new 1.5mL EP tube, adhering a library label on the tube wall, using Qsep400 to check the quality to be qualified, and storing the product in a special library storage position at the temperature of-20 ℃.

Pre-quality inspection (Qsep400) and sequencing results on the resulting library

FIG. 1 and FIG. 2 show the pre-machine Qsep400 quality inspection of the library, and the number LR201015-0100(A1) is a kit for extracting the competitive products(magenta, D3141) and kit for building a database from a competitive product: (

Ultra^TMII, DNA Library Prep Kit, E7645S) Library quality inspection results, and LR201015-0101(B1) is a quality inspection result of a Library part built by a competitive product extraction Kit (magenta, D3141) + a self-research Kit; no. LR201015-0102(C1) self-research kit extraction part and competitive product library-building kit (C201015-0102)

Ultra^TMII, DNA Library Prep Kit, E7645S) Library quality inspection results, and LR201015-0103(D1) is Library quality inspection results of an extraction part and a Library building part of the self-research Kit; LR201015-0104(E1) is a competitive extraction kit (assist in, 18820ES50) and a competitive library kit (E201015-E8932) ((R))

Ultra^TMII DNA Library Prep Kit, E7645S), LR201015-0105(F1), Library-building partial quality test results for competitive extraction Kit (assist in san-out, 18820ES50) and self-research Kit. FIG. 3 shows the results of the sequencing data confirmatory analysis, and the self-research kit is not significantly different from the competitive kit.

Claims (7)

1. A nucleic acid extraction kit for use in the construction of a genomic library of pathogenic microorganisms in human feces, comprising: a nucleic acid releasing agent LysisA, an Enhancer Lysis Enhancer, a de-inhibitor IRT, a Binding solution Binding Buffer, a washing solution 1 and a washing solution 2;

the nucleic acid releasing agent lysine A comprises 20 mM-40 mM EDTA, 40 mM-80 mM Tris-HCl, 2% -5% PVP-40 (mass fraction), a solvent DEPC water and a pH value of 9-10;

the Enhancer lysine Enhancer comprises 12-15% of SDS (mass fraction), 4-8% of PVP-40 (mass fraction) and DEPC water as a solvent.

2. The nucleic acid kit according to claim 1, wherein the nucleic acid releasing agent lysine A comprises 20mM EDTA, 50mM Tri-HCl, 2% PVP-40 (mass fraction), and DEPC water as a solvent, and the Enhancer lysine Enhancer comprises 15% SDS (mass fraction), 5% PVP-40 (mass fraction), and DEPC water as a solvent.

3. The nucleic acid kit of claim 1, wherein the de-inhibitor IRT comprises: 120mM NH₄Al(SO₄)₂.12H₂O, DEPC water is used as a solvent, and the pH value is 3.0-4.0;

the Binding solution Binding Buffer comprises: 3M guanidinium isothiocyanate, 16.7% isopropanol (V/V), 2% Triton X100(V/V), 10mM EDTA, 5mM Tris-HCl in DEPC water at pH 6.0-7.0;

the washing solution 1 comprises: 20% ethanol (V/V), 20% isopropanol (V/V), 70mM sodium chloride, 20mM Tris-HCl, DEPC water as solvent, and pH 6.0-7.0;

the washing liquid 2 comprises: 70% ethanol (V/V), 20mM Tris-HCl, DEPC water as solvent, pH 6.0-7.0.

4. A kit for the fragmentation treatment and end modification of a pathogenic microorganism genome library in human feces is characterized by comprising AC buffers and AC Enzymes;

the AC Buffer comprises: 500mM Tris-HCl, 200mM KCl, 100mM MgCl2, 1mM DTT, 10mM MATP, 10mM dATP, 2mM dTTP, 2mM dGTP, 2mM dCTP, 0.1% Triton X-100(v/v), and the solvent is DEPC water;

the AC Enzymes comprise: 1.5U/ul T4 Polynucleotide Kinase (Enzymatics, Y9040L), 1U/ul T4 DNApolymerase (Enzymatics, P7080L), 0.5U/ul Klenow Fragment (Enzymatics, P7060L), 1U/ul Taq-B DNApolymerase (Enzymatics, P7250L), 4mM Tris-HCl, 5mM KCl, 0.8mM DTT, 0.2mM EDTA, 22% (mass fraction) Glycerol, 0.5% Triton X-100(v/v), and DEPC water as a solvent.

5. A kit for connecting and enriching joints of a pathogenic microorganism genome library in human excrement is characterized by comprising Ligation XP and AD Buffer;

the Ligation XP comprises: 14% Glycerol (v/v), 20% PEG-6000 (mass fraction), 25% 1,2-Propanediol (v/v), 40mM Tris-HCl, 10mM MgCl2, 10mM MATP, 10mM DTT, 80U/ul Rapid 4 DNAIGAse (EnzyValley), and DEPC water as a solvent;

the AD Buffer comprises: 10mM Tris-HCl, 0.5mM EDTA, 50mM NaCl, and DEPC water as solvent.

6. A nucleic acid extraction method for a pathogenic microorganism genome library in human excrement is characterized by comprising the following steps:

nucleic acid release: transferring the excrement sample to a bead grinding tube for grinding beads, adding a nucleic acid releasing agent LysisA and an Enhancer Lysis Enhancer into the sample, performing vortex scattering on the sample, incubating, and putting the grinding tube into a bead grinding instrument for bead grinding;

inhibitor removal: transferring all the supernatant into a centrifuge tube, adding inhibitor-removed IRT into the supernatant, uniformly mixing by vortex, and centrifuging;

column adsorption, namely transferring the supernatant into a centrifuge tube, adding Binding solution Binding Buffer into the supernatant, reversing and uniformly mixing to obtain mixed solution, putting a purification column into a collection tube, transferring the mixed solution into a column, centrifuging to discard effluent, and putting the column back into the collection tube;

desalting: adding washing solution 1 into the column, centrifuging, pouring off the filtrate, putting the column back into the collecting pipe, adding washing solution 2 into the column, centrifuging, repeating the operation of washing solution 2 for several times, pouring off the filtrate, putting the column back into the collecting pipe, and centrifuging;

nucleic acid elution: the column was placed in a centrifuge tube, sterilized water was added to the center of the membrane of the column, and the column was left at room temperature and then centrifuged to obtain nucleic acid.

7. A method for building a database of pathogenic microorganism genomes in human excrement is characterized by comprising the following steps:

dna fragmentation:

preparing a breaking system in 50ul breaking pipes, and selecting corresponding breaking time according to different breaking systems:

B. end repair (A-Tailed Conversion):

preparing a terminal repair reaction system in a 0.2mL PCR tube:

Fragmentds_DNA 25μL ACBuffer 3μL ACEnzymes 2μL TotalVolume 30μL

blowing and beating for at least 10 times or carrying out vortex oscillation and uniform mixing, and carrying out short-time centrifugation to collect liquid on the pipe/hole wall;

the reaction system was placed on a PCR instrument with the hot lid set at 80 ℃ and the following procedure was run:

·20℃30min；

·65℃30min；

·4℃Forever

C. connecting joints:

5min before the ligation reaction, the Adapter working solution was prepared according to the following table and placed on ice for use:

DNAinput Dilution 50ng-500ng Nodilution 5ng-50ng 10-fold

the following reagents were added directly to the end-repair reaction system:

A-TailingcDNA 30μL LigationXP 20μL Adapter(forillumina) 5μL TotalVolume 55μL

placing the reaction system on a PCR instrument, setting the temperature of a hot cover as Off, and incubating for 20min at 20 ℃;

D. fragment selection and purification

InsertSize ～150bp 150-250bp 250-350bp 350-450bp 450-550bp >550bp 1stBeadsVol.(μL) 50 40 30 25 20 15 2ndBeadsVol.(μL) 25 20 15 10 10 10

Taking the Fapon DNA clean Beads out, standing to room temperature, and uniformly mixing in a vortex manner for later use;

to a 1.5mL tube or octal tube was added 30. mu.L ddH₂Adding O +30 mu LFapon DNA clean Beads into 55 mu L of the reaction product in the previous step, uniformly mixing by vortex, collecting the liquid on the tube wall instantly, and standing for 5min at room temperature;

placing a 1.5mL tube or an eight-connected tube on a magnetic frame until the solution in the tube is clarified;

carefully sucking the supernatant into a new 1.5mL EP tube or an eight-linked tube, adding 15 μ L Fapon DNA clean Beads, mixing well, and standing at room temperature for 5 min;

placing the EP tube or the eight-connected tube on a magnetic frame, and removing supernatant after the solution in the tube is clarified;

adding 200 mu L of freshly prepared 80% ethanol into an EP tube or an eight-connection tube, rotating a 1.5mL tube for one circle, standing the eight-connection tube for 30s, and then removing a supernatant;

repeating the steps, and removing the supernatant as much as possible;

placing a 1.5mL tube or an eight-connection tube on a magnetic frame, standing at room temperature for 10min until the magnetic beads are dried or opening the tube and placing the tube on a 45 ℃ metal bath until the surfaces of the magnetic beads are free of water;

removing the centrifugal tube from the magnetic frame, adding 22 mu L of ddH2O for resuspension of magnetic beads, uniformly mixing by vortex or blowing, collecting the liquid on the tube wall instantly, and standing at room temperature for 2 min;

placing the EP tube or the eight-connected tube on a magnetic frame, transferring 20 mu L of supernatant into a new PCR tube after the solution in the tube is clarified, and carrying out the next amplification;

C. library amplification:

preparing a library amplification reaction system on an ice box:

setting up a library amplification reaction program:

the cycle numbers are referenced as follows:

TotalDNAinput RecommendedPCRcycles TotalDNAinput RecommendedPCRcycles 1ug 5 50ng 10-11 500ng 6-8 10ng 12-13 100ng 8-9 5ng 13-15

D. and (3) purifying an amplification product:

vortex Fapon DNA clean Beads, resuspend them;

transferring the PCR product to a new 1.5mL tube or an eight-connected tube, adding 0.8X 40 mu L Fapon DNA clean Beads, mixing uniformly by vortex, collecting the tube wall liquid instantly, and standing at room temperature for 5 min;

placing 1.5mL tube or eight-connected tube on a magnetic frame, standing for 2min, and standing until the solution in the tube is clear;

carefully sucking and discarding supernatant liquid, adding 200 μ L of newly prepared 80% ethanol into the tube, rotating the tube on a magnetic frame, and discarding supernatant after the solution is clarified;

repeating the steps, and removing the supernatant as much as possible;

placing a 1.5mL tube or an eight-connection tube on a magnetic frame, standing at room temperature for 10min until the magnetic beads are dried or opening the tube and placing the tube on a 45 ℃ metal bath until the surfaces of the magnetic beads are free of water;

adding 28 μ L ddH2O into the EP tube or the eight-link tube, blowing, beating, mixing, standing for 2min, and placing the EP tube on a magnetic frame until the liquid becomes clear;

and sucking 25 mu L of supernatant into a new 1.5mL EP tube, adhering a library label on the tube wall, using Qsep400 to check the quality to be qualified, and storing the product in a special library storage position at the temperature of-20 ℃.

Images