CN111304300B - Method for detecting genome DNA copy number of each microorganism species in sample to be detected - Google Patents

Method for detecting genome DNA copy number of each microorganism species in sample to be detected Download PDF

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CN111304300B
CN111304300B CN201911299156.3A CN201911299156A CN111304300B CN 111304300 B CN111304300 B CN 111304300B CN 201911299156 A CN201911299156 A CN 201911299156A CN 111304300 B CN111304300 B CN 111304300B
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曹德盼
任若通
饶冠华
贾雪峰
蒋智
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Beijing Golden Spoon Gene Technology Co ltd
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Abstract

The invention discloses a method for detecting the total bacterial genome DNA content m in a sample to be detected in a high-throughput mannerbacGenomic DNA content m of respective microorganism speciesiGenomic DNA copy number N of the respective microorganism speciesiAnd the number of cells U of the respective microbial speciesiThe penalty of (1). The method and the qPCR method provided by the invention are adopted to detect the test product, the detected mass concentration of each species genome has extremely high linear correlation under different microorganism compositions and different microorganism nucleic acid ratios, and the method provided by the invention has higher accuracy. The method enables the results obtained by the clinical pathogenic microorganism metagenome NGS detection method to be comparable among different samples, improves the traditional clinical pathogenic microorganism metagenome NGS detection from a qualitative detection method to a quantitative detection method, and has important application value.

Description

Method for detecting genome DNA copy number of each microorganism species in sample to be detected
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a method for detecting the copy number of genome DNA of each microorganism species in a sample to be detected.
Background
At present, the second-generation sequencing microbial detection technology of the metagenome shotgun method is applied to pathogen detection of clinical samples, and mainly comprises a microbial detection method which randomly interrupts nucleic acid in the clinical samples by a physical method, a chemical method or a biological method, constructs a second-generation sequencing library, performs high-throughput sequencing by using a second-generation sequencer, analyzes species sources of sequencing short reads by using a bioinformatics method, and takes the number of the sequencing short reads as a detection index. The method can qualitatively detect the microorganisms, but cannot detect the quantity of the microorganisms, and the detection indexes are not comparable among different samples.
The Quantitative Real-time PCR (Quantitative Real-time PCR) technology is a method for adding fluorescent groups into a PCR reaction system, realizing Real-time monitoring of the whole PCR process through continuous accumulation of fluorescent signals and carrying out Quantitative analysis on unknown samples through a standard curve. The method comprises the following specific steps: (1) diluting standard samples with known concentration in a gradient manner, and detecting the Ct value of each diluted sample by using a real-time fluorescence quantitative PCR method; (2) taking the concentration or copy number of the standard substance as an abscissa and the corresponding Ct value as an ordinate, and drawing a standard curve; (3) and (3) detecting the Ct value of the unknown sample by using a real-time fluorescence quantitative PCR method, and then obtaining the concentration or copy number of the unknown sample according to the standard curve. However, one real-time fluorescence quantitative PCR reaction system can only detect one unknown sample, and the detection of a plurality of unknown samples is time-consuming and labor-consuming, i.e., high-throughput detection cannot be realized.
Disclosure of Invention
The invention aims to quantitatively detect the content m of the total bacterial genome DNA in a sample to be detectedbacGenomic DNA content m of respective microorganism speciesiGenomic DNA copy number N of the respective microorganism speciesiAnd the number of cells U of the respective microbial speciesi。
The invention firstly protects a method for detecting the content m of total bacterial genome DNA in a sample to be detectedbacThe method of (c), may comprise step (a) and step (b):
the step (a) includes the steps of:
(a-1) obtaining the types of bacteria which are usually contained according to the source of a sample to be detected, selecting a representative bacterial strain as a standard strain for each type of bacteria, and forming a standard strain group by each standard strain;
(a-2) comparing the sequences of the 16s rDNA of each standard strain in the standard strain group to obtain a specific DNA fragment; the specific DNA fragment is positioned in a conserved region of each standard strain 16s rDNA, and the homology of the specific DNA fragment and a matching region of each standard strain 16s rDNA is more than 90 percent (such as 90 to 95 percent, 95 to 100 percent, 90 percent, 95 percent or 100 percent);
(a-3) mixing the total DNA of each standard strain in the standard strain group to obtain the total DNA of the standard strain group; then diluting to obtain standard total DNA standard products of standard bacterial flora with different concentrations;
(a-4) carrying out fluorescence quantitative PCR on the standard total DNA standard substance of the standard bacterial strain group with different concentrations, and collecting corresponding Ct values; the target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment;
(a-5) drawing a standard curve by taking the concentration of the standard total DNA standard substance of the standard bacterial strain group as an abscissa and the corresponding Ct value as an ordinate;
the step (b) comprises the steps of:
(b-1) carrying out fluorescence quantitative PCR on the total DNA of the sample to be detected, and collecting a Ct value; the target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment;
(b-2) substituting the Ct value collected in the step (b-1) into the standard curve to obtain the concentration of the total bacterial genome DNA in the sample to be detected;
(b-3) obtaining the content m of the total bacterial genome DNA in the sample to be detected according to the concentration of the total bacterial genome DNA in the sample to be detectedbac。
The invention also provides a method for detecting the content mi of the genomic DNA of each microorganism species in a sample to be detected, which comprises the following steps (a), (b), (c) and (d):
the step (a) includes the steps of:
(a-1) obtaining the types of bacteria which are usually contained according to the source of a sample to be detected, selecting a representative bacterial strain as a standard strain for each type of bacteria, and forming a standard strain group by each standard strain;
(a-2) comparing the sequences of the 16s rDNA of each standard strain in the standard strain group to obtain a specific DNA fragment; the specific DNA fragment is positioned in a conserved region of each standard strain 16s rDNA, and the homology of the specific DNA fragment and a matching region of each standard strain 16s rDNA is more than 90 percent (such as 90 to 95 percent, 95 to 100 percent, 90 percent, 95 percent or 100 percent);
(a-3) mixing the total DNA of each standard strain in the standard strain group to obtain the total DNA of the standard strain group; then diluting to obtain standard total DNA standard products of standard bacterial flora with different concentrations;
(a-4) carrying out fluorescence quantitative PCR on the standard total DNA standard substance of the standard bacterial strain group with different concentrations, and collecting corresponding Ct values; the target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment;
(a-5) drawing a standard curve by taking the concentration of the standard total DNA standard substance of the standard bacterial strain group as an abscissa and the corresponding Ct value as an ordinate;
the step (b) comprises the steps of:
(b-1) carrying out fluorescence quantitative PCR on the total DNA of the sample to be detected, and collecting a Ct value; the target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment;
(b-2) substituting the Ct value collected in the step (b-1) into the standard curve to obtain the concentration of the total bacterial genome DNA in the sample to be detected;
(b-3) obtaining the content m of the total bacterial genome DNA in the sample to be detected according to the concentration of the total bacterial genome DNA in the sample to be detectedbac；
The step (c): carrying out metagenome second-generation sequencing on the total DNA of the sample to be detected to obtain the microorganism species contained in the sample to be detected and the genome DNA composition proportion P thereofi；
The step (d): the content m of the total bacterial genome DNA in a sample to be detectedbacAnd the genomic DNA composition ratio P of each microorganism species in the sample to be testediRespectively substituting the formula I to obtain the genome DNA content m of each microorganism species in the sample to be detectedi；
Figure BDA0002321417210000031
The method comprises the following steps of (1) finding a total of n microbial species in a sample to be detected, wherein n is a natural number more than 1; bacterial species marker numberIs [1, m ]](ii) a The genomic DNA composition ratio of the microbial species i is Pi。
The invention also provides a method for detecting the genome DNA copy number N of each microorganism species in a sample to be detectediThe method of (a), comprising step (a), step (b), step (c) and step (e):
the step (a) includes the steps of:
(a-1) obtaining the types of bacteria which are usually contained according to the source of a sample to be detected, selecting a representative bacterial strain as a standard strain for each type of bacteria, and forming a standard strain group by each standard strain;
(a-2) comparing the sequences of the 16s rDNA of each standard strain in the standard strain group to obtain a specific DNA fragment; the specific DNA fragment is positioned in a conserved region of each standard strain 16s rDNA, and the homology of the specific DNA fragment and a matching region of each standard strain 16s rDNA is more than 90 percent (such as 90 to 95 percent, 95 to 100 percent, 90 percent, 95 percent or 100 percent);
(a-3) mixing the total DNA of each standard strain in the standard strain group to obtain the total DNA of the standard strain group; then diluting to obtain standard total DNA standard products of standard bacterial flora with different concentrations;
(a-4) carrying out fluorescence quantitative PCR on the standard total DNA standard substance of the standard bacterial strain group with different concentrations, and collecting corresponding Ct values; the target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment;
(a-5) drawing a standard curve by taking the concentration of the standard total DNA standard substance of the standard bacterial strain group as an abscissa and the corresponding Ct value as an ordinate;
the step (b) comprises the steps of:
(b-1) carrying out fluorescence quantitative PCR on the total DNA of the sample to be detected, and collecting a Ct value; the target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment;
(b-2) substituting the Ct value collected in the step (b-1) into the standard curve to obtain the concentration of the total bacterial genome DNA in the sample to be detected;
(b-3) obtaining the total bacterial genome in the sample to be detected according to the concentration of the total bacterial genome DNA in the sample to be detectedDNA content mbac；
The step (c): carrying out metagenome second-generation sequencing on the total DNA of the sample to be detected to obtain the microorganism species contained in the sample to be detected and the genome DNA composition proportion P thereofi；
The step (e): the content m of the total bacterial genome DNA in a sample to be detectedbacAnd the genomic DNA composition ratio P of each microorganism species in the sample to be testediAnd genome size G of the respective microorganism speciesiRespectively substituting into formula II to obtain the genome DNA copy number N of each microorganism species in the sample to be detectedi；
Figure BDA0002321417210000041
The total number of microorganism species is n, wherein n is a natural number more than 1; bacterial species marker number [1, m](ii) a The genomic DNA composition ratio of the microbial species i is PiThe genome size of the microorganism species i is Gi。
The invention also provides a method for detecting the cell number U of each microorganism species in a sample to be detectediThe method of (a), comprising step (a), step (b), step (c) and step (f):
the step (a) includes the steps of:
(a-1) obtaining the types of bacteria which are usually contained according to the source of a sample to be detected, selecting a representative bacterial strain as a standard strain for each type of bacteria, and forming a standard strain group by each standard strain;
(a-2) comparing the sequences of the 16s rDNA of each standard strain in the standard strain group to obtain a specific DNA fragment; the specific DNA fragment is positioned in a conserved region of each standard strain 16s rDNA, and the homology of the specific DNA fragment and a matching region of each standard strain 16s rDNA is more than 90 percent (such as 90 to 95 percent, 95 to 100 percent, 90 percent, 95 percent or 100 percent);
(a-3) mixing the total DNA of each standard strain in the standard strain group to obtain the total DNA of the standard strain group; then diluting to obtain standard total DNA standard products of standard bacterial flora with different concentrations;
(a-4) carrying out fluorescence quantitative PCR on the standard total DNA standard substance of the standard bacterial strain group with different concentrations, and collecting corresponding Ct values; the target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment;
(a-5) drawing a standard curve by taking the concentration of the standard total DNA standard substance of the standard bacterial strain group as an abscissa and the corresponding Ct value as an ordinate;
the step (b) comprises the steps of:
(b-1) carrying out fluorescence quantitative PCR on the total DNA of the sample to be detected, and collecting a Ct value; the target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment;
(b-2) substituting the Ct value collected in the step (b-1) into the standard curve to obtain the concentration of the total bacterial genome DNA in the sample to be detected;
(b-3) obtaining the content m of the total bacterial genome DNA in the sample to be detected according to the concentration of the total bacterial genome DNA in the sample to be detectedbac；
The step (c): carrying out metagenome second-generation sequencing on the total DNA of the sample to be detected to obtain the microorganism species contained in the sample to be detected and the genome DNA composition proportion P thereofi；
The step (f): the content m of the total bacterial genome DNA in a sample to be detectedbacAnd the genomic DNA composition ratio P of each microorganism species in the sample to be testediGenome size G of respective microorganism speciesiAnd ploidy D of the respective microorganism speciesiRespectively substituting the microorganism species with the formula III to obtain the cell number U of each microorganism species in the sample to be detectedi；
Figure BDA0002321417210000051
The total number of microorganism species is n, wherein n is a natural number more than 1; bacterial species marker number [1, m](ii) a The genomic DNA composition ratio of the microbial species i is PiThe genome size of the microorganism species i is Gi(ii) a Ploidy of microbial species i is Di。
In any of the above steps (a-3), the dilution may be gradient dilution or non-gradient dilution. The gradient dilution may be a 2-20 fold (e.g., 2-10 fold, 10-20 fold, 2 fold, 10 fold, or 20 fold) gradient dilution.
The target of the primer for performing the fluorescent quantitative PCR in any one of the above-mentioned steps (a-4) may be the same as or different from the target of the primer for performing the fluorescent quantitative PCR in any one of the above-mentioned steps (b-1). When the target of the primer for performing the fluorescent quantitative PCR in any one of the above-mentioned steps (a-4) is the same as the target of the primer for performing the fluorescent quantitative PCR in any one of the above-mentioned steps (b-1), the primers for performing the fluorescent quantitative PCR may be the same or different. In the present example, the target of the primer for performing the quantitative fluorescence PCR in step (a-4) is the same as the target of the primer for performing the quantitative fluorescence PCR in step (b-1), and the primer for performing the quantitative fluorescence PCR is also the same.
In any one of the above steps (a-5), the abscissa is the concentration of the standard bacterial population total DNA standard, and specifically, the abscissa is the logarithm of the concentration of the standard bacterial population total DNA standard. In the embodiment of the present invention, the logarithmic value of the concentration of the standard strain group total DNA standard may specifically be a logarithmic value with the concentration of the standard strain group total DNA standard being base 10.
Any one of the above steps (c) comprises the following steps in sequence:
(c-1) taking the total DNA of a sample to be detected, and constructing a metagenome next-generation sequencing library;
(c-2) taking a metagenome next-generation sequencing library, and sequencing to obtain sequencing data;
(c-3) analyzing the sequencing data to obtain the microorganism species contained in the sample to be detected and the genomic DNA composition ratio P thereofi。
In the step (c-1), the construction of the metagenome next-generation sequencing library can be carried out by adopting a commercial kit, and the construction method is carried out according to the instruction of the kit. In the embodiment of the invention, a human pathogenic nucleic acid detection kit (Beijing gold spoon gene) is specifically adopted to construct a metagenome second-generation sequencing library of a sample.
In the step (c-2), "taking the metagenome second-generation sequencing library, sequencing and obtaining sequencing data" may be: and sequencing the metagenome second-generation sequencing library by using a sequencer, and then exporting off-line data according to the specification of the sequencer. The sequencer may specifically be the illuminaintextseq 500CN sequencer. The sequencing strategy during sequencing was SE75, the Index strategy was paired, and the sequencing amount was 20M reads.
In the step (c-3), "analyzing the sequencing data" to obtain the microorganism species contained in the sample to be tested and the genomic DNA composition ratio P thereofiThe method can be used for analyzing the data of a computer computing platform which selects a GNU/Linux operating system and is provided with biological information analysis tools such as trimmatic, bwa, samtools, NCBI-blast +, MEGAN and the like to obtain the microorganism species contained in the sample to be detected and the genomic DNA composition ratio P of the microorganism speciesi. Selecting a GNU/Linux operating system and a computer computing platform provided with a trimmatic tool, a bwa tool, a samtools tool, an NCBI-blast tool, a MEGAN tool and other biological information analysis tools to analyze the downloaded data, wherein the specific steps can be as follows in sequence:
1. and (5) performing quality control on the data by using a trimmatic tool and default parameters, and outputting clearreads.
2. Clean reads were host filtered using bwa tools, default parameters, with the reference genome being human genome version b 38.
3. Using samtools, the parameter "view-f 4", derives reads that do not align to the human genome version b38, and outputs microbe-reads.
4. Using NCBI-blast + tool, parameter "-m 8", comparing database as nt, carrying out species identification on micro-reads, and outputting blast result in tabular format.
5. Using the MEGAN tool, default parameters, blast results were imported into the analysis, outputting a species list of "microbial species id number-reads number".
6. The ratio of the number of reads of each microorganism species to the sum of the numbers of reads of each microorganism species was calculated using the Microsoft excel tool to obtain the genomic DNA composition ratio P of each microorganism speciesiTo obtain
Figure BDA0002321417210000061
And (4) matrix.
Any of the microorganisms described above may include bacteria, fungi and/or viruses.
Any of the above microorganisms may specifically consist of bacteria, fungi and/or viruses.
Any of the test samples described above may be a clinical sample. The clinical sample may specifically be bronchoalveolar lavage fluid.
In any of the above methods, when the sample to be tested is a clinical sample, the standard bacterial strain group may be the standard bacterial strain group 1; the standard strain group 1 comprises one or more of Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Staphylococcus epidermidis, Haemophilus influenzae, Ralstonia insoleosa and/or Escherichia coli.
In the embodiment of the present invention, when the sample to be tested is a clinical sample, the standard bacterial strain group 1 may specifically consist of strains of acinetobacter baumannii, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae, streptococcus pneumoniae, staphylococcus epidermidis, haemophilus influenzae, Ralstonia insoleosa, and/or escherichia coli.
In any of the above methods, when the sample to be tested is bronchoalveolar lavage fluid, the standard bacterial strain group can be standard bacterial strain group 2; standard strain group 2 may comprise one or more strains of Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Escherichia coli and/or Haemophilus influenzae.
In the embodiment of the present invention, when the sample to be tested is bronchoalveolar lavage fluid, the standard bacterial strain group 2 may specifically consist of strains of acinetobacter baumannii, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae and/or streptococcus pneumoniae.
The Acinetobacter baumannii can be at least one of drug-resistant Acinetobacter baumannii (Acinetobacter baumannii) ATCCBA-2800, multi-drug-resistant Acinetobacter baumannii (Acinetobacter baumannii Multidrug-resistant), pan-drug-resistant Acinetobacter baumannii-resistant, Acinetobacter baumannii (Acinetobacter baumannii) ATCC19606 and Acinetobacter baumannii-BNCC-337173-standard strains.
The Staphylococcus aureus can be at least one of Staphylococcus aureus (ATCC 6538), Staphylococcus aureus (ATCC 22004) and Staphylococcus aureus-ATCC 25923-standard strains.
The Pseudomonas aeruginosa can be at least one of Pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC 10145 and Pseudomonas aeruginosa standard strain ATCC 9027.
The Klebsiella pneumoniae can be at least one of Klebsiella pneumoniae (Klebsiella pneumoniae) ATCCBA-2783, Klebsiella pneumoniae CICC 10072 and Klebsiella pneumoniae (Klebsiella pneumoniae) LCT-KP182 strains.
The Streptococcus pneumoniae may be at least one of Streptococcus pneumoniae (Streptococcus pneumoniae) ATCC 49619 and Streptococcus pneumoniae-ZKCC-550.
The Staphylococcus epidermidis can be at least one of Staphylococcus epidermidis (Staphylococcus epidermidis) ATCC 12228, Staphylococcus epidermidis standard strain CMCC (B)26069 and Staphylococcus epidermidis NRRLY-2332.
The Haemophilus influenzae can be Haemophilus influenzae (Haemophilus influenzae) ATCC 49247.
The Ralstonia inertiosa may specifically be Ralstonia inertiosa ATCC 49129.
The Escherichia coli may be specifically at least one of Escherichia coli (Escherichia coli) ATCC 25909, EHEC enterohemorrhagic 0157 and EIEC invasive Escherichia coli.
The content m of the total bacterial genome DNA of any one of the standard bacterial strain group 1 in a clinical sample is detectedbacGenomic DNA content m of each microbial species in clinical samplesiGenomic DNA copy number N of individual microbial species in clinical samplesiOr the number of cells U of each microbial species in the clinical sampleiAlso belongs to the protection of the inventionAnd (4) protecting the scope.
The content m of the total bacterial genome DNA of any one of the standard bacterial strain groups 2 in the detection of bronchoalveolar lavage fluidbacThe genomic DNA content m of the respective microbial species in bronchoalveolar lavage fluidiGenomic DNA copy number N of individual microbial species in bronchoalveolar lavage fluidiOr the number of cells of each microbial species U in bronchoalveolar lavage fluidiAlso belongs to the protection scope of the invention.
The method provided by the invention can be used for detecting the total bacterial genome DNA content m in a sample to be detected in a high-throughput mannerbacGenomic DNA content m of respective microorganism speciesiGenomic DNA copy number N of the respective microorganism speciesiAnd the number of cells U of the respective microbial speciesi. The method and the qPCR method provided by the invention are adopted to detect the test product, the detected mass concentration of each species genome has extremely high linear correlation under different microorganism compositions and different microorganism nucleic acid ratios, and the method provided by the invention has higher accuracy. The method is applied to the detection of the clinical pathogenic microorganism metagenome NGS, can accurately calibrate the genome content, the genome DNA copy number and the cell number of each microorganism species in a sample to be detected, enables the results obtained by the detection method of the clinical pathogenic microorganism metagenome NGS to have comparability among different samples, improves the traditional detection method of the clinical pathogenic microorganism metagenome NGS from a qualitative detection method into a quantitative detection method, and has important application value.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The quantitative tests in the following examples, all set up three replicates and the results averaged.
In the following examples, the names, suppliers and product numbers of some of the test reagents are shown in Table 1.
TABLE 1
Figure BDA0002321417210000081
Figure BDA0002321417210000091
In the following examples, the genome size G of the microbial speciesiCan be inquired and obtained on a website (the website address is https:// www.ncbi.nlm.nih.gov/genome /), and the ploidy D of the microorganism speciesiCan be obtained by inquiring on a website (the website address is https:// www.ncbi.nlm.nih.gov/genome /).
In the following examples, the UCP DNA Micro Kit is used to extract total DNA of a sample, and the protease K, Buffer AUL, QIAamp UCP MinElute spin column, Buffer AUW1, Buffer AUW2 and Buffer AUE are all components in the UCP DNA Micro Kit, and the specific steps are as follows:
1. slowly thawing the sample on ice, and slightly swirling and mixing uniformly;
2. after step 1, 6 centrifuge tubes (specification 1.5mL) are taken, 100 muL of bronchoalveolar lavage fluid sample, 10 muL of protease K and 100 muL of Buffer AUL are added into each centrifuge tube, and vortex is carried out for 15 s;
3. after the step 2 is finished, taking the centrifuge tube, carrying out warm bath at 56 ℃ for 10min, and slightly centrifuging (aiming at collecting the liquid to the bottom of the tube);
4. after step 3 is completed, adding 50 μ L of ethanol into each centrifuge tube, mixing uniformly by vortexing for 15s, then incubating for 3min at room temperature, and slightly centrifuging (collecting the liquid to the bottom of the tube);
5. after completion of step 4, the systems in the centrifuge tubes were carefully transferred to 6 QIAamp UCP MinElute spin columns, respectively, and centrifuged at 6000g for 1 min;
6. after step 5, transferring the column to a new centrifuge tube, adding 500. mu.L Buffer AUW1 into the column, and centrifuging at 6000g for 1 min;
7. after step 6, transferring the column to a new centrifuge tube, adding 500. mu.L Buffer AUW2 into the column, and centrifuging at 6000g for 1 min;
8. after step 7, transferring the centrifuge column to a new centrifuge tube, and centrifuging for 3min at 20000 g;
9. after step 8, transferring the column to a new centrifuge tube, adding 25 μ L Buffer AUE into the column, incubating at room temperature for 1min, centrifuging at 20000g for 3min, and collecting the eluate;
10. after step 9 is completed, another new centrifugal column is taken and moved to a new centrifugal tube, then the eluent collected in step 9 is added into the centrifugal column, the mixture is incubated for 1min at room temperature, and then is centrifuged for 3min at 20000g, and the eluent is collected, wherein the eluent is the total DNA of the sample.
In the following examples, a human pathogenic nucleic acid detection kit is used to construct a metagenome second-generation sequencing library of a sample, and 10 × Fragmentation Buffer, 5 × WGS Fragmentation Mix, Enhance, Buffer EB, index adaptor, 5 × Ligation Buffer, DNA ligand and DNA Clean Beads are all components in the human pathogenic nucleic acid detection kit, and the specific steps are as follows:
1. preparing a reaction system 1; the reaction system 1 was 15.25. mu.L, consisting of 10. mu.L of total DNA of the sample, 1.5. mu.L of 10 Xfragmentation Buffer, 3. mu.L of 5 XWGS Fragmentation Mix, and 0.75. mu.L of LENhance;
2. placing the PCR tube filled with the reaction system 1 in a PCR instrument for reaction to obtain a terminal repair product;
the reaction procedure is as follows: 1min at 4 ℃; 35min at 32 ℃; 30min at 65 ℃; storing at 4 deg.C;
3. diluting an index joint (with the concentration of 10 mu M) by using Buffer EB to obtain a joint diluent;
if the sample of bronchoalveolar lavage fluid is more than 100ng, the index joint is diluted by 4 times, namely the concentration of the joint diluent is 2.5 mu M; if the bronchoalveolar lavage fluid sample is less than 100ng, the index linker is diluted by 10 times, i.e. the concentration of the linker diluent is 1 μ M;
4. preparing a reaction system 2; the reaction system 2 is 30 μ L, and consists of 10 μ L of the end repair product, 6 μ L of 5 × Ligation Buffer, 3 μ L of LDNA ligase, 5 μ L of linker diluent and 1 μ L of LBuffer EB;
5. placing the PCR tube with the reaction system 2 in a PCR instrument for reaction to obtain a joint connection reaction product;
the reaction procedure is as follows: 15min at 20 ℃; storing at 4 deg.C;
6. taking 30 mu L of the joint connection reaction product, adding 20 mu L of Buffer EB and 40 mu L of LDNA Clean Beads, performing vortex oscillation or gently blowing and beating for 10 times by using a pipettor, fully and uniformly mixing, and incubating for 5min at room temperature;
7. the PCR tube that completed step 6 was placed in a magnetic rack and after the solution cleared (about 5min), the supernatant was carefully removed;
8. after step 7 was completed, the following steps were repeated 2 times: keeping the PCR tube on a magnetic frame, adding 200 mu L of 80% (v/v) ethanol aqueous solution to rinse magnetic beads (namely AMPure XP beads), carefully moving the PCR tube to the opposite side of the magnetic strip, incubating at room temperature for 30s, carefully removing supernatant after the magnetic beads are completely moved to the tube wall close to the magnetic strip (note: if the magnetic beads cannot be moved, blowing and beating the magnetic beads for several times until the magnetic beads are completely separated from the original positions, standing until the alcohol is clarified, completely moving the magnetic beads to the tube wall close to the magnetic strip, and then removing the supernatant);
9. after the step 8 is finished, keeping the PCR tube in a magnetic frame, and opening the cover to naturally dry for 5-10 min;
10. after step 9, taking out the PCR tube from the magnetic frame, adding 23 μ L of Buffer EB for elution, performing vortex oscillation or gently blowing and beating by using a pipette to fully mix the mixture, standing the mixture at room temperature for 5min, centrifuging the PCR tube for a short time, placing the PCR tube on the magnetic frame for standing, carefully sucking about 20 μ L of supernatant into a new PCR tube after the solution is clarified (about 5min), and avoiding touching magnetic beads;
and the supernatant in the PCR tube is the metagenome second-generation sequencing library of the sample.
In the following embodiments, a computer computing platform with a GNU/Linux operating system and installed with biological information analysis tools such as trimmatic, bwa, samtools, NCBI-blast +, MEGAN, etc. is used to analyze the following computer data, and the specific steps are as follows:
1. and (5) performing quality control on the data by using a trimmatic tool and default parameters, and outputting clearreads.
2. Clean reads were host filtered using bwa tools, default parameters, with the reference genome being human genome version b 38.
3. Using samtools, the parameter "view-f 4", derives reads that do not align to the human genome version b38, and outputs microbe-reads.
4. Using NCBI-blast + tool, parameter "-m 8", comparing database as nt, carrying out species identification on micro-reads, and outputting blast result in tabular format.
5. Using the MEGAN tool, default parameters, blast results were imported into the analysis, outputting a species list of "microbial species id number-reads number".
6. The ratio of the number of reads of each microorganism species to the sum of the numbers of reads of each microorganism species was calculated using the Microsoft excel tool to obtain the genomic DNA composition ratio P of each microorganism speciesiTo obtain
Figure BDA0002321417210000111
And (4) matrix.
Genomic DNA content m of each microorganism species in the samples in the examples described belowiThe calculation formula of (2) is as follows:
Figure BDA0002321417210000112
the total number of microorganism species is n, wherein n is a natural number more than 1; bacterial species marker number [1, m](ii) a The genomic DNA composition ratio of the microbial species i is Pi。
Genomic DNA copy number N of each microbial species in the samples in the examples belowiThe calculation formula of (2) is as follows:
Figure BDA0002321417210000113
the total number of microorganism species is n, wherein n is a natural number more than 1; bacterial species marker number [1, m](ii) a The genomic DNA composition ratio of the microbial species i is PiThe genome size of the microorganism species i is Gi。
The number of cells U of each microorganism species in the samples in the examples described belowiThe calculation formula of (2) is as follows:
Figure BDA0002321417210000114
the total number of microorganism species is n, wherein n is a natural number more than 1; bacterial species marker number [1, m](ii) a The genomic DNA composition ratio of the microbial species i is PiThe genome size of the microorganism species i is Gi(ii) a Ploidy of microbial species i is Di。
Example 1 detection of Total bacterial genomic DNA content m in test samplesbacAnd the genome content m of each microorganism species in the sample to be testediGenome copy number NiAnd cell number (number of viral particles) UiEstablishment of the method
The inventor establishes the content m of the total bacterial genome DNA in a sample to be detected through a large number of experimentsbacAnd the genome content m of each microorganism species in the sample to be testediGenome copy number NiAnd cell number (number of viral particles) UiThe method comprises the following specific steps:
1. and extracting the total DNA of the sample to be detected by adopting a UCP DNA Micro Kit.
2. And (3) after the step (1) is completed, taking the total DNA of the sample to be detected, and constructing a metagenome next-generation sequencing library of the sample to be detected by adopting a human pathogenic nucleic acid detection kit.
3. After the step 2 is completed, taking a metagenome second-generation sequencing library of a sample to be tested, and sequencing by adopting an IlluminaNextseq500CN sequencer, wherein a sequencing strategy selects SE75, an Index strategy selects double ends, and the sequencing quantity is 20M reads; the off-machine data was derived according to the instructions of the illuminainnextseq 500CN sequencer; selecting a GNU/Linux operating system and a computer computing platform provided with a trimmatic, bwa, samtools, NCBI-blast +, MEGAN and other biological information analysis tools to analyze the off-line data, and obtaining the microbial species contained in the sample to be detected and the genomic DNA composition ratio P of the microbial speciesi。
4. Drawing of real-time fluorescence quantitative PCR standard curve of total DNA of bacteria
(1) Obtaining commonly-contained bacterial species according to the source of a sample to be detected, selecting a representative bacterial strain as a standard strain for each bacterial species, and forming a standard strain group by each standard strain; and then, extracting the total DNA of each standard strain in the standard strain group by adopting a UCP DNA Micro Kit.
Comparing the sequences of the standard strains 16s rDNA in the standard strain group to obtain a specific DNA fragment; the specific DNA fragment is positioned in a conserved region of each standard strain 16s rDNA and has homology of more than 90 percent with a matching region of each standard strain 16s rDNA.
(2) Mixing the total DNA of each standard strain to obtain the total DNA of a standard strain group; and then carrying out gradient dilution to obtain the standard total DNA standard products of the standard bacterial strain groups with different concentrations.
(3) And (3) taking the standard total DNA standard substance of the standard bacterial flora with different concentrations, carrying out fluorescence quantitative PCR, and collecting corresponding Ct values. The target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment.
(4) And (3) taking the concentration logarithm value of the total DNA standard substance of the standard bacterial strain group as an abscissa and the corresponding Ct value as an ordinate, and drawing a real-time fluorescence quantitative PCR standard curve of the total DNA of the bacteria.
5. Taking the total DNA of a sample to be detected, carrying out fluorescence quantitative PCR (the primer for carrying out the fluorescence quantitative PCR is completely the same as the primer for carrying out the fluorescence quantitative PCR in the step (3) in the step 4), and collecting a Ct value; substituting the Ct value into a bacterial total DNA real-time fluorescence quantitative PCR standard curve to obtain the concentration of total bacterial genome DNA in a sample to be detected; further obtaining the total bacterial genome DNA content m in the sample to be detectedbac。
6. The content m of the total bacterial genome DNA in a sample to be detectedbacAnd the microorganism species contained in the sample to be detected obtained in the step 3 and the genomic DNA composition ratio P thereofiRespectively substituting the formula I to obtain the genome content m of each microorganism species in the sample to be detectedi。
7. The content m of the total bacterial genome DNA in a sample to be detectedbacAnd 3, the microorganism species contained in the sample to be detected obtained in the step 3 and the genomic DNA composition proportion P thereofiAnd genome size G of the respective microorganism speciesiRespectively substituting the genes in the formula II to obtain the genome of each microorganism species in the sample to be detected.
8. The content m of the total bacterial genome DNA in a sample to be detectedbacAnd 3, the microorganism species contained in the sample to be detected obtained in the step 3 and the genomic DNA composition proportion P thereofiGenome size G of respective microorganism speciesiAnd ploidy D of the respective microorganism speciesiRespectively substituting the formula III to obtain the cell number (virus particle number) U of each microorganism species in the sample to be detectedi。
Example 2 detection of Total bacterial genomic DNA content m in bronchoalveolar lavage fluid Using the method established in example 1bacThe genome content m of each microbial species in bronchoalveolar lavage fluidiGenome copy number NiAnd cell number (number of viral particles) Ui
First, obtaining total DNA of bronchoalveolar lavage fluid
The provider of bronchoalveolar lavage fluid in this example had informed consent.
Taking 600 mu L of bronchoalveolar lavage fluid (namely a sample), and extracting total DNA by using a UCP DNA Micro Kit to obtain the total DNA of the bronchoalveolar lavage fluid.
A total of 20. mu.L of eluate was collected. The total DNA concentration of bronchoalveolar lavage fluid was determined to be 5.3 ng/. mu.L.
Construction of metagenome next-generation sequencing library of bronchoalveolar lavage fluid
Taking total DNA of the bronchoalveolar lavage fluid, and adopting a human pathogenic nucleic acid detection kit to construct a metagenomic next-generation sequencing library of the bronchoalveolar lavage fluid.
Third, computer sequencing and metagenome second generation sequencing data analysis
1. Sequencing on machine
(1) The second generation of metagenomic sequencing library of bronchoalveolar lavage fluid was sequenced using illumina nextseq500CN sequencer. The sequencing strategy selected SE75, the Index strategy selected both ends, and the sequencing amount was 20M reads.
(2) After completion of step (1), the off-line data was derived according to the instructions of the illuminainnextseq 500CN sequencer.
The result shows that in the metagenome second-generation sequencing library of the bronchoalveolar lavage fluid, the number of reads obtained by on-machine sequencing is 21746813, and the proportion of the reads from human sources in off-machine data is 99.99%.
2. Data analysis
Selecting a GNU/Linux operating system and installing a computer computing platform of biological information analysis tools such as trimmatic, bwa, samtools, NCBI-blast +, MEGAN and the like to analyze the data of the computer.
The results show that the microbial species contained in the bronchoalveolar lavage fluid and the genomic DNA composition ratio P thereofiAs shown in table 2, column 6.
TABLE 2
Figure BDA0002321417210000141
Fourthly, drawing the real-time fluorescence quantitative PCR standard curve of the total DNA of the bacteria
1. Since clinical bronchoalveolar lavage fluid samples often contain acinetobacter baumannii, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae, and streptococcus pneumoniae, the respective strains in table 3 were selected as standard strains for each bacterium, and a standard strain group was composed of the respective standard strains.
TABLE 3
Figure BDA0002321417210000142
2. After the step 1 is completed, the UCP DNA Micro Kit is adopted to respectively extract the total DNA of the standard strains shown in the table 3, and then a Qubit instrument is used to perform operation according to the instruction of the Qubit dsDNA HS Assay Kit, so as to respectively determine the concentration of the total DNA of each standard strain. The results are shown in Table 4.
TABLE 4
Figure BDA0002321417210000143
Figure BDA0002321417210000151
3. After the step 2 is completed, the centrifuge tube is taken out, the total DNA of each standard strain is added according to the taking amount in the table 4, the Buffer AUE is used for complementing the total DNA to 50 mu L, and the mixture is slightly swirled to obtain the total DNA1 of the mixed strain group with the concentration of 1000 pg/mu L. The concentration of the total DNA of each standard strain in the mixed bacterial flora total DNA1 was 200 pg/. mu.L.
The total DNA1 of the mixed strain group is taken and diluted by Buffer AUE in a 10-fold gradient manner, and the total DNA2 of the mixed strain group with the concentration of 100 pg/mu L, the total DNA3 of the mixed strain group with the concentration of 10 pg/mu L, the total DNA4 of the mixed strain group with the concentration of 1 pg/mu L, the total DNA5 of the mixed strain group with the concentration of 0.1 pg/mu L and the total DNA6 of the mixed strain group with the concentration of 0.01 pg/mu L are obtained in sequence.
4. Taking the total DNA of the mixed strain group (the total DNA1 of the mixed strain group, the total DNA2 of the mixed strain group, the total DNA3 of the mixed strain group, the total DNA4 of the mixed strain group, the total DNA5 of the mixed strain group or the total DNA6 of the mixed strain group), preparing a reaction system according to the instruction of a Femto Bacterial DNA Quantification Kit, then placing the reaction system in a fluorescent quantitative PCR instrument, setting a PCR program according to the instruction of the Kit, carrying out fluorescent quantitative PCR and collecting the Ct value.
And replacing the total DNA of the mixed bacterial flora with sterilized double distilled water, and taking the obtained product as a negative control, wherein the obtained product is unchanged in other steps.
The total DNA of the mixed Bacterial strain group is replaced by a PCR amplicon positive product (a component in a Femto Bacterial DNA Quantification Kit), and other steps are not changed and are used as positive control.
5. And (3) taking the concentration logarithm value of the total DNA of the mixed bacterial flora as an abscissa and the corresponding Ct value as an ordinate, and drawing a real-time fluorescence quantitative PCR standard curve of the total DNA of the bacteria.
The result shows that the real-time fluorescence quantitative PCR standard curve of the total DNA of the bacteria is as follows: y ═ 3.567logX + 25.437.
Fifthly, the total bacterial genome DNA content m in the bronchoalveolar lavage fluidbacThe genomic DNA content m of the respective microbial species in bronchoalveolar lavage fluidiGenomic DNA copy number NiAnd thinNumber of cells (number of viral particles) UiMeasurement of (2)
1. Total bacterial genome DNA content m in bronchoalveolar lavage fluidbacMeasurement of (2)
(1) Taking 1 mu L of total DNA of the bronchoalveolar lavage fluid obtained in the first step, preparing a reaction system according to the instruction of a Femto Bacterial DNA Quantification Kit, then placing the reaction system in a fluorescent quantitative PCR instrument, setting a PCR program according to the instruction of the Kit, carrying out fluorescent quantitative PCR, and collecting a Ct value.
(2) Substituting the Ct value collected in the step (1) into the bacterial total DNA real-time fluorescence quantitative PCR standard curve drawn in the step four to obtain the bacterial total DNA concentration of the bronchoalveolar lavage fluid; multiplying the total bacterial DNA concentration of the bronchoalveolar lavage fluid by the elution volume to obtain the total bacterial genome DNA content m in the bronchoalveolar lavage fluidbac。
The results showed that the bacterial total DNA concentration of bronchoalveolar lavage fluid was 133.267pg/μ L, and the total bacterial genomic DNA content of bronchoalveolar lavage fluid was mbac2665.34 pg.
2. Genome content m of each microbial species in bronchoalveolar lavage fluidi
The total bacterial genome DNA content m in the bronchoalveolar lavage fluidbacAnd the microbial species contained in the bronchoalveolar lavage fluid obtained in the step three and the genomic DNA composition ratio P thereofiRespectively substituting the formula I to obtain the genome DNA content m of each microorganism species in the bronchoalveolar lavage fluidi。
The results are shown in column 5 of Table 5.
TABLE 5
Figure BDA0002321417210000161
3. Genomic DNA copy number N of individual microbial species in bronchoalveolar lavage fluidi
The total bacterial genome DNA content m in the bronchoalveolar lavage fluidbacAnd step three, the microbial species contained in the bronchoalveolar lavage fluid obtained in the step threeAnd the genomic DNA composition ratio P thereofiAnd genome size G of the respective microorganism speciesiRespectively substituting into formula II to obtain genome DNA copy number N of each microorganism species in bronchoalveolar lavage fluidi。
The results are shown in column 6 of Table 5.
4. Cell number (viral particle number) U of each microbial species in bronchoalveolar lavage fluidi
The total bacterial genome DNA content m in the bronchoalveolar lavage fluidbacAnd the microbial species contained in the bronchoalveolar lavage fluid obtained in the step three and the genomic DNA composition proportion P of the microbial speciesiGenome size G of respective microorganism speciesiAnd ploidy D of the respective microorganism speciesiRespectively substituting into formula III to obtain the cell number (virus particle number) U of each microorganism species in the bronchoalveolar lavage fluidi。
The results are shown in Table 5, column 7.
Example 3, example 1 accuracy test of the established method
Preparation of test article
Staphylococcus epidermidis, Acinetobacter baumannii, Candida albicans and EB virus are all ATCC products with the product numbers 12228, 19606, 14053 and VR-1492 respectively.
1. And respectively extracting total DNA of staphylococcus epidermidis, acinetobacter baumannii, candida albicans and EB virus by adopting a UCP DNA Micro Kit.
2. The mass concentrations of total DNA of Staphylococcus epidermidis, total DNA of Acinetobacter baumannii, total DNA of Candida albicans, total DNA of EB virus and host nucleic acid were determined using the Qubit dsDNA HS Assay Kit with reference to the Qubit instrument instructions. The host nucleic acid is human nucleic acid.
As a result, the concentration of total DNA of Staphylococcus epidermidis was 2.96 ng/. mu.L, that of Acinetobacter baumannii was 7.36 ng/. mu.L, that of Candida albicans was 0.498 ng/. mu.L, that of Epstein-Barr virus was 1e5 copy/. mu.L, and that of human nucleic acid was 7.74 ng/. mu.L.
3. After completion of step 2, total DNA of Staphylococcus epidermidis, total DNA of Acinetobacter baumannii, total DNA of Candida albicans, total DNA of EB virus and host nucleic acid were mixed to obtain test articles S01-S16 each having a volume of 80. mu.L. Test article S01-the nucleic acid mass ratio/copy number of test article S16 is detailed in Table 6.
TABLE 6
Figure BDA0002321417210000171
Secondly, the concentration of genomic DNA of each microorganism species in 16 samples was determined by the method established in example 1
1. And (4) taking the test product, and extracting the total DNA by adopting a UCP DNA Micro Kit to obtain the total DNA of the test product.
2. And (3) after the step 1 is completed, taking the total DNA of the test article, and constructing a metagenome next-generation sequencing library of the test article by adopting a human pathogenic nucleic acid detection kit.
3. After the step 2 is completed, taking a metagenome second-generation sequencing library of a test article, and sequencing by adopting an IlluminaNextseq500CN sequencer, wherein a sequencing strategy selects SE75, an Index strategy selects double ends, and the sequencing quantity is 20M reads; the off-machine data was derived according to the instructions of the illuminainnextseq 500CN sequencer; selecting a GNU/Linux operating system and installing a computer computing platform of biological information analysis tools such as trimmatic, bwa, samtools, NCBI-blast +, MEGAN and the like to analyze the off-line data, and obtaining the microorganism species contained in the test sample and the genomic DNA composition ratio P thereofi。
4. Drawing of real-time fluorescence quantitative PCR standard curve of total DNA of bacteria
(1) The strains shown in Table 7 were selected as standard strains, and a standard strain group was composed of the respective standard strains. The bacteria corresponding to the standard strains of the standard strain group are common infectious bacteria of clinical samples, so that the standard strain group can detect any clinical sample.
TABLE 7
Figure BDA0002321417210000181
(2) After the step (1) is completed, extracting total DNA of the standard strains shown in the table 7 by using a UCP DNA Micro Kit, and then performing operation by using a Qubit instrument according to the instruction of the Qubit dsDNA HS Assay Kit to determine the concentration of the total DNA of each standard strain respectively.
(3) And (3) after the step (2) is completed, taking a centrifuge tube, adding the total DNA of each standard strain, complementing the total DNA to 50 mu L by Buffer AUE, and slightly vortex mixing to obtain the total DNA1 of the mixed strain group with the concentration of 1000 pg/mu L. The concentration of the total DNA of each standard strain in the mixed bacterial flora total DNA1 was 200 pg/. mu.L.
The total DNA1 of the mixed strain group is taken and diluted by Buffer AUE in a 10-fold gradient manner, and the total DNA2 of the mixed strain group with the concentration of 100 pg/mu L, the total DNA3 of the mixed strain group with the concentration of 10 pg/mu L, the total DNA4 of the mixed strain group with the concentration of 1 pg/mu L, the total DNA5 of the mixed strain group with the concentration of 0.1 pg/mu L and the total DNA6 of the mixed strain group with the concentration of 0.01 pg/mu L are obtained in sequence.
(4) Taking the total DNA of the mixed strain group (the total DNA1 of the mixed strain group, the total DNA2 of the mixed strain group, the total DNA3 of the mixed strain group, the total DNA4 of the mixed strain group, the total DNA5 of the mixed strain group or the total DNA6 of the mixed strain group), preparing a reaction system according to the instruction of a Femto Bacterial DNA Quantification Kit, then placing the reaction system in a fluorescent quantitative PCR instrument, setting a PCR program according to the instruction of the Kit, carrying out fluorescent quantitative PCR and collecting the Ct value.
And replacing the total DNA of the mixed bacterial flora with sterilized double distilled water, and taking the obtained product as a negative control, wherein the obtained product is unchanged in other steps.
The total DNA of the mixed Bacterial strain group is replaced by a PCR amplicon positive product (a component in a Femto Bacterial DNA Quantification Kit), and other steps are not changed and are used as positive control.
(5) And (3) taking the concentration logarithm value of the total DNA of the mixed bacterial flora as an abscissa and the corresponding Ct value as an ordinate, and drawing a real-time fluorescence quantitative PCR standard curve of the total DNA of the bacteria.
The result shows that the real-time fluorescence quantitative PCR standard curve of the total DNA of the bacteria is as follows: y-3.2934 logX + 23.215.
5. Genomic DNA content m of each microorganism species in the test articleiMeasurement of (2)
(1) Total bacterial genome DNA content m in test samplebacMeasurement of (2)
(1-1) preparing a reaction system from 1 mu L of total DNA of a test article according to the instruction of a Femto Bacterial DNA Quantification Kit, placing the reaction system in a fluorescent quantitative PCR instrument, setting a PCR program according to the instruction of the Kit, performing fluorescent quantitative PCR, and collecting a Ct value.
(1-2) substituting the Ct value collected in the step (1-1) into the bacterial total DNA real-time fluorescence quantitative PCR standard curve drawn in the step (4) to obtain the bacterial total DNA concentration of the test sample; multiplying the total bacterial DNA concentration of the test sample by the elution volume to obtain the total bacterial genome DNA content m in the test samplebac。
(2) Genomic DNA content m of each microorganism species in the test articleiMeasurement of (2)
The content m of the total bacterial genome DNA in the test samplebacAnd the microorganism species contained in the test sample obtained in the step 3 and the genomic DNA composition ratio P thereofiRespectively substituting the formula I to obtain the genome DNA content m of each microorganism species in the test samplei. And further obtaining the concentration of the genomic DNA of each microorganism species in the test sample.
The results are shown in columns 3-6 of Table 8.
TABLE 8
Figure BDA0002321417210000191
Figure BDA0002321417210000201
In Table 7, Q16S is the genomic DNA concentration of each microorganism species in each test sample, which was calibrated by the method established in example 1; qPCR is to use qPCR method to calibrate the genomic DNA concentration of each microorganism species in each test sample.
Thirdly, detecting the concentration of the genome DNA of each microorganism species in 16 test samples by adopting a qPCR method
1. Detection of genomic DNA concentration of Staphylococcus epidermidis in test article
(1) Drawing a staphylococcus epidermidis DNA real-time fluorescence quantitative PCR standard curve
(1-1) taking 1 mu L of total DNA of the staphylococcus epidermidis, adding 9 mu L of LBuffer AUE, and uniformly mixing to obtain the total DNA1 of the staphylococcus epidermidis.
(1-2) taking total staphylococcus epidermidis DNA1, and performing 10-fold gradient dilution by using Buffer AUE to obtain staphylococcus epidermidis DNA standard products with different concentrations.
(1-3) taking staphylococcus epidermidis DNA standard products with different concentrations, carrying out fluorescence quantitative PCR in an ABI7500 fluorescence quantitative PCR instrument by adopting a staphylococcus epidermidis nucleic acid determination kit (a fluorescence PCR method) kit (a product of Zhijiang biology company), and collecting corresponding Ct values.
And (1-4) drawing a real-time fluorescence quantitative PCR standard curve of the staphylococcus epidermidis DNA by taking the concentration logarithm value of the staphylococcus epidermidis DNA standard substance as an abscissa and the corresponding Ct value as an ordinate.
(2) Taking total DNA of a test product, performing fluorescent quantitative PCR in an ABI7500 fluorescent quantitative PCR instrument by adopting a staphylococcus epidermidis nucleic acid determination kit (fluorescent PCR method), and collecting a Ct value; substituting the Ct value into a staphylococcus epidermidis DNA real-time fluorescence quantitative PCR standard curve to obtain the concentration of staphylococcus epidermidis genome DNA in the test sample.
2. Detection of genomic DNA concentration of Acinetobacter baumannii in test sample
According to the method of the step 1, the total DNA of the staphylococcus epidermidis is replaced by the total DNA of the acinetobacter baumannii, the staphylococcus epidermidis nucleic acid determination kit (fluorescence PCR method) is replaced by the acinetobacter baumannii nucleic acid detection kit, and the concentration of the acinetobacter baumannii genome DNA in the test sample is obtained without changing other steps.
3. Detection of genomic DNA concentration of Candida albicans in test sample
According to the method of the step 1, the total DNA of the staphylococcus epidermidis is replaced by the total DNA of the candida albicans, the nucleic acid determination kit (fluorescence PCR method) of the staphylococcus epidermidis is replaced by the nucleic acid determination kit (fluorescence PCR method) of the candida albicans, and the concentration of the genomic DNA of the candida albicans in the test sample is obtained without changing other steps.
4. Detection of genomic DNA concentration of EB Virus in test article
Replacing the total DNA of the staphylococcus epidermidis with the total DNA of the EB virus according to the method in the step 1, replacing a nucleic acid determination kit (fluorescence PCR method) of the staphylococcus epidermidis with a nucleic acid amplification (PCR) fluorescence quantitative detection kit of the EB virus, and obtaining the concentration of the EB virus genome DNA in the test sample without changing other steps.
The results are shown in columns 3-6 of Table 8.
Correlation coefficient of Pearson
Linear correlations of the concentration values measured by the Q16S method and the qPCR method in the same sample were calculated using the pearson () function in microsoft excel.
The results are shown in Table 8, column 7.
The results show that the mass concentration of each species genome measured by the method established in example 1 and the qPCR method has extremely high linear correlation under different microorganism compositions and different microorganism nucleic acid ratios. It can be seen that the method established in example 1 has high accuracy in detecting the genomic DNA concentration of each of the microbial species in the 16 samples.

Claims (12)

1. Detecting genome DNA content m of each microorganism species in sample to be detectediThe method of (a), comprising step (a), step (b), step (c) and step (d):
the step (a) includes the steps of:
(a-1) obtaining the types of bacteria which are usually contained according to the source of a sample to be detected, selecting a representative bacterial strain as a standard strain for each type of bacteria, and forming a standard strain group by each standard strain;
(a-2) comparing the sequences of the 16s rDNA of each standard strain in the standard strain group to obtain a specific DNA fragment; the specific DNA fragment is positioned in a conserved region of each standard strain 16s rDNA and has homology of more than 90 percent with a matching region of each standard strain 16s rDNA;
(a-3) mixing the total DNA of each standard strain in the standard strain group to obtain the total DNA of the standard strain group; then diluting to obtain standard total DNA standard products of standard bacterial flora with different concentrations;
(a-4) carrying out fluorescence quantitative PCR on the standard total DNA standard substance of the standard bacterial strain group with different concentrations, and collecting corresponding Ct values; the target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment;
(a-5) drawing a standard curve by taking the concentration of the standard total DNA standard substance of the standard bacterial strain group as an abscissa and the corresponding Ct value as an ordinate;
the step (b) comprises the steps of:
(b-1) carrying out fluorescence quantitative PCR on the total DNA of the sample to be detected, and collecting a Ct value; the target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment;
(b-2) substituting the Ct value collected in the step (b-1) into the standard curve to obtain the concentration of the total bacterial genome DNA in the sample to be detected;
(b-3) obtaining the content m of the total bacterial genome DNA in the sample to be detected according to the concentration of the total bacterial genome DNA in the sample to be detectedbac；
The step (c): carrying out metagenome second-generation sequencing on the total DNA of the sample to be detected to obtain the microorganism species contained in the sample to be detected and the genome DNA composition proportion P thereofi；
The step (d): the content m of the total bacterial genome DNA in a sample to be detectedbacAnd the genomic DNA composition ratio P of each microorganism species in the sample to be testediRespectively substituting the formula I to obtain the genome DNA content m of each microorganism species in the sample to be detectedi；
Figure FDA0003363290090000011
The method comprises the following steps of (1) finding a total of n microbial species in a sample to be detected, wherein n is a natural number more than 1; bacterial species marker number [1, m](ii) a The genomic DNA composition ratio of the microbial species i is Pi；
The method is used for screening a sample containing microorganisms;
the microorganism is bacteria, fungi and/or virus;
the method is useful for diagnosis and treatment of non-diseases.
2. The method of claim 1, wherein: the step (c) comprises the following steps in sequence:
(c-1) taking the total DNA of a sample to be detected, and constructing a metagenome next-generation sequencing library;
(c-2) taking a metagenome next-generation sequencing library, and sequencing to obtain sequencing data;
(c-3) analyzing the sequencing data to obtain the microorganism species contained in the sample to be detected and the genomic DNA composition ratio P thereofi。
3. The method of claim 1 or 2, wherein: the sample to be detected is a clinical sample.
4. The method of claim 3, wherein:
when the sample to be detected is a clinical sample, the standard bacterial strain group is a standard bacterial strain group 1; the standard strain group 1 comprises one or more of Acinetobacter baumannii, staphylococcus aureus, pseudomonas aeruginosa, Klebsiella pneumoniae, streptococcus pneumoniae, staphylococcus epidermidis, haemophilus influenzae, Ralstonia insoleosa and/or Escherichia coli;
when the sample to be detected is bronchoalveolar lavage fluid, the standard bacterial strain group is a standard bacterial strain group 2; the standard strain group 2 comprises one or more of Acinetobacter baumannii, staphylococcus aureus, pseudomonas aeruginosa, Klebsiella pneumoniae, streptococcus pneumoniae, Escherichia coli and/or Haemophilus influenzae.
5. Detection of genome DNA copy number N of each microorganism species in sample to be detectediThe method of (a), comprising step (a), step (b), step (c) and step (e):
the step (a) includes the steps of:
(a-1) obtaining the types of bacteria which are usually contained according to the source of a sample to be detected, selecting a representative bacterial strain as a standard strain for each type of bacteria, and forming a standard strain group by each standard strain;
(a-2) comparing the sequences of the 16s rDNA of each standard strain in the standard strain group to obtain a specific DNA fragment; the specific DNA fragment is positioned in a conserved region of each standard strain 16s rDNA and has homology of more than 90 percent with a matching region of each standard strain 16s rDNA;
(a-3) mixing the total DNA of each standard strain in the standard strain group to obtain the total DNA of the standard strain group; then diluting to obtain standard total DNA standard products of standard bacterial flora with different concentrations;
(a-4) carrying out fluorescence quantitative PCR on the standard total DNA standard substance of the standard bacterial strain group with different concentrations, and collecting corresponding Ct values; the target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment;
(a-5) drawing a standard curve by taking the concentration of the standard total DNA standard substance of the standard bacterial strain group as an abscissa and the corresponding Ct value as an ordinate;
the step (b) comprises the steps of:
(b-1) carrying out fluorescence quantitative PCR on the total DNA of the sample to be detected, and collecting a Ct value; the target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment;
(b-2) substituting the Ct value collected in the step (b-1) into the standard curve to obtain the concentration of the total bacterial genome DNA in the sample to be detected;
(b-3) obtaining the content m of the total bacterial genome DNA in the sample to be detected according to the concentration of the total bacterial genome DNA in the sample to be detectedbac；
The step (c): carrying out metagenome second-generation sequencing on the total DNA of the sample to be detected to obtain the microorganism species contained in the sample to be detected and the genome DNA composition proportion P thereofi；
The step (e): the content m of the total bacterial genome DNA in a sample to be detectedbacAnd the genomic DNA composition ratio P of each microorganism species in the sample to be testediAnd genome size G of the respective microorganism speciesiRespectively substituting into formula II to obtain the genome DNA copy number N of each microorganism species in the sample to be detectedi；
Figure FDA0003363290090000031
The total number of microorganism species is n, wherein n is a natural number more than 1; bacterial species marker number [1, m](ii) a The genomic DNA composition ratio of the microbial species i is PiThe genome size of the microorganism species i is Gi；
The method is used for screening a sample containing microorganisms;
the microorganism is bacteria, fungi and/or virus;
the method is useful for diagnosis and treatment of non-diseases.
6. The method of claim 5, wherein: the step (c) comprises the following steps in sequence:
(c-1) taking the total DNA of a sample to be detected, and constructing a metagenome next-generation sequencing library;
(c-2) taking a metagenome next-generation sequencing library, and sequencing to obtain sequencing data;
(c-3) analyzing the sequencing data to obtain the microorganism species contained in the sample to be detected and the genomic DNA composition ratio P thereofi。
7. The method of claim 5 or 6, wherein: the sample to be detected is a clinical sample.
8. The method of claim 7, wherein:
when the sample to be detected is a clinical sample, the standard bacterial strain group is a standard bacterial strain group 1; the standard strain group 1 comprises one or more of Acinetobacter baumannii, staphylococcus aureus, pseudomonas aeruginosa, Klebsiella pneumoniae, streptococcus pneumoniae, staphylococcus epidermidis, haemophilus influenzae, Ralstonia insoleosa and/or Escherichia coli;
when the sample to be detected is bronchoalveolar lavage fluid, the standard bacterial strain group is a standard bacterial strain group 2; the standard strain group 2 comprises one or more of Acinetobacter baumannii, staphylococcus aureus, pseudomonas aeruginosa, Klebsiella pneumoniae, streptococcus pneumoniae, Escherichia coli and/or Haemophilus influenzae.
9. Detecting cell number U of each microorganism species in sample to be detectediThe method of (a), comprising step (a), step (b), step (c) and step (f):
the step (a) includes the steps of:
(a-1) obtaining the types of bacteria which are usually contained according to the source of a sample to be detected, selecting a representative bacterial strain as a standard strain for each type of bacteria, and forming a standard strain group by each standard strain;
(a-2) comparing the sequences of the 16s rDNA of each standard strain in the standard strain group to obtain a specific DNA fragment; the specific DNA fragment is positioned in a conserved region of each standard strain 16s rDNA and has homology of more than 90 percent with a matching region of each standard strain 16s rDNA;
(a-3) mixing the total DNA of each standard strain in the standard strain group to obtain the total DNA of the standard strain group; then diluting to obtain standard total DNA standard products of standard bacterial flora with different concentrations;
(a-4) carrying out fluorescence quantitative PCR on the standard total DNA standard substance of the standard bacterial strain group with different concentrations, and collecting corresponding Ct values; the target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment;
(a-5) drawing a standard curve by taking the concentration of the standard total DNA standard substance of the standard bacterial strain group as an abscissa and the corresponding Ct value as an ordinate;
the step (b) comprises the steps of:
(b-1) carrying out fluorescence quantitative PCR on the total DNA of the sample to be detected, and collecting a Ct value; the target of the primer for performing the fluorescent quantitative PCR is the specific DNA fragment or a part of the specific DNA fragment;
(b-2) substituting the Ct value collected in the step (b-1) into the standard curve to obtain the concentration of the total bacterial genome DNA in the sample to be detected;
(b-3) obtaining the content m of the total bacterial genome DNA in the sample to be detected according to the concentration of the total bacterial genome DNA in the sample to be detectedbac；
The step (c): carrying out metagenome second-generation sequencing on the total DNA of the sample to be detected to obtain the microorganism species contained in the sample to be detected and the genome DNA composition proportion P thereofi；
The step (f): the content m of the total bacterial genome DNA in a sample to be detectedbacAnd the genomic DNA composition ratio P of each microorganism species in the sample to be testediGenome size G of respective microorganism speciesiAnd ploidy D of the respective microorganism speciesiRespectively substituting the microorganism species with the formula III to obtain the cell number U of each microorganism species in the sample to be detectedi；
Figure FDA0003363290090000041
The total number of microorganism species is n, wherein n is a natural number more than 1; bacterial species marker number [1, m](ii) a The genomic DNA composition ratio of the microbial species i is PiThe genome size of the microorganism species i is Gi(ii) a Ploidy of microbial species i is Di；
In the step (a-5), the concentration of the standard total DNA standard substance of the standard bacterial strain group is taken as an abscissa, and specifically, the concentration logarithm value of the standard total DNA standard substance of the standard bacterial strain group is taken as the abscissa;
the method is used for screening a sample containing microorganisms;
the microorganism is bacteria, fungi and/or virus;
the method is useful for diagnosis and treatment of non-diseases.
10. The method of claim 9, wherein: the step (c) comprises the following steps in sequence:
(c-1) taking the total DNA of a sample to be detected, and constructing a metagenome next-generation sequencing library;
(c-2) taking a metagenome next-generation sequencing library, and sequencing to obtain sequencing data;
(c-3) analyzing the sequencing data to obtain the microorganism species contained in the sample to be detected and the genomic DNA composition ratio P thereofi。
11. The method of claim 9 or 10, wherein: the sample to be detected is a clinical sample.
12. The method of claim 11, wherein:
when the sample to be detected is a clinical sample, the standard bacterial strain group is a standard bacterial strain group 1; the standard strain group 1 comprises one or more of Acinetobacter baumannii, staphylococcus aureus, pseudomonas aeruginosa, Klebsiella pneumoniae, streptococcus pneumoniae, staphylococcus epidermidis, haemophilus influenzae, Ralstonia insoleosa and/or Escherichia coli;
when the sample to be detected is bronchoalveolar lavage fluid, the standard bacterial strain group is a standard bacterial strain group 2; the standard strain group 2 comprises one or more of Acinetobacter baumannii, staphylococcus aureus, pseudomonas aeruginosa, Klebsiella pneumoniae, streptococcus pneumoniae, Escherichia coli and/or Haemophilus influenzae.
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