从复杂样本中富集真菌的方法及其在真菌研究中的应用

Method for enriching fungi from complex sample and application of method in fungus research

文档序号：3205 发布日期：2021-09-17 浏览：42次中文

1. A method for enriching a fungus from a complex sample, said complex sample comprising a fungus and a bacterium, said method comprising:

1) removing substances with the particle size of more than 100 mu m from the complex sample, and concentrating the complex sample through a filter membrane with the particle size of 0.8-1.0 mu m to obtain a concentrated sample;

2) the bacteria in the concentrated sample are lysed and the bacterial nucleic acids are then removed by DNase.

2. The method of claim 1, wherein the complex sample is enriched for fungi in a higher abundance of bacteria than fungi; preferably the complex sample is human or animal faeces.

3. The method for enriching fungi from a complex sample according to claim 1 or 2, wherein the bacteria content in the concentrated sample is 1x10⁴～1x10⁸copies/uL, fungal content 1x10²～1x10⁵copies/uL。

4. The method for enriching fungi from a complex sample according to any of claims 1 to 3, wherein step 1) comprises in particular:

after the complex sample is resuspended by phosphate buffer solution, removing substances with the particle size larger than 100 mu m, then filtering by adopting a filter membrane with the particle size of 0.8-1.0 mu m, discarding the filtrate, flushing the filter membrane by using the phosphate buffer solution, and then carrying out centrifugal concentration on the flushing solution at 10000-12000g to obtain a concentrated sample.

5. The method for enriching a fungus from a complex sample according to any one of claims 1 to 4,

in step 2), the bacteria in the concentrated sample are lysed using lysozyme and NaOH-SDS.

6. The method for enriching fungi from a complex sample according to claim 5, wherein the operation of lysing bacteria in the concentrated sample in step 2) comprises:

i) mixing the concentrated sample with NaOH-SDS, processing at room temperature, and centrifuging to remove supernatant;

ii) resuspending the pellet obtained in step i), mixing it with a lysozyme solution, treating it at 36-38 ℃ and discarding the supernatant;

wherein the NaOH-SDS contains 0.15-0.25N NaOH and 0.8-1.2 wt% SDS; the concentration of the lysozyme solution is 40-60 mg/mL;

the volume ratio of the concentrated sample to NaOH-SDS is 1: 0.8-1.2; the volume ratio of the precipitation resuspension to the lysozyme solution was 1: 0.5-0.8;

preferably, the treatment time in step i) is 4-6min, and the treatment time in step ii) is 0.8-1.2 h;

preferably, the centrifugation in steps i), ii) is carried out at 10000-.

7. The method for enriching fungi from a complex sample according to any of claims 1 to 6, wherein in step 2) bacterial nucleic acids are removed using non-specific endonucleases/nucleases;

preferably, the operation of removing bacterial nucleic acid using HL-SAN DNase in step 2) specifically comprises:

mixing the sample after bacteria lysis with HL-SAN buffer and HL-SAN DNase, and incubating for 13-17min at 36-38 ℃ and 700-; adding HL-SAN DNase again, and incubating for 13-17min at the temperature of 36-38 ℃ and the speed of 700-; then centrifuged at 7000g of 5000-.

8. A method for obtaining fungal nucleic acid from a complex sample, the method comprising:

obtaining an enriched sample using the method of enriching a fungus from a complex sample according to any one of claims 1 to 7, followed by extracting DNA and/or RNA from the enriched sample.

9. A method for high throughput sequencing and analysis of fungi, comprising:

performing high-throughput sequencing and analysis after obtaining a DNA sample by using the method for obtaining fungal nucleic acid from a complex sample according to claim 8;

wherein the high throughput sequencing comprises amplicon sequencing and/or metagenomic sequencing.

10. A method for analyzing a macrotranscriptome of a fungus, comprising:

after obtaining an RNA sample using the method of claim 8 for obtaining fungal nucleic acid from a complex sample, the mRNA is captured and inverted into cDNA, followed by macrotranscriptome analysis.

Background

The intestinal tract provides a growing and breeding environment for various microorganisms, including prokaryotes, bacteria, archaea, eukaryotic viruses, bacteriophages, fungi, protozoa and other eukaryotic organisms. These gut microorganisms play an important role in maintaining the healthy homeostasis of the host and affect a variety of host functions including gut barrier integrity, vitamin synthesis, metabolic activity, nervous system and inflammation and immunity.

With the development of high-throughput sequencing technology in recent 15 years, the identification and gene function research of complex flora in specific environment are receiving wide attention. The current study of the intestinal flora focuses on the changes in the composition, characteristics and functions of the bacteria, mainly based on the quantitative dominance of the bacteria in the intestinal tract (on average 10 per gram of fecal sample)¹¹Individual bacterial cells). The intestinal fungus is small in quantity (only 10 fungal cells per gram of fecal sample)⁵-10⁶One), and database imperfections, etc., have received less attention from previous studies to gut fungi. Some recent studies on intestinal fungi have shown that fungi exist on the skin and almost all mucosal surfaces in the human body, mainly including unicellular yeast, multicellular mold and bimodal fungi in the form of yeast and filamentous cells, interact with the host in symbiotic, parasitic and other ways, and have an important function in maintaining the human health homeostasis.

The current research methods for intestinal fungi mainly comprise:

(1) the fungus strain is identified by observing the form of the fungus and analyzing physiological and biochemical indexes, and the metabolism and functional characteristics of the fungus strain are researched. However, many species cannot be detected due to the unclear suitable culture conditions; meanwhile, the single strain separated by culture needs to be identified by sanger sequencing or flight mass spectrometry. High throughput is also more difficult to achieve with longer overall cycle times.

(2) Species identification is achieved at the nucleic acid level by high throughput sequencing methods. After the method extracts the sample genome nucleic acid, the composition and the function of the fungi in a specific environment are researched in a mode of sequencing a PCR product obtained by amplifying a characteristic fragment on the genome nucleic acid (amplicon sequencing) or directly establishing a library for sequencing (metagenome sequencing). Wherein amplicon sequencing primarily targets the 2 hypervariable regions ITS1 and ITS2 transcriptional interspacers in the fungal ribosome operator region. Based on the short read length characteristic of the next generation sequencing platform, the ITS1 or ITS2 region is independently used as a DNA barcode for fungus identification at present, and the fungus identification at the species level can be basically realized. The method has the advantages that the amplification step does not exist in the sequencing of the metagenome and the macrotranscriptome, the species composition and the structure in a sample can be visually analyzed, meanwhile, the key functional path can be analyzed, and the new gene function is excavated, so that the method is an important strategy for researching the intestinal flora.

As the number of the fungus cells in the intestinal tract sample is less than that of the fungus cells, sequencing information can be covered by high-level bacteria and host DNA, and the detection rate of the intestinal tract fungus is very low, the proportion of the detected fungus is only 0.01 percent in the previous research of carrying out metagenome sequencing on the intestinal tract sample, so that the metagenome and the macrotranscriptome are difficult to apply in the research of focusing the intestinal tract fungus. Meanwhile, the amplicon sequencing of the intestinal fungus has the condition of low abundance of detected species. However, no enrichment for intestinal fungi has been published.

Disclosure of Invention

The invention aims to provide a method for enriching fungi from a complex sample and application thereof in fungus research, which realizes the enrichment of the fungus nucleic acid in the complex sample (especially the bacteria with dominant quantity) for the first time by effectively enriching the fungi before nucleic acid extraction, is favorable for promoting the research of the sequencing of the fungus metagenome and the macrotranscriptome in the complex sample (such as feces), and improves the abundance of the fungus species detection in the traditional amplicon sequencing.

Specifically, the present invention provides a method for enriching fungi from a complex sample, wherein the complex sample contains fungi and bacteria, the method comprises the following steps:

2) the bacteria in the concentrated sample are lysed and the bacterial nucleic acids are then removed by DNase.

The invention discovers that the method can efficiently enrich the fungi from the complex sample, thereby being beneficial to the follow-up research of the fungi.

When the complex sample contains bacteria in a higher abundance than fungi, and further, when the complex sample is human or animal feces, the fungus enrichment effect can still be well achieved by using the method of the invention.

Preferably, the bacteria content in the concentrated sample is 1x10⁴～1x10⁸(copies/uL) fungal content 1X10²～1x10⁵(copies/uL). When the contents of bacteria and fungi are within the above range, the subsequent lysis and purification effects are more excellent.

Preferably, step 1) specifically comprises:

As a preferred embodiment, the operation of step 1) is specifically as follows:

1. mixing the complex sample according to the weight ratio of 1 g: mixing with phosphate buffer (such as PBS) at a ratio of 50-70mL to obtain sample suspension;

2. and (3) passing the complex sample through a 100-micron filter membrane to remove other substances, collecting filtrate, filtering by using a 0.8-1.0-micron (more preferably 0.8-micron) filter membrane, discarding the effluent, repeatedly washing the filter membrane by using a phosphate buffer solution until the washing solution is colorless, and concentrating the filtrate by centrifugation at 10000-12000g (more preferably 11000 g).

Preferably, in step 2), the bacteria in the concentrated sample are lysed using lysozyme and NaOH-SDS.

The invention further discovers that compared with other cracking modes, by combining lysozyme and NaOH-SDS, the cell wall of gram-positive bacteria and gram-negative bacteria can be better destroyed and the cells can be cracked while fungi are prevented from being damaged, so that the DNA of the gram-positive bacteria and the gram-negative bacteria can be more favorably released to the outside of the cells to facilitate digestion and removal. Wherein, the lysis effect of NaOH-SDS on gram-negative bacteria is better, and the lysis effect of lysozyme on gram-positive bacteria is better.

More preferably, in step 2), the operation of lysing bacteria in the concentrated sample specifically comprises:

i) mixing the concentrated sample with NaOH-SDS, processing at room temperature, and centrifuging to remove supernatant;

ii) resuspending the pellet obtained in step i), mixing it with a lysozyme solution, treating it at 36-38 ℃ and centrifuging to remove the supernatant;

wherein the NaOH-SDS contains 0.15-0.25N NaOH and 0.8-1.2 wt% SDS; the concentration of the lysozyme solution is 40-60 mg/mL;

the volume ratio of the concentrated sample to NaOH-SDS is 1: 0.8-1.2; the volume ratio of the precipitation resuspension to the lysozyme solution was 1: 0.5-0.8.

In the above amount, it is more favorable to realize an ideal cracking effect. Preferably, the treatment time in step i) is 4-6min and the treatment time in step ii) is 0.8-1.2 h.

Preferably, the centrifugation in steps i), ii) is carried out at 10000-.

More preferably, the NaOH-SDS contains 0.2N NaOH and 1 wt% SDS; the concentration of the lysozyme solution is 50 mg/mL.

Through the mode, the method is more favorable for improving the cracking effect on bacteria while avoiding damaging fungal cells.

Preferably, when the bacterial nucleic acid is removed by using the non-specific endonuclease/nuclease in the step 2), the removal effect on the bacterial nucleic acid is better.

The skilled person can select nuclease and corresponding nuclease buffer to treat and operate the sample after bacterial lysis according to common knowledge to remove bacterial nucleic acid therein, which can achieve good nucleic acid removal effect on the sample after bacterial lysis in the present invention.

As a preferred embodiment, the procedure of removing bacterial nucleic acid using the non-specific endonuclease/nuclease HL-SAN DNase in step 2) specifically comprises:

mixing the sample after bacterial lysis with a high-salt nuclease buffer HL-SAN buffer (5.5 MNaCl and 100mM MgCl2in nucleic-free water) and a non-specific endonuclease/nuclease HL-SAN DNase (25U/. mu.l), and incubating for 13-17min at 36-38 ℃ and 700-900 rpm; adding nonspecific endonuclease/nuclease HL-SAN DNase (25U/mul), and incubating at 36-38 deg.C and 700-; then centrifuged at 7000g of 5000-.

Furthermore, it is within the scope of the present invention that the product may be washed separately after lysis and removal of bacterial nucleic acid by one skilled in the art based on common general knowledge.

As a preferred embodiment, the operation of step 2) is specifically as follows:

1. concentrating the sample according to the volume ratio of 1: 0.8-1.2, mixing with NaOH-SDS (0.2N NaOH, 1% SDS), standing at room temperature for 4-6min, centrifuging at 10000-.

2. After resuspending the pellet with TE buffer, the pellet resuspension was performed in a volume ratio of 1: 0.5-0.8, mixing with lysozyme solution (40-60mg/mL), treating at 36-38 deg.C for 0.8-1.2h, and centrifuging at 12000g for 4-6min at room temperature of 10000-. The supernatant was discarded.

3. Washing the precipitate with phosphate buffer solution, centrifuging at 10000-12000g for 3-4min, and discarding the supernatant (the washing process can be repeated 1-2 times); resuspend the pellet with 90-110. mu.L phosphate buffer.

4. 90-110. mu.L of HL-SAN buffer (5.5M NaCl and 100mM MgCl2in null-free water), 4-6. mu.L of HL-SAN DNase (25U/. mu.l) were added and incubated at 36-38 ℃ and 700-.

5. Adding 1-3 μ L HL-SAN DNase (25U/. mu.l), incubating at 36-38 deg.C and 700-900rpm for 13-17min, and centrifuging at 7000g at 5000-38 deg.C to remove supernatant.

6. Resuspend the pellet with phosphate buffer and remove the supernatant by centrifugation at 7000g of 5000-; repeating for 2-3 times.

The above preferred embodiments can be combined by the person skilled in the art according to common general knowledge to obtain a preferred embodiment of the method of the invention for enriching fungi from a complex sample.

Further, the present invention provides a method for obtaining fungal nucleic acid from a complex sample, the method comprising:

the method for enriching the fungi from the complex sample is used for obtaining an enriched sample, and then DNA and/or RNA are extracted from the enriched sample.

Further, the invention also provides a high-throughput sequencing and analyzing method for fungi, which comprises the following steps:

after a DNA sample is obtained by using the method for obtaining the fungal nucleic acid from the complex sample, high-throughput sequencing and analysis are carried out;

wherein the high throughput sequencing comprises amplicon sequencing and/or metagenomic sequencing.

Further, the present invention also provides a method for analyzing a macrotranscriptome of a fungus, comprising:

after obtaining an RNA sample by using the method for obtaining fungal nucleic acid from a complex sample, mRNA in the RNA sample is captured and inverted into cDNA, and then macro-transcriptome analysis is carried out.

In some preferred embodiments, mRNA can be captured by an oligo dT primer.

Based on the technical scheme, the invention has the following beneficial effects:

the invention can realize the enrichment of fungi and nucleic acid thereof in human or animal waste samples, reduce the proportion of bacterial nucleic acid, and ensure that the enriched samples can be applied to the analysis of amplicons, metagenomes and macrotranscriptomes of targeted fungi. In addition, the invention can also be applied to any complex sample for covering the fungal information due to high abundance of bacteria, so as to realize enrichment of the fungi.

According to the invention, the fungi are enriched before the nucleic acid is extracted, so that the fungal copy number in the nucleic acid sample is effectively increased, the application of the metagenome and macrotranscriptome sequencing technology in the research of the intestinal mycological group becomes possible, and the dilemma of the metagenome and the macrotranscriptome in the research of the intestinal fungal flora is solved. Meanwhile, in the conventional amplicon sequencing, the enriched fungi increase the richness of the fungal species and improve the detection rate of the fungal population.

Drawings

FIG. 1 is a flow chart of the method for enriching and extracting fungal nucleic acid in the intestinal fungus group according to the embodiment of the invention.

FIG. 2 is a standard curve of bacterial copy number and fungal copy number in real-time fluorescent quantitative PCR of example 1 of the present invention, wherein A is a standard curve of bacterial copy number in real-time fluorescent quantitative PCR; panel B is a standard curve of fungal copy number in real-time fluorescent quantitative PCR.

FIG. 3 shows the results of 6 samples according to the example of the present invention, wherein A is the fungal copy number of the nucleic acid sample extracted at different enrichment stages from the 6 samples according to the example of the present invention; panel B shows the fungal and bacterial copy number in nucleic acid samples after 6 sample enrichment treatments performed according to the present invention.

FIGS. 4-9 show the top20 species composition of 6 samples from the present example after sequencing the amplicons at various stages of processing.

In view of the fact that the colors of the species in this type of treatment map cannot be distinguished under black and white, the species in each map are further described below:

specifically, in fig. 4, each histogram includes, from top to bottom: other, Torulaspora, Candida, Chaetomium, Trichosporon, Kazachstania, Alternaria, Paraphoma, Penicillium, Cystobasidium, Fusarium, Cypheropora, Aspergillus, Talaromyces, Nothophora, Gibberella, Rhodotorula, Cladosporium, Wallemia, Unidentified, Saccharomyces.

In fig. 5, each histogram includes, from top to bottom: other, Alternaria, Candida, Sistotrema, Ascotricha, Chaetomium, Trichoderma, Tranzscheliella, Rhodotorula, Myrothecium, Coprinus, Aspergillus, Agrocybe, Wallemia, Nakaseomyces, Filobasidium, Nigrospora, Unidentified, Nothophoma, Cladosporium, Saccharomyces.

In fig. 6, each histogram includes, from top to bottom: other, Trichosporon, Knufia, Fomitopsis, Gibberella, Exophiala, Candida, Acremonium, Wallemia, Reinicum, Nothophoma, Talaromyces, Pithioascus, Fusarium, Wickerhamomyces, Penicillium, Alternaria, Cladosporium, Unidentified, Aspergillus, Saccharomyces.

In fig. 7, each histogram includes, from top to bottom: other, Fomitopsis, Phoalophora, Meyerozyma, Psathyrella, Nakaseomyces, Talaromyces, Cladosporum, Aspergillus, Fusarium, Torulaspora, Ceratocysis, Knufia, Tulostoma, Unidentified, Cladosporum, Rhodotorula, Candida, Nothophoma, Saccharomyces.

In fig. 8, each histogram includes, from top to bottom: other, Penicillium, Nakaseomyces, Trichosporon, Aureobasidium, Naganishia, Visnoniacozyma, Candida, Exophiala, Ogataea, Kurtzmaniella, Botrytis, Wallemia, Aspergillus, Rhodotorula, Talaromyces, Nothophoma, Cladosporium, Unidentified, Trichosporon, Saccharomyces.

In fig. 9, each histogram includes, from top to bottom: other, Naganishia, Cladosporium, Fusarium, Trichoderma, Nakaseomyces, Penicillium, Tilletia, Talaromyces, Cordyceps, Aureobasidium, Unidentified, Aspergillus, Pleurotus, Hypersizygus, Alternaria, Cladosporium, Candida, Saccharomyces, Dictyophora, Phallus.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

In the following examples, aqueous PES filters of 50mm diameter and 0.8 μm pore size were obtained from Tianjingtiang laboratory instruments and membrane filters (STV6) were purchased from Beijing green field energy-producing electromechanical devices, Inc.

Lysozyme was purchased from Tiangen, and HL-SAN DNase was purchased from Arcticzymes.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1

The present example provides a method for enriching and extracting fungal nucleic acid in a group of intestinal fungi, and the flow chart refers to fig. 1.

1. As an experimental subject, 6 samples of feces of adult healthy human (designated as F) stored at-80 ℃ were taken, and 0.5g of the feces was weighed into 50mL centrifuge tubes, and then filled into 30mL of sterilized PBS buffer solution, and sufficiently and uniformly suspended.

2. The fecal suspension was filtered through a 100 μm filter to remove food debris, the filtrate was collected and filtered through a 0.8 μm (50 mm diameter) filter using a membrane filter, the effluent was discarded, the filter was repeatedly washed with PBS buffer until the wash was colorless, 11000g, and the filtrate was concentrated to 1mL (sample was denoted FV herein) by centrifugation for 5 min.

3. 200 μ L of the enriched filtrate was added to an equal volume of NaOH-SDS (0.2N NaOH, 1% SDS), allowed to stand at room temperature for 5min, centrifuged at 11000g at room temperature for 5min, and the supernatant was discarded.

4. To the precipitate were added 110. mu.L of TE buffer, 70. mu.L of lysozyme solution (50mg/mL), and treated at 37 ℃ for 1 hour. 11000g was centrifuged at room temperature for 5 min. The supernatant was discarded.

5. Washed twice with 500. mu.L PBS buffer, centrifuged at 11000g for 3min at room temperature, and the supernatant discarded (here sampled from the pellet as FVGJ). The pellet was resuspended in 100. mu.L of phosphate buffer.

If step 3 was not carried out, steps 4,5 were carried out directly, i.e. when lysozyme treatment alone was used, the precipitate was sampled FVJ.

6. mu.L of HL-SAN buffer (5.5M NaCl and 100mM MgCl2in nuclear-free water), 5. mu.L of HL-SAN DNase, at 37 ℃ and 800rpm, was added and incubated for 15 min.

7. Add 2. mu.L HL-SAN DNase,37 ℃, 800rpm, incubate for 15 min.

8.6000g, and centrifuging for 3 min. The supernatant was discarded and 300. mu.L of PBS buffer was used to resuspend the pellet.

9.6000g, and centrifuging for 3 min. The supernatant was discarded and 150. mu.L of PBS buffer was used to resuspend the pellet.

10.6000g, and centrifuging for 3 min. The supernatant was discarded and the precipitate was retained.

11. The precipitated sample collected in step 10 was subjected to simultaneous extraction of DNA and RNA using the AllPrep Power focal DNA/RNA Kit (Qiagen, cat # 80244).

And 12, carrying out quality detection and quantification on the DNA and RNA samples by using Nanodrop and Qubit.

DNA samples bacterial and fungal copy number were quantified using an ABI QuantStudio 7 quantitative PCR instrument using KAPA SYBR FAST kit (cat # KK 4601). The specific primers, reaction system and reaction program are as follows:

(1) bacterial part:

specific primers are shown in table 1 below:

TABLE 1

Reaction system:

reaction procedure: at 95 ℃ for 3 min; (95 ℃,15s, 55 ℃,30 s)40 cycles.

(2) Fungi part:

specific primers are shown in table 2 below:

TABLE 2

Note: "n" mentioned in SEQ ID No.3 of the sequence Listing represents "i".

Reaction system:

reaction procedure: at 95 ℃ for 10 min; (95 ℃,15s, 55 ℃,30 s; 70 ℃, 60 s; 40 cycles).

14. After capturing mRNA from the RNA sample, mRNA was enriched by oligo (dT)15Primer (Promega, cat # C1101) and inverted to give cDNA. The reaction system and reaction conditions were as follows:

a) oligo (dT) capture of mRNA

RNA(1ug) 26μL

Oligo(dT)15(0.1ug/μL) 2μL

Total 28. mu.L

Gently mix well and centrifuge briefly. Metal bath at 65 deg.C for 5 min; ice bath for 2 min.

b) One-chain reaction

Mixing, and centrifuging for a short time;

at 25 ℃ for 10 min; 60min at 37 ℃; 95 ℃ for 5 min; and preserving at 4 ℃.

c) Two chain reaction

Mixing, and centrifuging for a short time;

at 25 ℃ for 10 min; 60min at 37 ℃; 75 ℃ for 10 min; and preserving at 4 ℃.

d) Magnetic bead purification

Placing the Agencourt AMPure XP magnetic beads (Beckman Coulter) at room temperature in advance, and fully shaking and uniformly mixing.

Transferring the cDNA samples to 1.5mL centrifuge tubes respectively, adding equal volume of resuspended AMPure XP magnetic beads, and mixing the mixture by gentle blowing and sucking.

Incubating for 5min at room temperature, placing the centrifuge tube on a magnetic bead rack, and standing at room temperature until the solution becomes clear. Discard the supernatant and take care to avoid aspirating the beads.

200 μ L of 70% ethanol solution prepared in situ is added, and the mixture is kept standing for 30s at room temperature, and the ethanol solution is discarded. And repeating the previous step, washing with an ethanol solution for two times, and drying the magnetic beads at room temperature to avoid cracks of the magnetic beads.

The tube was removed and 32. mu.L of nuclear-free water was added and gently shaken to suspend the beads. Incubate at room temperature for 5 min.

The centrifuge tube was placed on a magnetic bead stand and allowed to stand at room temperature until the eluate became clear. Aspirate 31. mu.L of the supernatant into a new 1.5mL centrifuge tube to obtain a cDNA sample.

15. And (3) carrying out amplicon and metagenome sequencing analysis on the DNA sample obtained in the step (11), and carrying out macrotranscriptome library construction and sequencing analysis on the cDNA sample obtained in the step (14).

The detection and analysis of samples F, FV, FVJ, and FVGJ collected from the 6 sample extraction processes were carried out by the following specific methods:

QPCR detection of fungal and bacterial copy number

1) The ct values were obtained by bacterial Qpcr reaction using the starting amount of 100ng/uL, 5-fold gradient diluted e.coil DNA as template according to step 13 above, and a standard curve of bacterial ct values versus 16s rDNA copy number per uL DNA was plotted (fig. 2, panel a is a standard curve of bacterial copy number for 1 sample).

2) A standard curve of fungal ct values versus 18s rDNA copy number per uL DNA was prepared according to the above step 13 by reacting fungal Qpcr with 1ng/uL of Saccharomyces cerevisiae 18srDNA as template (FIG. 2B is a standard curve of fungal copy number for example 1).

3) The bacterial and fungal Qpcr reactions were performed on the collected samples F, FV, FVJ, and FVGJs according to the aforementioned step 13, and the bacterial and fungal copy numbers were quantified by the obtained ct values and the corresponding standard curves (FIG. 3, Panel A, Panel B).

ITS amplicon library construction and sequencing

DNA samples of collected samples F, FV, FVJ, FVGJ were amplified using primers for the ITS2 region of fungus, and the gITS7/ITS4 primer pair was selected. gITS7(SEQ ID No.5, 5 '-GTGARTCATCGARTCTTTG-3'), ITS4(SEQ ID No.6, 5'-TCCTCCGCTTATTGATATGC-3').

Library construction involves a total of two rounds of amplification.

1) First round amplification System:

reaction procedure: at 95 ℃ for 3 min; (95 ℃,15 s; 55 ℃,30 s; 72 ℃,30 s; 40 cycles); 72 ℃ for 5 min; storing at 4 ℃.

2) After PCR reaction, PCR product purification is carried out by adopting the operation steps of an Agencour AMPure XP kit:

and taking out 22uL of PCR products of the first round, transferring the PCR products into a new 1.5mL centrifuge tube, adding 18uL of AMPure XP magnetic beads, carrying out vortex oscillation, mixing uniformly, instantly separating, standing at room temperature for 15min, and fully combining with the magnetic beads.

And placing the centrifuge tube containing the magnetic beads and the PCR product on a magnetic frame, standing for 5min, and removing supernatant by using a pipette after the solution becomes clear. Twice with 80% ethanol: the beads were washed with 200uL of 80% ethanol and after 30sec of standing, the supernatant was quickly aspirated. After repeating the operation once, standing the mixture at room temperature for 5min to completely dry the magnetic beads and avoid the magnetic beads from drying and cracking.

Adding 25uL RB elution solution into the centrifuge tube, repeatedly blowing and uniformly mixing by using a pipette gun, standing for 5min at room temperature, putting the centrifuge tube back onto a magnetic frame, standing until the solution becomes clear, and transferring about 23uL DNA sample by using the pipette gun to elute to a new 1.5mL centrifuge tube.

3) Second round of PCR amplification:

an amplification system:

reaction procedure: at 95 ℃ for 3 min; (95 ℃,30 s; 55 ℃,30 s; 72 ℃,30 s; 8 cycles); 72 ℃ for 5 min; storing at 4 ℃.

4) Next, steps 4-8 are repeated, a second round of PCR products are purified and the constructed library concentration is checked with a Nanodrop and library integrity is checked using 1.5% agarose gel electrophoresis (120V, 35 min).

5) And (3) carrying out concentration detection on the constructed intestinal flora ITS2 gene sequencing library by using a Qubit nucleic acid fluorescence quantitative instrument. DNA library samples of different concentrations were mixed in equal amounts and subjected to PE250 paired-end sequencing using an Illumina HiSeq 2500 sequencer.

ITS2 amplicon sequencing data analysis

1) For paired-end sequencing data obtained by Illumina HiSeq 2500 sequencing platform, we first merged the paired-end sequences of the original off-line data using fastq _ mergepages command of vsearch software.

2) Quality control is carried out on the spliced sequence by using a fastx _ filter command of VSEARCH software, and a low-quality sequence is filtered, wherein the parameter is that the fastq _ maxee _ rate is 0.01, namely, the read error rate does not exceed 1%.

3) And extracting the double-ended sequences after quality control by adopting ITSx software, and randomly extracting the sequences with the corresponding number of each sequencing data by using a fastx _ subsample command in the USEARCH software for flattening by taking the lowest sequence number of all samples as a standard in order to ensure that the different samples can be subjected to transverse comparative analysis. And combining the normalized data, utilizing a sequence _ full length command of VSEARCH software to remove redundancy of the sequence, and then utilizing a UCHIME command of USEARCH software to remove chimeras by adopting an ITS database.

4) Generating an abundance spectrum matrix by using the sequence through a USEARCH software otutab command; species annotation was performed using the UNITE database using BLAST software. The next step of species composition analysis was performed.

5) Species composition analysis the species composition of top20 was analyzed and displayed using the ggalluvial package in the R language.

The results are shown in fig. 2-9, and it can be seen from the results that after 6 adult fecal samples are treated by the fungus enrichment method provided by the invention, the fungus copy number is increased, and the species abundance of ITS amplicon sequencing is increased. Specifically, the method comprises the following steps:

(one) the increase in fungal copy number per unit DNA (ng) after enrichment was about 2 orders of magnitude (18s rDNA average copy number from 2.87 x 10) compared to control (untreated fecal samples)⁴Increase 1.63 x10⁶) (Panel A in FIG. 3); the fungal nucleic acid copy number after enrichment treatment is predominant compared to bacteria (16s rDNA average copy number is 1.42 x 10)⁵Less than 1.63 x10 fungal copy number⁶). (in FIG. 3, B view.)

And (II) comparing with the control group, after 6 samples are enriched and analyzed by ITS amplicon sequencing, the abundance of the composition of the fungus species with the abundance of Top20 is increased. (FIGS. 4-9)

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> method for enriching fungi from complex sample and application thereof in fungus research

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