Method for absolute quantification of fungi in sample by using internal reference sequence

Abstract

The invention belongs to the technical field of microbial amplicon detection, and particularly relates to a method for absolute quantification of fungi in a sample by using an internal reference sequence. According to the method for absolutely quantifying the fungi in the sample by using the internal reference sequence, the absolute quantification of the fungi microorganisms is realized by designing the specific internal reference sequence, and the technical problem that only relative abundance can be obtained and the absolute content cannot be obtained in the prior art is solved. Besides, the internal reference sequence has no homology with a DNA sequence in a natural organism, so that the internal reference sequence can be accurately separated from a sequencing result, interference with other sequences in a sample is avoided, meanwhile, the internal reference sequence has similar amplification efficiency with a target sequence, and the accuracy of the internal reference sequence is improved. And quantitative deviation caused by amplification efficiency difference is reduced. In terms of operation, the method is simple to operate, accurate in detection and wide in application, and has a wide market prospect in microbial diversity research.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial amplicon detection, and particularly relates to a method for absolutely quantifying fungi in a sample by using an internal reference sequence. BACKGROUND

[0002] ITS1 is located between 18S and 5.8S of the ribosomal rDNA sequence of eukaryotes, and ITS2 is located between 5.8S and 28S of the ribosomal rDNA sequence of eukaryotes. Since the ITS region is cut off during the processing of ribosomal RNA and does not play a functional role, the selection pressure is small in the evolution process, and the evolution rate is about 10 times that of 18S rDNA, which belongs to a moderately conserved region, and it can be used to study the classification hierarchy below the species. At present, second-generation sequencing still occupies a large market share due to its high throughput, low cost, short cycle and optimized algorithm, and second-generation sequencing usually selects ITS1 or ITS2 variable regions for sequencing.

[0003] The absolute abundance of microorganisms has been proven to be essential for analyzing the diversity of microbial communities in natural environments, and currently, the analysis of microbial abundance in the environment mainly relies on high-throughput sequencing technology. Amplicon sequencing is sequencing of PCR products or captured fragments of a specific length, and currently, high-throughput sequencing technology is mainly used. However, due to technical limitations, only the classification of microorganisms and their relative abundance can be obtained from high-throughput sequencing results, and the absolute content of microorganisms at different classification levels cannot be obtained, and the analysis of relative abundance ignores the differences in the total amount of microorganisms between different samples, resulting in errors in the analysis results.

[0004] Therefore, absolute quantitative analysis can measure the gene copy number of each microorganism in the sample, obtain the real number of each microorganism and the real difference between samples, and further reflect the real diversity of the microbial community. SUMMARY

[0005] Based on this, an embodiment of the application provides a method for absolutely quantifying fungi in a sample by using an internal reference sequence.

[0006] In one aspect, the application provides a method for absolutely quantifying fungi in a sample by using an internal reference sequence, comprising the following steps: S1, designing a nucleotide sequence artificially synthesized and not existing in a natural environment, the artificially synthesized sequence containing the binding sites of ITS full-length primers ITS1F and LR3 and ITS1 region primers ITS5 and ITS2aR, and having no homology with DNA sequences in organisms in nature; S2, extracting microbial genomic DNA from the sample; S3, mix the designed chimeric internal standard plasmids according to a certain proportion and add to the DNA sample obtained in S2, and use ITS full-length primers or ITS1 region primers to perform PCR amplification; S4, perform high-throughput sequencing on the PCR product obtained in S3; S5, draw a standard curve according to the relative content of the internal standard, and calculate the absolute abundance of fungi in the sample.

[0007] In some embodiments, the number of artificially synthesized sequences is 10-30. In some embodiments, the number of artificially synthesized sequences is 18.

[0008] In some embodiments, the length and GC content of the artificially synthesized sequences are the same as those of the target fungal sequences.

[0009] In some embodiments, the sequence of the artificially synthesized nucleotide sequence is shown in SEQ ID NO. 1-SEQ ID NO. 18.

[0010] In some embodiments, the ITS full-length primer is shown in SEQ ID NO. 19-SEQ ID NO. 20.

[0011] In some embodiments, the ITS1 region primer is shown in SEQ ID NO. 21-SEQ ID NO. 22.

[0012] In some embodiments, the mixing ratio of the chimeric internal standard plasmid and the DNA sample is 1:20.

[0013] In some embodiments, after S4, the step of performing sequence clarification and quality analysis on the sequencing data to screen out qualified data meeting the requirements is further included.

[0014] In some embodiments, after S4, the step of denoising, removing chimeras, and removing sequencing errors or errors caused by PCR amplification is further included.

[0015] In some embodiments, after S4, the step of performing species annotation on the sequencing data to obtain species classification annotation information at different classification levels is further included.

[0016] In some embodiments, after S4, the step of performing diversity analysis on the sequencing data is further included.

[0017] The application provides a method for absolute quantification of fungi in a sample by using an internal reference sequence, and realizes absolute quantification of fungal microorganisms by designing a specific internal reference sequence, and solves the technical problem that only relative abundance can be obtained and absolute content cannot be obtained in the prior art. In addition, the internal reference sequence of the application has no homology with the DNA sequence in the organism in nature, which ensures that the internal reference sequence can be accurately separated from the sequencing result, avoids interference with other sequences in the sample, and at the same time ensures that the internal reference sequence and the target sequence have similar amplification efficiency, and reduces the quantitative deviation caused by the difference in amplification efficiency. In terms of operation, the application is also simple to operate, accurate in detection, widely applicable, and has a broad market prospect in the research of microbial diversity. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the application, more completely understand the application and its beneficial effects, the drawings needed to be used in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0019] Figure 1 The plasmid map of the recombinant plasmid used in an embodiment of the application; Figure 2 The agarose gel electrophoresis identification result of the product of the internal standard plasmid provided by an embodiment of the application by using ITS1 region primers for PCR amplification; Figure 3 The agarose gel electrophoresis identification result of the product of the internal standard plasmid provided by an embodiment of the application by using ITS full-length primers for PCR amplification; Figure 4 The standard curve obtained by taking the LG value of the initial reads number of each internal standard ITS1 region as the abscissa and the CT value measured by high-throughput sequencing as the ordinate for an embodiment of the application; Figure 5 The standard curve obtained by taking the LG value of the initial reads number of each internal standard ITS full-length as the abscissa and the CT value measured by high-throughput sequencing as the ordinate for an embodiment of the application; Figure 6 The fungal microorganism abundance graph of an embodiment of the application. DETAILED DESCRIPTION

[0020] The application will be described in further detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are only used to explain the application and not intended to limit the scope of the application. The purpose of providing these embodiments and examples is to make the disclosure of the application more thoroughly and comprehensively understood. It should also be understood that the application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the application, and the equivalent forms obtained by such changes or modifications also fall within the protection scope of the application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the application. It should be understood that the application can be implemented without one or more of these details.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0022] The term Unless otherwise indicated or contradictory, the terms or phrases used herein have the following meanings: The selection scope of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, which includes any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in this application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0023] In this application, "multiple", "various", "multiple times", "multiple" and the like are used without specific limitation, which means greater than or equal to 2 in quantity. For example, "one or more" means one or more than two.

[0024] The "combination thereof", "any combination thereof", "any combination thereof" and the like used herein include all suitable combinations of any two or more listed items.

[0025] In the present application, the term "suitable" in "suitable combination", "suitable manner", "any suitable manner" and the like means that the technical solution of the present application can be implemented, the technical problem of the present application can be solved, and the intended technical effect of the present application can be achieved.

[0026] In the present application, "further", "still further", "in particular" and the like are used for description purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present application.

[0027] In the present application, "optionally", "optional" and "optional" mean that it can or can not be present, i.e. it means that it is selected from either of the two parallel schemes "has" or "has not". If there are multiple "options" in a technical solution, and there is no special instruction and no contradictory relationship or mutual restriction, each "option" is independent.

[0028] In the present application, the technical features described in an open manner include both the closed technical solution consisting of the listed features and the open technical solution of the listed features.

[0029] In the present application, when referring to a numerical interval (i.e. a numerical range), if not otherwise specified, the optional numerical distribution within the above numerical interval is considered to be continuous, and includes both numerical endpoints (i.e. the minimum value and the maximum value) of the numerical range and every numerical value between the two numerical endpoints. If not otherwise specified, when a numerical interval refers only to integers within the numerical interval, including both endpoint integers of the numerical range and every integer between the two endpoints, in the present application, it is equivalent to directly listing each integer, for example, t is an integer selected from 1 to 10, which means that t is any integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in the present application should be understood to include any and all sub-ranges subsumed therein.

[0030] In the present application, the temperature parameter, if not otherwise specified, allows for constant temperature treatment and also allows for fluctuations within a certain temperature interval. It should be understood that the constant temperature treatment allows for fluctuations within the accuracy range controlled by the instrument. Fluctuations within a range of, for example, ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0031] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) means volume percentage, and % (w / v) means mass volume percentage.

[0032] All the documents mentioned in the present application are cited in the present application as references, as if each document is cited as a reference individually. Unless and to the extent that the cited documents conflict with the purpose and / or technical solutions of the present application, the cited documents are cited in the present application in their entirety and for all purposes. When the present application refers to the cited documents, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited in the present application. When the present application refers to the cited documents, the examples and preferred modes of the cited relevant technical features can also be incorporated into the present application as references, but are limited to the implementation of the present application. It should be understood that when the cited content conflicts with the description in the present application, the present application is for reference or is modified adaptively according to the description in the present application.

[0033] The term "artificially synthesized sequence" is an artificially designed DNA sequence that does not exist in a natural environment, which has the function of a reference standard substance in the art for absolute quantification of microorganisms in a sample. The specific sub-positions of the artificially synthesized sequence include 18 specific sequences such as ITS-spike1, its-spike5, its-spike7, which are synthesized by a gene synthesis company and cloned into a plasmid for use as an internal standard plasmid.

[0034] The term "ITS full-length primer and ITS1 region primer", refers to a specific primer pair designed for fungal ribosomal DNA internal transcribed spacer (ITS) in the art. The ITS full-length primer ITS1F / LR3 can amplify the complete ITS region (including ITS1, 5.8S and ITS2), while the ITS1 region primer ITS5 / ITS2aR only amplifies the ITS1 region.

[0035] The term "chimeric internal standard plasmid" refers to a recombinant plasmid DNA molecule containing artificially synthesized sequences in the art. This plasmid is constructed by genetic engineering technology and has the following functions: as a reference standard substance, it is co-amplified with sample DNA during PCR amplification; through its known concentration and sequence characteristics, it realizes the absolute quantification of microorganisms in the sample. The specific sub-positions of the chimeric internal standard plasmid include internal standard mixtures (internal standard MIX) containing different concentration gradients.

[0036] The term "high-throughput sequencing" refers to a technical platform that can simultaneously sequence a large number of DNA molecules in the art. In the present application, the specific sub-positions of high-throughput sequencing include second-generation sequencing platforms (such as Illumina MiSeq, Illumina NovaSeq) and third-generation sequencing platforms (such as PacBio Sequel II, Oxford Nanopore PromethION), which can generate a large amount of sequence data for microbial community analysis.

[0037] The term "absolute quantification" refers to an analysis method that measures the number of gene copies of each microorganism in a sample to obtain the actual number of each microorganism. Unlike relative quantification, absolute quantification can reflect the actual difference in the total amount of microorganisms between different samples, and thus reflect the actual diversity of the microbial community. In the present application, the absolute abundance of each fungus in the sample is calculated by the number of internal standard plasmids.

[0038] The term "OTU (Operational Taxonomic Unit)" refers to an operational taxonomic unit, which is the basic unit of clustering and classification of sequencing sequences based on sequence similarity. In the present application, sequencing results are clustered into OTUs by comparing with a database, and the relative abundance and absolute abundance of the microbial community are further analyzed.

[0039] The term "standard curve" refers to a linear relationship curve between concentration and detection signal established by a standard with a known concentration. In the present application, a standard curve is plotted with the LG value of the initial reads number of each internal standard as the abscissa and the CT value measured by high-throughput sequencing as the ordinate, which is used to calculate the absolute abundance of fungi in the sample.

[0040] The term "diversity analysis" refers to a comprehensive evaluation method for the structure and functional characteristics of a microbial community. In the present application, diversity analysis includes alpha diversity (species richness and evenness) and beta diversity (species composition difference between different samples) analysis, which is used to comprehensively evaluate the ecological characteristics of the microbial community.

[0041] In one aspect, the present application provides a method for absolute quantification of fungi in a sample using an internal reference sequence, comprising the following steps: S1. Designing a nucleotide sequence that does not exist in nature, which contains the binding sites of ITS full-length primers ITS1F and LR3 and ITS1 region primers ITS5 and ITS2aR, and has no homology with DNA sequences in organisms in nature; S2. Extracting microbial genomic DNA from a sample; S3. Mixing the designed and synthesized chimeric internal standard plasmids according to a certain proportion and adding them to the DNA sample obtained in S2, and performing PCR amplification using ITS full-length primers or ITS1 region primers; S4. High-throughput sequencing of the PCR product obtained in S3; S5. Drawing a standard curve according to the relative content of the internal standard to calculate the absolute abundance of fungi in the sample.

[0042] The present application realizes absolute quantification of fungal microorganisms by designing specific internal reference sequences and co-amplifying them with sample DNA, and solves the technical problem in the prior art that only relative abundance can be obtained and absolute content cannot be obtained.

[0043] In some embodiments, the number of artificially synthesized sequences is 10-30; In some embodiments, the number of artificially synthesized sequences is 18.

[0044] The specific source of the artificially synthesized sequences includes synthesis by a gene synthesis company and cloning into a plasmid as an internal reference plasmid; the optional number of artificially synthesized sequences is 10-30, for example, 18; the length and GC content of the artificially synthesized sequences are the same as those of the fungal target sequences, ensuring consistency of amplification efficiency.

[0045] In some embodiments, the length and GC content of the artificially synthesized sequences are the same as those of the fungal target sequences. This technical feature ensures that the internal reference sequences have similar amplification efficiency to the target sequences, reducing quantitative deviation caused by differences in amplification efficiency.

[0046] In some embodiments, the sequence of the artificially synthesized nucleotide sequence is shown in SEQ ID NO. 1-SEQ ID NO. 18.

[0047] In some embodiments, the ITS full-length primer is shown in SEQ ID NO. 19-SEQ ID NO. 20.

[0048] In some embodiments, the ITS1 region primer is shown in SEQ ID NO. 21-SEQ ID NO. 22.

[0049] In some embodiments, the mixing ratio of the chimeric internal reference plasmid and the DNA sample is 1:20. This technical feature avoids experimental errors caused by too large differences in the ratio by optimizing the mixing ratio, ensuring the accuracy of quantification.

[0050] In some embodiments, after S4, the step of performing sequence clarification and quality analysis on the sequencing data to screen out qualified data meeting the requirements is further included. This technical feature improves the accuracy and reliability of subsequent analysis through data quality control.

[0051] In some embodiments, after S4, the step of denoising, removing chimeras, and removing errors caused by sequencing errors or PCR amplification is further included. This technical feature reduces the influence of error data on the analysis results through data purification processing, improving the accuracy of quantification.

[0052] In some embodiments, after S4, a step of species annotation of the sequencing data is further included to obtain species classification annotation information at different classification levels. This technical feature provides more detailed microbial community composition information through species classification annotation, enhancing the biological significance of the analysis results.

[0053] In some embodiments, after S4, a step of diversity analysis of the sequencing data is further included. This technical feature comprehensively evaluates the structural and functional characteristics of the microbial community through diversity analysis, providing important data support for ecological research.

[0054] The application also provides a preparation method of a method for absolute quantification of fungi in a sample using an internal reference sequence, comprising the following steps: designing an artificially synthesized sequence that does not exist in nature; synthesizing the artificial sequence through a gene synthesis company and cloning it into a plasmid as an internal standard plasmid; diluting the internal standard plasmid according to different concentration gradients to prepare an internal standard mixture. The preparation method ensures the quality and stability of the internal standard plasmid through a standardized process, providing reliable standard materials for absolute quantification.

[0055] Further, the preparation of the internal standard mixture includes diluting the internal standard plasmid so that the final concentration presents multiple concentration gradients from high to low. This technical feature constructs a more perfect standard curve through the design of multiple concentration gradients, improving the accuracy of quantification.

[0056] The embodiments of the application will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application. The experimental methods in the following examples are not specified, and the priority is given to the instructions given in the application. The experimental methods can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0057] In the following specific examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range if not specifically stated. With respect to temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0058] It should be understood that in various embodiments of the application, the size of the sequence number of the above-mentioned processes does not mean the order of execution, and the execution order of the processes should be determined by their functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the application.

[0059] Example 1: This example provides a method for absolute quantification of fungi in a sample using an internal reference sequence, taking the ITS1 region as an example.

[0060] S1: Collect water, fermentation and soil samples of natural environment species for standby; S2: Design internal standard sequence, containing primer binding site of identifying fungal sequence, the length and GC content are the same as the target in vivo, and there is no homology with DNA sequence in organism in natural environment; >ITS-spike1 (SEQ ID NO. 1) > ITS-spike5 (SEQ ID NO. 2) > ITS-spike7 (SEQ ID NO. 3) >ITS-spik9 (SEQ ID NO. 4) >ITS-spik12 (SEQ ID NO. 5) >ITS-spik13 (SEQ ID NO. 6) >ITS-spik17 (SEQ ID NO. 7) >ITS-spik21 (SEQ ID NO. 8) >ITS-spik26 (SEQ ID NO. 9) >ITS-spik28 (SEQ ID NO. 10) >ITS-spik29 (SEQ ID NO. 11) >ITS-spik30 (SEQ ID NO. 12) > ITS-spik31 (SEQ ID NO. 13) > ITS-spik32 (SEQ ID NO. 14) > ITS-spik34 (SEQ ID NO. 15) > ITS-spik35 (SEQ ID NO. 16) >ITS-spik38 (SEQ ID NO. 17) >ITS-spik40 (SEQ ID NO. 18) S3: The internal standard plasmid was synthesized by a gene company, and the recombinant plasmid map is shown in Figure 1 ; S4: The internal standard plasmid was amplified using ITS1 region universal primers ITS5 / ITS2aR to ensure that the internal standard sequence could be amplified, and the agarose gel electrophoresis identification result of the internal standard plasmid PCR product is shown in Figures 2-3 .

[0061] S5: Preparation of internal standard mixture (internal standard MIX). Dilute 18 internal standard plasmids so that the final concentration presents 5 groups from high to low (22455), then take equal amount of internal standard mixture to internal standard MIX.

[0062] S6: Weigh 300 mg of the sample to be tested, and use the kit FastDNA™ Spin Kit (SKU: 116560200-CF) to extract the total DNA of the microorganism, and the experimental steps are as follows: (1) Add 500 mg of sample to LysingMatrixE; (2) Add 980 µL of Lysis Buffer S1, 120 µL of Lysis Buffer S2 and 10 µL of RNas Ea Solution to LysingMatrixE, use FastPrep-24TM5G sample preparation instrument (Cat. No. 116005500), 6.0 m / s, grind for 40 s; 14,000 x g, centrifuge for 5 mins; (3) Transfer the supernatant (800 µL) to a new 1.5 mL centrifuge tube (self-provided), add 250 μL of Inhibitor Removal MS, invert mix 20 times, 14,000 x g, centrifuge for 5 min; (4) Transfer the supernatant (800 µL) to a new 2.0 mL centrifuge tube (self-provided), add an equal volume of Binding Buffer MS and 5 μL of Magnetic Beads, vortex mix well; (5) Place the centrifuge tube on a shaker and shake for 5 min; (6) Place the centrifuge tube on a magnetic stand and magnetize for 5 min until the magnetic beads are completely adsorbed, discard the supernatant; (7) Add 800 μL of Wash Buffer S to the centrifuge tube, resuspend the magnetic beads, and place the centrifuge tube on a shaker and shake for 3 min; (8) Place the centrifuge tube on a magnetic stand and magnetize for 1 min until the magnetic beads are completely adsorbed, discard the supernatant; (9) Repeat steps 7 and 8 once; (10) Place the magnetic beads at 55°C and dry for 10 min; (11) Add 100 μL DES Buffer to the centrifuge tube, resuspend the magnetic beads, and place the centrifuge tube at 55°C for 5 min for elution; (12) Place the centrifuge tube on a magnetic stand and magnetize for 5 min until the magnetic beads are completely adsorbed. Transfer the supernatant (eluted DNA) to a new 1.5 mL centrifuge tube (self-provided) and it can be used for downstream experiments. For long-term storage, place it at -20°C to avoid repeated freezing and thawing. S7: Add the internal standard MIX prepared in S6 to the DNA sample obtained in S7 at a proportion of 5%, and then use the primer pairs ITS1F / LR3 and ITS5 / ITS2aR for amplification, and recover and purify the PCR product. The primers are shown in Table 1.

[0063] Table 1 S8: After quality identification of the PCR product obtained in S7, perform high-throughput sequencing.

[0064] S9: Compare the sequencing results with the corresponding database and cluster them into operational taxonomic units (OTU in English).

[0065] S10: Draw a standard curve according to the relative content of the internal standard, and calculate the absolute abundance of the soil microbial community. The standard curve obtained by taking the LG value of the initial reads number of each internal standard as the abscissa and the CT value measured by high-throughput sequencing as the ordinate is shown in Figures 4-5 .

[0066] The analysis results are shown in Figure 6 , which reveals the absolute abundance of fungal microorganisms.

[0067] The results show that in the present application, 18 artificially synthesized sequences that do not exist in the natural environment are designed as internal standards. After adding the internal standards to the sample for PCR amplification and high-throughput sequencing, the internal standard sequences can be accurately separated from the sequencing results, thereby playing the role of quantitative internal reference.

[0068] The above-described embodiments only express several implementation manners of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but cannot be understood as a limitation on the patent protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. In addition, it should be understood that, after reading the above teaching content of the present application, the skilled person in the art can make various modifications or modifications to the present application, and the equivalent forms obtained are also within the protection scope of the present application. It should also be understood that, on the basis of the technical solutions provided by the present application, the skilled person in the art obtains the technical solutions through logical analysis, reasoning or limited experiments, and all of them are within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application should be subject to the content of the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A method for absolute quantification of fungi in a sample using an internal reference sequence, characterized in that, Includes the following steps: S1. Design a synthetic nucleotide sequence that does not exist in the natural environment. The synthetic sequence contains binding sites for the full-length ITS primers ITS1F and LR3 and the ITS1 region primers ITS5 and ITS2aR, and has no homology with DNA sequences in organisms in nature. S2. Extract microbial genomic DNA from the sample; S3. Mix the designed and synthesized chimeric internal standard plasmid in a certain proportion and add it to the DNA sample obtained in S2. Perform PCR amplification using full-length ITS primers or ITS1 region primers. S4. Perform high-throughput sequencing on the PCR products obtained in S3. S5. Plot a standard curve based on the relative content of the internal standard and calculate the absolute abundance of fungi in the sample.

2. The method for absolute quantification of fungi in a sample using an internal reference sequence according to claim 1, characterized in that, The number of artificially synthesized sequences is 10-30; Optionally, the number of artificially synthesized sequences is 18.

3. The method for absolute quantification of fungi in a sample using an internal reference sequence according to claim 1, characterized in that, The length and GC content of the artificially synthesized sequence are the same as those of the fungal target sequence.

4. The method for absolute quantification of fungi in a sample using an internal reference sequence according to claim 1, characterized in that, The sequences of the artificially synthesized nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.

18.

5. The method for absolute quantification of fungi in a sample using an internal reference sequence according to claim 1, characterized in that, The full-length ITS primers are shown in SEQ ID NO.19-SEQ ID NO.20; and / or The primers for the ITS1 region are shown in SEQ ID NO.21-SEQ ID NO.

22.

6. The method for absolute quantification of fungi in a sample using an internal reference sequence according to claim 1, characterized in that, The mixing ratio of the chimeric internal standard plasmid to the DNA sample is 1:100-100:

1.

7. The method according to any one of claims 1 to 6, characterized in that, Following S4, the process also includes sequence clarity and quality analysis of the sequencing data to screen out qualified data that meet the requirements.

8. The method according to claim 7, characterized in that, Following S4, steps are also included to denoise the sequencing data, remove chimeras, and remove sequencing errors or errors caused by PCR amplification.

9. The method according to claim 7, characterized in that, Following S4, the steps also include species annotation of sequencing data to obtain species classification annotation information at different taxonomic levels.

10. The method according to claim 7, characterized in that, Following S4, a step of performing diversity analysis on the sequencing data is also included.

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