CN114395614A - Method for rapidly detecting body fluid metagenome pathogenic microorganisms in high flux - Google Patents
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Abstract
A method for rapidly detecting the metagenome pathogenic microorganism of body fluid with high flux belongs to the field of biotechnology. The method comprises the whole process of extracting and establishing the library of the body fluid pathogenic microorganisms. The reagents used in the extraction process comprise proteinase K solution, magnetic beads, lysis solution, lysis binding solution, primary washing solution, secondary washing solution and eluent, and the reagents used in the library construction process comprise terminal repair enzyme, joint reagent and pcr reagent. In the process of extracting pathogenic microorganisms, the formulas of cell lysis solution, lysis binding solution, primary washing solution and secondary washing solution are optimized, and the experimental method for building a low-sample-volume library is optimized in the process of building a library of the pathogenic microorganisms. The invention improves the experimental method for detecting the body fluid metagenome microorganisms, shortens the detection time and lays the experimental foundation for the sequencing of the body fluid samples.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to extraction of metagenome pathogenic microorganisms, nucleic acid extraction, library construction of nucleic acid of the metagenome pathogenic microorganisms and final metagenome sequencing.
Background
Circulating free DNA or cell free DNA (cfDNA), is a degraded DNA fragment that is released into the plasma. cfDNA is present in various body fluids of the human body at concentrations that vary with tissue damage, cancer, inflammatory responses, and the like. The cfDNA of normal human is mainly generated by small and uniform 185-bp 200bp small fragment DNA generated during apoptosis. In certain diseases and specific conditions, other cells also release free DNA into the blood, including circulating tumor DNA (ctdna) derived from tumor cells and cfDNA derived from fetuses, etc., resulting in the production of large fragments of DNA of different sizes and greater than 200 bp. Blood is not the only bodily fluid available for fluid biopsy, e.g., urine, feces, cerebrospinal fluid (CSF), saliva, pleural fluid, and ascites are all potential sources from which certain disease or infectious nucleic acid material (including cfDNA) can be obtained.
The extraction of free nucleic acid becomes a key step of detection, reagents with certain toxicity such as phenol, chloroform and the like are required in a reagent precipitation method used in the traditional extraction, the amount of the reagents required to be prepared in the experimental process is large, the pollution risk is high, and the extraction concentration of nucleic acid is low; rare pathogens cannot be known through conventional library detection, and the detection of fragment length has certain limitation, so that the difficulty is brought to subsequent clinical detection.
With the continuous development of high-throughput sequencing technology in recent years, particularly the appearance of a new generation sequencer, the development of metagenomics is particularly rapid. The sequencing technology has low cost, high flux, high speed and large data volume generated by single operation, can simultaneously sequence hundreds of thousands to millions of DNA molecules, has wider sequencing fragments, and can detect pathogenic microorganisms which cannot be predicted by clinical routine means.
Disclosure of Invention
In order to solve the problems of the prior art, the invention aims to provide a method for rapidly detecting the metagenomic pathogenic microorganisms in body fluid. Establishing an efficient nucleic acid extraction method aiming at a body fluid sample, and constructing a library of extracted nucleic acid products.
In order to achieve the above purpose, the invention provides the following technical scheme:
a method for rapidly detecting the metagenome pathogenic microorganism of body fluid with high flux optimizes the formulas of cell lysate and washing solution, wherein the lysate LSBuffer comprises the following components: the concentration of the ethylene diamine tetraacetic acid is 10-30mmol/L, the concentration of the sodium chloride is 100-350mmol/L, the concentration of the triton X-100 is 3-10% of volume fraction, the concentration of the guanidinium isothiocyanate is 1-8mol/L, and the concentration of the guanidinium isothiocyanate is 5-8 wt% of Tween 20. The components of the lysis binding solution LBBuffer comprise: 50-300g/L of guanidinium isothiocyanate, 2-8g/L of sodium chloride, 20-50% of triton X-100 by volume, 5-40% of isopropanol by volume, 5-15g/L of citric acid and tris (hydroxymethyl) aminomethane. The composition of the washing solution H1 included: the concentration of the ethylene diamine tetraacetic acid is 5-100mmol/L, the concentration of the sodium chloride is 100-300mmol/L, the concentration of the absolute ethyl alcohol is 20-60% volume fraction, the concentration of the isopropanol is 25-80% volume fraction, and the concentration of the triton X-100 is 2-5% volume fraction; 3-7 mol/L guanidine hydrochloride, 1-10 wt% Tween 20. The secondary washing liquid H2 comprises the following components: 75% of ethanol. The eluent comprises the following components: 10-20 mmol/LTris-Hcl and 0.5-1.5 mmol/LEDTA.
A method for quickly detecting the pathogenic microbe of the metagenome of body fluid in high flux includes such steps as separating and extracting DNA from the body fluid sample to obtain nucleic acid extract, constructing the library of its nucleic acid, and sequencing the metagenome to obtain the result of sequencing the metagenome related to said sample. The whole detection steps are as follows:
s1 sample pretreatment: the resulting samples were centrifuged and the supernatants transferred to 15ml fresh centrifuge tubes.
S2 cell lysis: adding ProteinaseKSolution and lysis solution LSBuffer into the centrifuge tube in S1, and mixing by vortexing at maximum speed. And putting the whirled centrifugal tube into a constant-temperature water bath kettle for incubation for 20-40 min. And standing for 10-15min after incubation.
Lysis and binding of the S3 microorganism: adding the magnetic beads and the lysis binding solution LBbuffer into a centrifuge tube of S2, reversing and mixing evenly, and continuously mixing evenly for 5-20min at room temperature. Placing the centrifugal tube on a magnetic separation frame, waiting at room temperature until the solution becomes completely clear, and completely adsorbing magnetic beads on one side in the tube; the adsorption state was maintained, the supernatants were carefully aspirated, and after all the supernatants were aspirated, the magnetic separation rack was removed.
S4 one wash: adding primary washing liquid H1, and mixing for 3-7 min; and placing the centrifugal tube on a magnetic separation frame, and waiting at room temperature until the magnetic beads are completely adsorbed on one side in the tube. Keeping the adsorption state, carefully absorbing the supernatant and removing the magnetic separation rack; this step was repeated once.
S5 secondary washing: adding secondary washing liquid H2, and mixing for 3-7 min; and placing the centrifugal tube on a magnetic separation frame, and waiting at room temperature until the magnetic beads are completely adsorbed on one side in the tube. Keeping the adsorption state, carefully absorbing the supernatant and removing the magnetic separation rack; this step was repeated once.
S6 dried magnetic beads: the tube was placed on a magnetic stand for 20-30min to dry the beads.
S7DNA elution: adding 50-100ul of eluent into the centrifugal tube, vortexing to fully resuspend the magnetic beads, and standing at room temperature for 3-7 min.
S8, DNA collection, namely placing the centrifuge tube on a magnetic separation frame, and waiting at room temperature until the solution becomes completely clear, wherein magnetic beads are completely adsorbed on one side in the tube. Carefully transfer the supernatant DNA product to a 1.5mL centrifuge tube.
And (4) measuring the concentration of S9: the sample DNA concentration was determined using EQUALBITDSDNAHSKIT reagent and the Qubit instrument.
S10 is to use polymerase to repair the free DNA fragment end and phosphorylate the 5 'end and add dA tail to the 3' end to obtain DNA plus A product. And introducing exogenous genes when a sample with the extraction concentration less than or equal to 0.517ng/ul is determined to be constructed, so that the library construction quality is improved.
S11 the DNA plus A product in step S10 is ligated with the DNA linker using ligase to obtain a DNA fragment containing a linker form.
S12 the DNA fragment obtained in step S11 is subjected to purification treatment.
S13 PCR amplification is carried out using the DNA fragment purified in step S12 as a template.
S14 sorting the PCR amplified products in step S13.
S15, purifying the product obtained after sorting in the step S14 to obtain a final product.
If the sample type in the step S1 is blood, the sample type should be processed at 3000rpm for 5-10min and the supernatant obtained after centrifugation is transferred to a new 15ml centrifuge tube for centrifugation again, wherein the centrifugation condition is 10000-16000g for 5-10 min. Transfer 1-6ml of the supernatant to a new 15ml centrifuge tube for use. If the sample is a non-blood sample, 10000-.
The amount of the ProteinaseKSsolution added in the step S2 is 10-30 ul.
The components of the cracking solution LSBuffer in the step S2 include that the concentration of ethylene diamine tetraacetic acid is 10-30mmol/L, the concentration of sodium chloride is 100-350mmol/L, the concentration of Triton X-100 is 3-10% volume fraction, the concentration of guanidinium isothiocyanate is 1-8mol/L, and 5-8 wt% Tween 20.
The ingredients of the lysis binding solution LBBuffer in the step S3 are 50-300g/L guanidinium isothiocyanate, 2-8g/L sodium chloride, 20-50% triton X-100 by volume, 5-40% isopropanol by volume, 5-15g/L citric acid and tris (hydroxymethyl) aminomethane.
The washing liquid H1 in the step S4 comprises the components of 5-100mmol/L of ethylenediamine tetraacetic acid, 100-300mmol/L of sodium chloride, 20-60% of absolute ethyl alcohol by volume fraction, 25-80% of isopropanol by volume fraction, and 2-5% of triton X-100 by volume fraction; 3-7 mol/L guanidine hydrochloride, 1-10 wt% Tween 20.
The secondary washing liquid H2 in the step S5 contains 75% ethanol.
The eluent in the step S7 comprises 10-20 mmol/LTris-Hcl and 0.5-1.5 mmol/LEDTA.
If the sample extraction concentration is low in step S10, 20ng of foreign gene is introduced into the extracted product and the library is created.
The reaction conditions of phosphorylation at the end S105 'and dA tail addition at the end 3' are 10-20 ℃,15-20min, 55-65 ℃ and 15-20 min.
The PCR reaction condition in the step S11 is 15-20 ℃ and 10-17 min.
The PCR amplification reaction condition in the step S13 is 95 ℃ and 3 min; 20s at 98 ℃; 60 ℃ for 15 s; 72 ℃ for 30 s; 72 ℃ for 5min, the number of cycles is determined according to the data volume of the database.
The method optimizes the formulas of cell lysis solution, lysis binding solution, primary washing solution and secondary washing solution for the extraction step, and can better release sample nucleic acid; the database building step optimizes the experimental method for building the database with low sample volume, ensures that the experimental sample is smoothly loaded on the computer and obtains accurate data volume. Compared with the prior art, the method has the advantages that the extraction time of the sample is obviously shortened compared with the conventional extraction time, the extraction concentration is uniform, the purity is good, the method can at least meet 50ng library construction, if the library construction amount is extremely low, for example, less than 20ng library construction amount, the exogenous gene is introduced to construct the library together, and the subsequent library construction operation by using unknown library construction amount is avoided. The invention optimizes the formulas of lysis solution, lysis binding solution, primary washing solution and secondary washing solution extracted from the body fluid sample, and fully releases genetic materials in the body fluid sample; and the experimental method for constructing the database with low sample amount is solved in the database construction process of the pathogenic microorganisms.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.
Example 1
A method for rapidly detecting the metagenome pathogenic microorganism of body fluid in high flux is characterized by comprising the following steps:
(1) sample pretreatment: the resulting samples were centrifuged and the supernatants transferred to 15ml fresh centrifuge tubes.
(2) Cell lysis: adding ProteinaseKSolution and lysis solution LSBuffer into the centrifuge tube in (1), and mixing uniformly by vortexing at maximum speed. And putting the whirled centrifugal tube into a constant-temperature water bath kettle for incubation for 30 min. After incubation, the mixture was allowed to stand for 10 min.
(3) Lysis and binding of microorganisms: adding the magnetic beads and the lysis binding solution LBbuffer into the centrifuge tube in the step (2), reversing and mixing evenly, and continuously mixing evenly for 10min at room temperature. Placing the centrifugal tube on a magnetic separation frame, waiting at room temperature until the solution becomes completely clear, and completely adsorbing magnetic beads on one side in the tube; the adsorption state was maintained, the supernatants were carefully aspirated, and after all the supernatants were aspirated, the magnetic separation rack was removed.
(4) Primary washing: adding primary washing liquid H1, and vortex mixing for 5 min; and placing the centrifugal tube on a magnetic separation frame, and waiting at room temperature until the magnetic beads are completely adsorbed on one side in the tube. Keeping the adsorption state, carefully absorbing the supernatant and removing the magnetic separation rack; this step was repeated once.
(5) And (3) secondary washing: adding secondary washing liquid H2, and mixing for 5 min; and placing the centrifugal tube on a magnetic separation frame, and waiting at room temperature until the magnetic beads are completely adsorbed on one side in the tube. Keeping the adsorption state, carefully absorbing the supernatant and removing the magnetic separation rack; this step was repeated once.
(6) And (3) drying the magnetic beads: the tube was placed on a magnetic stand for 25min to dry the beads.
(7) DNA elution: 50-100ul of eluent is added into the centrifugal tube, the magnetic beads are fully resuspended by vortex, and the mixture is placed for 5min at room temperature.
(8) And (3) DNA collection, namely placing the centrifuge tube on a magnetic separation frame, and waiting at room temperature until the solution becomes completely clear, and completely adsorbing magnetic beads on one side in the tube. Carefully transfer the supernatant DNA product to a 1.5mL centrifuge tube.
(9) And (3) concentration determination: the sample DNA concentration was determined using EQUALBITDSDNAHSKIT reagent and the Qubit instrument.
Comparative example 1
(1)1-10ml of body fluid sample is centrifuged at 3000-5000 Xg for 5-10min, then the supernatant is taken and placed in a clean centrifuge tube, and the supernatant is collected after centrifugation at 10000-16000rpm for 5-10 min.
(2) Adding proteinase K and unoptimized lysis buffer LS into the collected supernatant, mixing uniformly, and performing incubation treatment to obtain a lysis mixture.
(3) Adding an adsorption binding solution BufferLB mixed with isopropanol into the cracking mixture, uniformly mixing, carrying out ice bath for 3-5min, and transferring the mixed solution into a silica gel adsorption column for negative pressure suction filtration;
(4) after the filtration is finished, washing the silica gel adsorption column by using a first unaptimized cleaning solution BufferH1, a second unaptimized cleaning solution BufferH2 and absolute ethyl alcohol in sequence, and drying the silica gel adsorption column at room temperature after centrifugal treatment to obtain the impurity-removed silica gel adsorption column with cfDNA adsorbed thereon;
(5) and (3) adding 20-150ul of eluent to elute the silica gel adsorption column, and centrifuging and collecting the eluent to obtain the cfDNA.
The results are shown in Table 1:
TABLE 1 comparison of the effect of extracting the concentration of the metagenomic pathogenic microorganism from body fluid
Note: comparative example 1 shows the effect of extraction of components of cell lysate and wash solution without optimization.
The results in table 1 show that the DNA concentration of the body fluid sample obtained by optimally extracting the components of the cell lysate and the washing solution in the kit is higher than that of the body fluid sample obtained by the traditional experimental method, the concentration is uniform, 100ng of library construction can be met, and the ratio of A260/A280 is in the range of 1.8-2.0.
Example 2
(1) And adding 20ng of exogenous gene into the sample with low extraction concentration, and then performing end repair, DNA 5 'end phosphorylation and DNA 3' end A addition by using end repair enzyme to obtain a DNA A addition product.
(2) And (2) using ligase to link the DNA plus A product in the step (1) and the DNA adaptor together to obtain the DNA fragment containing the adaptor form.
(3) And (3) purifying the DNA fragment obtained in the step (2).
(4) And (4) performing PCR amplification by using the DNA fragment purified in the step (3) as a template.
(5) And (4) sorting the products obtained after PCR amplification in the step (4).
(6) And (5) purifying the product obtained after sorting in the step (5) to obtain a final product.
Comparative example 2
(1) And directly carrying out end repair, DNA 5 'end phosphorylation and DNA 3' end A addition on the sample with low extraction concentration by using end repair enzyme to obtain a DNA A addition product.
(2) And (2) using ligase to link the DNA plus A product in the step (1) and the DNA adaptor together to obtain the DNA fragment containing the adaptor form.
(3) And (3) purifying the DNA fragment obtained in the step (2).
(4) And (4) performing PCR amplification by using the DNA fragment purified in the step (3) as a template.
(5) And (4) sorting the products obtained after PCR amplification in the step (4).
(6) And (5) purifying the product obtained after sorting in the step (5) to obtain a final product. The results are shown in Table 2:
TABLE 2 comparison of the effects of the concentration of the metagenomic pathogenic microorganisms in the body fluid in the reservoir
Note: comparative example 2 is an experimental result that the extraction concentration is low and no foreign gene is introduced during library construction.
The results in table 2 show that the library building concentration of the sample is uniform and the stability is good after 20ng of exogenous gene is introduced in example 2, while the library building concentration of the low-concentration sample without exogenous gene in comparative example 2 is not uniform, and some samples are too high and some samples are too low, so that the high reads background bacteria can be caused by the too high concentration, and the risk that the machine cannot be operated can be caused by the too low concentration.
The above description is only a preferred embodiment of the present invention, and any person skilled in the art may modify the present invention or modify it into an equivalent technical solution by using the technical solution described above. Therefore, any simple modifications or equivalent substitutions made in accordance with the technical solution of the present invention are within the scope of the claims of the present invention.
Claims (5)
1. A method for rapidly detecting the metagenome pathogenic microorganism of body fluid with high flux is characterized in that:
separating and extracting DNA from a body fluid sample to obtain a nucleic acid extract; constructing a library for nucleic acid of the sample, and finally performing metagenome sequencing to obtain a metagenome sequencing result related to the sample;
s1, sample pretreatment: centrifuging the obtained sample, and transferring the supernatant into a centrifuge tube;
s2, cell lysis: adding 10-30ul of protease K solution and LS Buffer of lysis solution into the centrifuge tube in S1, and uniformly mixing by vortexing at maximum speed; putting the whirled centrifugal tube into a constant-temperature water bath kettle for incubation for 20-40 min; standing for 10-15min after incubation;
the concentration of ethylene diamine tetraacetic acid in the lysis solution LS Buffer is 10-30mmol/L, the concentration of sodium chloride is 100-350mmol/L, the concentration of Triton X-100 is 3-10% volume fraction, and the concentration of guanidinium isothiocyanate is 1-8mol/L, Tween20 and is 5-8 wt%;
s3, cracking and combining microorganisms: adding the magnetic beads and the lysis binding solution LB Buffer into the centrifugal tube of S2, reversing and uniformly mixing, and continuously and uniformly mixing for 5-20min at room temperature; placing the centrifugal tube on a magnetic separation frame, waiting at room temperature until the solution becomes completely clear, and completely adsorbing magnetic beads on one side in the tube; keeping the adsorption state, carefully sucking the supernatant, and removing the magnetic separation rack after sucking all the supernatant;
the cracking binding solution LB Buffer comprises 50-300g/L of guanidinium isothiocyanate, 2-8g/L of sodium chloride, 20-50% of triton X-100 by volume, 5-40% of isopropanol by volume, 5-15g/L of citric acid and 10-50 mM of trihydroxymethyl aminomethane;
s4, primary washing: adding washing solution H1, and mixing for 3-7 min; placing the centrifugal tube on a magnetic separation frame, and waiting at room temperature until the magnetic beads are completely adsorbed on one side in the tube; keeping the adsorption state, sucking the supernatant and removing the magnetic separation rack; repeating the steps once;
the concentration of the ethylene diamine tetraacetic acid in the washing liquid H1 is 5-100mmol/L, the concentration of the sodium chloride is 100-300mmol/L, the concentration of the absolute ethyl alcohol is 20-60% volume fraction, the concentration of the isopropyl alcohol is 25-80% volume fraction, and the concentration of the triton X-100 is 2-5% volume fraction; the concentration of guanidine hydrochloride is 3-7 mol/L, Tween20 and the concentration is 1-10 wt%;
s5, secondary washing: adding washing solution H2, and mixing for 3-7 min; placing the centrifugal tube on a magnetic separation frame, and waiting at room temperature until the magnetic beads are completely adsorbed on one side in the tube; keeping the adsorption state, sucking the supernatant and removing the magnetic separation rack; repeating the steps once;
the secondary washing solution H2 contains 75% ethanol;
s6, drying magnetic beads: placing the centrifuge tube on a magnetic separation rack for 20-30min to dry the magnetic beads;
s7, DNA elution: adding 50-100ul of eluent into the centrifugal tube, vortexing to fully resuspend the magnetic beads, and standing at room temperature for 3-7 min;
the concentration of Tris-HCl in the eluent is 10-20 mmol/L and the concentration of EDTA is 0.5-1.5 mmol/L;
s8, DNA collection, namely placing the centrifugal tube on a magnetic separation frame, and waiting at room temperature until the solution becomes completely clear and magnetic beads are completely adsorbed on one side in the centrifugal tube; carefully transferring the supernatant DNA product to a centrifuge tube;
s9, concentration determination: using EQUALBIT DSDNA HS KIT reagent and a Qubit instrument to determine the DNA concentration of the sample;
s10, performing end repair on the free DNA fragment by using polymerase: phosphorylating the 5 'end and adding dA tail to the 3' end to obtain a DNA and A product; the reaction condition for phosphorylation at the 5' end is 10-20 ℃ and 15-20 min; the reaction conditions for adding dA tail to the 3' end are as follows: 55-65 deg.C for 15-20 min;
s11, using ligase to connect the DNA plus A product in the step S10 with a DNA adaptor to obtain a DNA fragment containing an adaptor form;
s12, purifying the DNA fragment obtained in the step S11;
s13, carrying out PCR amplification by taking the DNA fragment purified in the step S12 as a template; the PCR amplification reaction condition is 95 ℃ and 3 min; 20s at 98 ℃; 60 ℃ for 15 s; 72 ℃ for 30 s; at 72 ℃ for 5min, and the cycle number is determined according to the database building data volume;
s14, sorting products obtained after PCR amplification in the step S13;
s15, purifying the product obtained after sorting in the step S14 to obtain a final product.
2. The method for rapidly detecting the metagenomic pathogenic microorganism of the body fluid in high throughput according to claim 1, which is characterized in that: and when the sample extraction concentration in the step S10 is less than or equal to 0.517ng/ul, introducing an exogenous gene into the extracted product to establish a library.
3. The method for rapidly detecting the metagenomic pathogenic microorganism of the body fluid in high throughput according to claim 1, which is characterized in that: the body fluid comprises blood, urine, thoracic drainage fluid, abdominal drainage fluid, and cerebrospinal fluid.
4. The method for rapidly detecting the metagenomic pathogenic microorganism of the body fluid in high throughput according to claim 1, which is characterized in that: the sample in the step S1 is blood, and centrifugation is performed at 3000rpm for 5-10 min; transferring the supernatant obtained after centrifugation to a new centrifuge tube for centrifugation again, wherein the centrifugation condition is 10000-; 5-10 min; transferring the supernatant into a centrifuge tube for later use.
5. The method for rapidly detecting the metagenomic pathogenic microorganism of the body fluid in high throughput according to claim 1, which is characterized in that: the sample in the step S1 is a non-blood sample, 10000-.
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