CN113637788A

CN113637788A - Method for absolute quantification of fungi in sample by constructing recombinant plasmid

Info

Publication number: CN113637788A
Application number: CN202110870832.9A
Authority: CN
Inventors: 王莉; 杜海; 吕锡斌; 郝飞; 陈良强; 杨帆; 王和玉; 徐岩; 周天慈
Original assignee: Kweichow Moutai Co Ltd
Current assignee: Kweichow Moutai Co Ltd
Priority date: 2021-07-30
Filing date: 2021-07-30
Publication date: 2021-11-12

Abstract

The application belongs to the field of bioengineering, and particularly relates to a method for constructing recombinant plasmids to absolutely quantify fungi in a sample. The method comprises the steps of firstly obtaining the types of fungi in a sample to be detected, then respectively constructing recombinant plasmids by using ITS2 genes of the fungi, finally mixing the recombinant plasmids, and directly calculating the concentration of each fungus in the sample to be detected by using the reads number of each recombinant plasmid in the mixed plasmids as a basis, so that the step of microbial counting is avoided, the operation is simpler and more convenient, and the method has stronger purpose and higher accuracy in absolute quantification of the fungi in the sample.

Description

Method for absolute quantification of fungi in sample by constructing recombinant plasmid

Technical Field

The application belongs to the field of bioengineering, and particularly relates to a method for constructing recombinant plasmids to absolutely quantify fungi in a sample.

Background

The Chinese white spirit has a long brewing history, and the unique taste is deeply favored by the Chinese. Historically, the specialty wine culture formed in connection with drinking has even become part of the traditional culture of china. Nowadays, white spirit is a daily consumer product, and the sales volume is increased year by year. The Daqu is an essential component for traditional Chinese brewing, and plays an important role in saccharifying, liquefying and providing flavor substances in the brewing production of white spirit. In the process of brewing white spirit, Daqu is also an important index for controlling the product. The brewing ancestors concluded from the brewing practice that "the yeast is the bone of the wine" and "good yeast must be present if there is good wine". Common yeast raw materials include wheat, barley and peas, and are formed by mixing the crushed raw materials with a certain amount of water and then pressing. The growth and metabolism of microorganisms in the process of culturing the yeast provide abundant and unique microbial populations, enzymes and fermentation precursor substances for the brewing production of later-stage products. According to the difference of the highest yeast temperature in the solid fermentation process, the Daqu can be divided into low-temperature Daqu (40-50 ℃), medium-temperature Daqu (50-60 ℃) and high-temperature Daqu (60-65 ℃).

In the traditional solid-state liquor brewing process, microorganisms are used throughout the liquor production process. To a certain extent, brewing is the process of culturing microorganisms to obtain metabolites. The variety and proportion of the brewing microorganisms determine the variety and content of metabolites, and also determine the variety and content of aroma substances in the wine, and also determine the taste and style of the wine. The yeast contains abundant microbial community such as mold, yeast, and acetic acid bacteria. Among them, the action of fungi is not negligible. For example, fission yeast schizosaccharomyces pombe has good alcohol production capacity as a core alcohol production flora, and can be added into fermented grains as an enhanced yeast in Maotai-flavor liquor, so that the yield of ethanol can be improved, and the generation of other flavor metabolites is not influenced. Therefore, the method has great practical significance for revealing the diversity of the microbial communities of the Daqu fungi, analyzing the microbial driving force behind the change of flavor substances, improving the white spirit brewing process and producing the white spirit with stable quality.

In recent years, high throughput sequencing technology has been widely used in the study of microbial community structures in white spirit samples. The high-throughput sequencing technology can greatly reduce the sequencing cost, can realize large-scale parallel sequencing of microorganisms in a sample, and greatly expands the understanding of the white wine microbial community structure. We can characterize the relative abundance of each species in the microbial community based on the ratio of the reads measured by the OTU relative to the total reads for a single sample. However, due to the technical limitations of high-throughput sequencing, the microbial composition is only characterized by relative abundance, and the absolute content index of the microbes is lacked in the sequencing result. Relative abundance can be used to judge the dominance of a species, but cannot be used to compare the amount of a species in different samples. Characterization of relative abundance makes cross-sample, cross-domain comparisons difficult, and misleading conclusions can result if the relative abundance is misused for cross-sample comparison when the total microbial load is inconsistent between samples. Thus, absolute content as a constant quantitative indicator, independent of total volume variation, has irreplaceable advantages when compared across samples.

However, few methods for absolute quantification of fungi in liquor samples by constructing recombinant plasmids exist, and the relative abundance of fungi in liquor samples is over-estimated or under-estimated compared with the absolute abundance. Therefore, many problems to be solved exist in the aspect of fungus quantification in liquor samples. The method for absolutely quantifying the fungi in the liquor sample is necessary and has important significance for filling the blank of the field, improving the depth of understanding the Daqu microbial community structure, making up the deficiency of absolute content in a high-throughput sequencing technology and further analyzing the Daqu microbial community structure.

Disclosure of Invention

The invention makes up the deficiency of the existing absolute fungus quantification method in the white spirit sample (such as Daqu and fermented grains), and provides another fungus quantification method by combining recombinant plasmids and internal references on the basis of the existing fungus quantification method in the white spirit sample.

In one aspect, the present application provides a method for absolute quantification of fungi in a sample, comprising the steps of:

s1, extracting the genome DNA of the sample to be detected;

s2, sequencing the genomic DNA extracted in the step S1 to obtain the fungus species in the sample;

s3, adding an internal reference into the sample to be detected, wherein the internal reference is shown as SEQ ID No.: 1 is shown in the specification;

s4, respectively introducing ITS2 of each fungus in the sample to be detected into plasmids, and constructing recombinant plasmids carrying ITS2 genes of the fungi;

s5, mixing each recombinant plasmid constructed in the step S4 with the internal reference in the step S3 to obtain a mixture;

s6, detection:

s6.1, detecting the following parameters of the sample to be detected containing the internal reference prepared in S3: the reads number R1 of the fungus in the sample to be detected containing the internal reference and the reads number R2 of the internal reference in the sample to be detected containing the internal reference;

s6.2, the following parameters were checked in the mixture prepared in S5: the reads number r1 of a recombinant plasmid carrying a fungal ITS2 gene in a mixture and the reads number r2 of an internal reference in the mixture;

s7, calculating the concentration of each fungus in the sample to be detected, which is measured in the step S2, wherein the calculation formula is as follows:

(ii) the concentration of a fungus in the sample (R1/R2) x the correction factor for a fungus;

the concentration c × (r1/r2) of a fungal recombinant plasmid carrying ITS2 gene in the mixture.

In some embodiments, the concentration of the recombinant plasmid carrying this fungal ITS2 gene in the mixture is determined using a protein nucleic acid determination spectrophotometer.

In some embodiments, the step S2 is performing high throughput sequencing; in some embodiments, the high throughput sequencing primer hybridizes to SEQ ID No.: 2 and 3.

In some embodiments, the step S3 includes adding an internal reference to the sample to be tested, until the concentration of the internal reference is 1 × 10⁶-1×10⁸copies/mL; in some embodiments, the internal reference is added to a concentration of 1 × 10⁷copies/mL。

In some embodiments, the fungus is selected from at least one of Pichia kudriavzevii, Rhodotorula mucilaginosa, han's yeast with abnormal gram (Wickerhamomyces anomalus), Saccharomyces fibuligerus (Saccharomyces fibuligera), torula delbrueckii, Rhizopus microsporum (Rhizopus microsporus), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Paecilomyces variotii (Paecilomyces variotiii), and Schizosaccharomyces pombe (Schizosaccharomyces pombe).

In some embodiments, the IT2 of step S4 is as set forth in at least one of SEQ ID No. 4-12.

In some embodiments, each recombinant plasmid in step S5 is mixed with an internal reference in equal proportions.

In some embodiments, the sample is selected from at least one of a Daqu, a fermented grain; in some embodiments, the sample is selected from a daqu.

In some embodiments, the recombinant plasmid vector is selected from at least one of the pET-28a (+) plasmid, pET-21a (+), and PGEX-6P plasmid; in some embodiments, the vector of the recombinant plasmid is a pET-28a (+) plasmid.

In some embodiments, the recombinant feed-transformed host cell is selected from the group consisting of a fungus or a bacterium; in some embodiments, the host cell is selected from the group consisting of a bacterium; in some embodiments, the host cell is selected from e.

In some embodiments, the reads number is determined by high throughput sequencing.

The method comprises the steps of firstly obtaining the types of fungi in a sample to be detected, then respectively constructing recombinant plasmids by using ITS2 genes of the fungi in a targeted manner, finally mixing the recombinant plasmids, and directly calculating the concentration of each fungus in the sample to be detected by using the reads number of each recombinant plasmid in the mixed plasmids as a basis, so that the step of counting microorganisms is avoided, the operation is simpler and more convenient, and the purpose is stronger.

Compared with the previous method for quantifying all fungi in a sample by using an internal standard, the method constructs a recombinant plasmid for each fungus, introduces the concept of a correction factor, quantifies one fungus by using one recombinant plasmid, can accurately evaluate the absolute abundance of the target fungus, and has higher accuracy.

The method makes up the problem that only the relative abundance of the sample can be obtained by utilizing a high-throughput sequencing technology, and can accurately judge the absolute abundance of the fungus, thereby effectively avoiding the phenomenon that the abundance value of the fungus is overestimated or underestimated. Accurate assessment of the abundance of the fungi is beneficial to improving the depth of understanding the microbial community structure and making up for the deficiency of absolute content in a high-throughput sequencing technology.

Drawings

FIG. 1: the pET-28a (+) plasmid and the synthesized internal reference DNA fragment are respectively used for detecting the absolute contents of Pichia kudriavzevii, Rhodotorula mucilaginosa, Hanm yeast anomala, Saccharomycoporia fibuligera, Torulopsis delbrueckii, Rhizopus microsporum, Saccharomyces cerevisiae, Paecilomyces variotii and Schizosaccharomyces pombe.

FIG. 2: respectively used for detecting the PCR verification results of the recombinant plasmids of absolute contents of Pichia kudriavzevii, Rhodotorula mucilaginosa, Hanjim yeast anomala, Saccharomycopsis fibuligera, Torulaspora delbrueckii, Rhizopus microsporum, Saccharomyces cerevisiae, Paecilomyces variotii and Schizosaccharomyces pombe.

FIG. 3: respectively used for detecting the absolute contents of recombinant plasmids of Pichia kudriaz, Rhodotorula mucilaginosa, Hanjim yeast anomala, Saccharomycopsis fibuligera, Torulopsis delbrueckii, Rhizopus microsporum, Saccharomyces cerevisiae, Paecilomyces varioti and Schizosaccharomyces pombe.

FIG. 4: comparison of relative abundance to absolute abundance.

FIG. 5: absolute quantitative sequencing results.

Table 4: the abundance difference.

FIG. 6: a standard curve.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

The invention is further illustrated with reference to specific examples.

Coli JM109 and E.coli BL21(DE3) referred to in the examples below were obtained from North Nay organisms, and the pET-28a (+) plasmid was obtained from Novagen (E.coli BL21(DE3) of the above strain is commercially available and does not require preservation for patent procedures); the yeast referred to in the following examples was purchased from Maotai-flavor liquor, Inc. in Guizhou; the Plant Genomic DNA Kit Plant Genomic DNA extraction Kit referred to in the following examples was purchased from root Biochemical technology (Beijing) Ltd.

The media involved in the following examples are as follows:

LB liquid medium: yeast powder 5.0g L^-1Tryptone 10.0g L^-1、NaCl 10.0g L^-1Ampicillin 100. mu. g L^-1。

LB solid medium: yeast powder 5.0g L^-1Tryptone 10.0g L^-1、NaCl 10.0g L^-1Agar powder 15g L^-1Ampicillin 100. mu. g L^-1。

Example 1: construction of method for detecting absolute content of fungi in sample

The method comprises the following specific steps:

extracting the genomic DNA of a sample to be tested according to the reference, "fungal design the architectural Ocean: specific microorganisms in North Atlantic modified wood as modified by HPerforming high-Throughput Sequencing on the mixture by using the upstream primer and the downstream primer provided by igh-Throughput amplification Sequencing, wherein the upstream primer is a DNA fragment with a nucleotide sequence shown as SEQ ID NO.2, and the downstream primer is a DNA fragment with a nucleotide sequence shown as SEQ ID NO.3, so as to obtain the kind of fungi in the sample to be detected; adding the internal reference into the sample to be detected until the concentration of the internal reference in the sample to be detected is 1 multiplied by 10⁷Obtaining a sample to be detected containing the internal reference by copies/mL; respectively introducing ITS2 genes of each fungus in a sample to be detected into the constructed recombinant plasmids to obtain recombinant plasmids respectively carrying ITS2 genes of each fungus; mixing each recombinant plasmid with an internal reference to obtain a mixture; detecting the reads number of each recombinant plasmid and the reads number of the internal reference in the mixture, detecting the reads number of each fungus and the reads number of the internal reference in a sample to be detected containing the internal reference, and calculating the concentration of each fungus in the sample to be detected by using a formula according to the concentration of each recombinant plasmid in the mixture, wherein the formula is as follows:

the concentration of a certain fungus in the sample (reads number of the fungus in the sample to be detected containing the internal reference/reads number of the internal reference in the sample to be detected containing the internal reference) multiplied by the correction factor of the fungus;

the concentration of recombinant plasmid carrying the fungal ITS2 gene in the mixture/(the number of reads in the mixture of recombinant plasmid carrying the fungal ITS2 gene/the number of reads in the mixture of the internal parameters) was determined as the correction factor for a given fungus.

The concentration of the recombinant plasmid carrying this fungal ITS2 gene in the mixture was determined using a protein nucleic acid determination spectrophotometer and the plasmid copy number was calculated according to the method described by Dhanasekaran.

Example 2: application of method for detecting absolute content of fungi in sample

The method of example 1 is used for detecting the absolute content of the fungus in the yeast, and the specific steps are as follows:

extracting the Genomic DNA of the Daqu to be tested by using a Plant Genomic DNA Kit Plant Genomic DNA extraction Kit, performing high-throughput sequencing on the mixture by using a DNA fragment with a nucleotide sequence shown as SEQ ID NO.2 as an upstream primer and a DNA fragment with a nucleotide sequence shown as SEQ ID NO.3 as a downstream primer (the high-throughput sequencing is completed by Beijing Ovweisen Genscience and technology Co., Ltd.), and selecting Pichia pastoris (Pichia kurarivizevii), Rhodotorula mucilaginosa (Rhodotorula mula), Saccharomyces anomala (Wickerhamomyces anomalus), Saccharomyces cerevisiae (Saccharomyces fibuligera), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces cerevisiae (Paulomyces japonicus and Pichia anomala), Rhizopus oligosporus (Pichia anomala), Pichia pastoris (Pichia pastoris), and Pichia anomala (Pichia anomala), and selecting the high-throughput sequencing results, The ITS2 genes (nucleotide sequences are respectively shown as SEQ ID NO. 4-SEQ ID NO. 12) of the enveloped yeast, the Torulopsis delbrueckii, the Rhizopus microsporus, the saccharomyces cerevisiae, the paecilomyces variotii and the schizosaccharomyces pombe and the Multiple Cloning Site (MCS) in the pET-28a (+) plasmid determine that the enzyme cutting sites are BamHI and SalI.

Preparing internal reference according to the patent application with the publication number of CN 111172256A; adding internal reference into Daqu to be tested until the concentration of the internal reference in the Daqu to be tested is 1 × 10⁷And (5) obtaining the Daqu to be detected containing the internal reference by copies/mL.

And (2) chemically synthesizing ITS2 genes of the Pichia kudriavzevii, the rhodotorula mucilaginosa, the Hanjim yeast with abnormal Wei, the covered yeast with microcapsule, the torula delbrueckii, the rhizopus microsporus, the saccharomyces cerevisiae, the paecilomyces varioti and the schizosaccharomyces pombe, and adding enzyme cutting sites of BamH and SalI at two ends of the ITS2 gene to obtain internal reference DNA fragments for detecting the absolute contents of the Pichia kudriavzevii, the rhodotorula mucilaginosa, the Hanjim yeast with abnormal Wei, the covered yeast with microcapsule, the torula delbrueckii, the rhizopus microsporus, the saccharomyces cerevisiae, the paecilomyces varioti and the schizosaccharomyces pombe respectively.

The pET-28a (+) plasmid and the synthesized internal reference DNA fragments which are respectively used for detecting the absolute contents of Pichia kudriavzevii, Rhodotorula mucilaginosa, Hanm yeast abnormity, Saccharomycoporia fibuligera, Torulopsis delbrueckii, Rhizopus microsporum, Saccharomyces cerevisiae, Paecilomyces variotii and Schizosaccharomyces pombe are taken as templates, and double digestion is carried out by using BamH I and Sal I (the digestion system is shown in Table 1, after the double digestion, the double digestion products of 5 mu L of the internal reference DNA fragments are identified by 2 percent agarose gel electrophoresis, and the identification result is shown in FIG 1).

TABLE 1 double enzyme digestion System

Reaction mixture	Volume(μL)
		Buffer	5
BamhⅠ	1
		SalⅠ	1
Template DNA	25
		dd H₂O	18
Total volume	50

Adding the double enzyme digestion product into CP buffer with 4 times volume, adding into an upper layer tube, centrifuging at 12000r/min for 2min, and repeatedly loading once; adding 700 mu L of DNA Wash Buffer, centrifuging at 12000r/min for 2min, pouring out the liquid in the lower collecting pipe, and repeating once; the empty adsorption column is separated once, centrifuged at 12000r/min for 2min, replaced with a new centrifuge tube of 1.5mL and mounted on the column; heating to volatilize alcohol after air separation, and heating at 60 ℃ for 1-2 min(ii) a Inhale 30-50 μ L ddH₂O (preheating at 60 ℃), and centrifuging at 12000r/min for 2min to elute DNA; the column purified product was ligated for 16h at 16 ℃ using T4 ligase (see Table 2 for ligation);

TABLE 2 connection System

Reaction mixture	Volume(μL)
		Buffer	2
Fragment 1 (fungal ITS2 sequence)	2
		Fragment 2(pET-28a (+) sequence)	1
T4 ligase	1
		dd H₂O	14
Total volume	20

And (2) transforming the Escherichia coli JM109 with the ligation product, coating the transformation product on an LB solid culture medium, culturing for 8h at 37 ℃, selecting a transformant on the LB solid culture medium, inoculating the transformant into an LB liquid culture medium for culturing, culturing for 10h at 37 ℃, extracting a plasmid, and performing plasmid transformation on the plasmid by using ITS 2F: 5'-GCATCGATGAAGAACGCAGC-3' (nucleotide sequence shown as SEQ ID NO. 2) as an upstream primer, and ITS 2R: 5'-TCCTCCGCTTATTGATATGC-3' (nucleotide sequence is shown as SEQ ID NO. 3) as a downstream primer to carry out PCR verification and sequencing, and obtain correctly verified recombinant plasmids which are respectively used for detecting the absolute contents of Pichia kudriavzevii, Rhodotorula mucilaginosa, Hanjim abnormal yeast, Saccharomycoporia fibuligera, Torulopsis delbrueckii, Rhizopus microsporus, Saccharomyces cerevisiae, Paecilomyces varioti and Schizosaccharomyces pombe, wherein the PCR reaction parameters are as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 30s, 25 cycles, extension at 72 ℃ for 30s, and PCR products were identified by 2% agarose gel electrophoresis (see Table 3 for validation system and results in FIG. 2).

TABLE 3 PCR verification System

Reaction mixture	Volume(μL)
		Mixture	10
Upstream primer	1
		Downstream primer	1
Template DNA	1
		dd H₂O	7
Total volume	20

Coli BL21(DE3) is transformed by the correctly verified recombinant plasmid, thus obtaining the recombinant escherichia coli containing the recombinant plasmids for detecting the absolute contents of Pichia kudriaz, Rhodotorula mucilaginosa, abnormal Wilken Han's yeast, Saccharomycopsis fibuligera, Torulopsis delbrueckii, Rhizopus microsporus, Saccharomyces cerevisiae, Paecilomyces varioti and Schizosaccharomyces pombe respectively; coating the obtained recombinant escherichia coli on an LB solid culture medium, and culturing at 37 ℃ for 8-10 h to obtain a single colony; selecting a single colony, inoculating the single colony into an LB liquid culture medium, and culturing at 37 ℃ for 12-14 h to obtain a seed solution; inoculating the seed solution into LB liquid culture medium according to the inoculum size of 4% (v/v), and culturing at 37 deg.C and 200rpm for 8h to obtain fermentation liquid; centrifuging the fermentation liquor at 4 deg.C and 1000rpm for 20min, and collecting thallus; the recombinant plasmids are extracted and amplified from the thalli and are respectively used for detecting the absolute contents of Pichia kudriavzevii, Rhodotorula mucilaginosa, abnormal Wilkhur yeast, Microdochium fibuligerum, Torulopsis delbrueckii, Rhizopus microsporus, Saccharomyces cerevisiae, Paecilomyces variotii and Schizosaccharomyces pombe (a plasmid map is shown in figure 3).

Mixing the obtained recombinant plasmids respectively used for detecting absolute contents of Pichia kudriavzevii, Rhodotorula mucilaginosa, Hanm anomala, Saccharomyces cerevisiae, Torulopsis fragilis, Rhizopus microsporum, Saccharomyces cerevisiae, Paecilomyces variotii and Schizosaccharomyces pombe with internal ginseng according to equal quantitative ratio (1:1:1:1:1:1:1:1: 1) to obtain a mixture, wherein the contents of the recombinant plasmids and the internal ginseng in the mixture are 1 × 10⁷copies/g; the mixture was mixed with ITS 2F: 5'-GCATCGATGAAGAACGCAGC-3' (nucleotide sequence shown as SEQ ID NO. 2) as an upstream primer, and ITS 2R: 5'-TCCTCCGCTTATTGATATGC-3' (nucleotide sequence shown in SEQ ID NO. 3) as downstream primer to perform high-throughput sequencing (the high-throughput sequencing is completed by Beijing Ovwison Gene science and technology Co., Ltd.) to obtain the reads number of each recombinant plasmid and the reads number of the internal reference in the mixture, and subjecting the Daqu to be detected containing the internal reference to ITS 2F: 5'-GCATCGATGAAGAACGCAGC-3' (nucleotide sequence shown as SEQ ID NO. 2) as an upstream primer, and ITS 2R: 5' -TCCTCCGCTTATTGATATGC-3' (the nucleotide sequence is shown in SEQ ID NO. 3) is used as a downstream primer to carry out high-throughput sequencing (the high-throughput sequencing is completed by Beijing Ovweisen Gene science and technology Co., Ltd.) to obtain the reads number of each fungus in the yeast to be detected containing the internal reference and the reads number of the internal reference, and the concentration of each fungus in the yeast to be detected is obtained by calculating according to the concentration of each recombinant plasmid in the mixture by using a formula, wherein the formula is as follows:

the concentration of a certain fungus in the sample (reads number R1 of the fungus in the sample to be tested containing the internal reference/reads number R2 of the internal reference in the sample to be tested containing the internal reference) x the correction factor of the fungus;

the concentration c/(the reads number r1 of the recombinant plasmid carrying the ITS2 gene in the mixture/the reads number r2 of the internal parameter in the mixture) of the recombinant plasmid carrying the ITS2 gene in a certain fungus in the mixture.

Through sequencing, the yeast for making hard liquor contains Pichia kudriavzevii, saccharomycete enveloped yeast, rhizopus microsporus, saccharomyces cerevisiae, rhodotorula mucilaginosa, abnormal Wilkholderia, Torulopsis delbrueckii and Paecilomyces variotii, while the fission yeast for making hard liquor does not cover the yeast for making hard liquor.

TABLE 4 calculation of r1, r2 values in recombinant plasmid mixtures, and correction factors

TABLE 5R 1, R2 values in Daqu samples, and the results of quantification of the concentration of each fungus

After absolute quantitative calculation, we regarded the 8 fungi contained in the yeast as a whole, and we compared the difference between their relative and absolute abundances according to the following formula (the abundance comparison is shown in fig. 4).

The difference in abundance of a fungus-the relative abundance of this fungus in 8 fungi-the absolute abundance of this fungus in 8 fungi.

Wherein the abundance difference value is a positive number, and the relative abundance is overestimated compared with the absolute abundance; the abundance difference value is a negative number indicating how much the relative abundance underestimates compared to the absolute abundance. We found that Pichia kudriaz was overestimated by 50.73%, whereas Saccharomyces cerevisiae was underestimated by 13.42% and Saccharomyces cerevisiae by 28.62% compared to absolute abundance (see Table 6 for detailed results).

TABLE 6

Meanwhile, the quantitative result shows that the absolute content of the saccharomyces cerevisiae in the yeast for making hard liquor is 5.89 multiplied by 10⁷The absolute content of colpis/g and Saccharomycopsis fibuligera in the yeast is 4.85X 10⁷The absolute content of copees/g and rhizopus microsporus in the yeast is 1.93 multiplied by 10⁷The absolute content of pichia kudriavzevii in yeast is 8.59 multiplied by 10⁶copies/g (quantitative results see Table 5, FIG. 5).

Example 3: verification of methods that can be used to detect absolute amounts of fungi in samples

Saccharomyces cerevisiae has good ethanol production capacity and is the core fungus of a white spirit brewing system, so that the Saccharomyces cerevisiae is selected from 8 kinds of quantitative fungi for verification of a quantitative method.

The specific implementation method comprises the following steps: screening to obtain Saccharomyces cerevisiae, and checking cell number to be 1.0 × 10 by using blood count plate method⁹cells/g, which were diluted to 10 cells⁸、10⁷、10⁶、10⁵、10⁴After cells/g, Genomic DNA of Saccharomyces cerevisiae was extracted using Plant Genomic Kit Plant Genomic DNA extraction Kit, and Saccharomyces cerevisiae was designedSpecific primers, ScaIRIF-CACAATGGGGCAAAGGCTTC, ScAIRIR-GCCAGAACTGAAGCACAAGC, were followed by fluorescent quantitative PCR of the extracted genome by means of a StepOneplus instrument (purchased from Saimer fly). The reaction system is as follows: the total 20. mu.L of the system was 2 XFast qPCR Master Mix 10. mu.L, upstream and downstream primers 0.4. mu.L each, genome 1. mu.L, and ultrapure water 8.2. mu.L. The reaction conditions are as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 10s, annealing at 50 deg.C for 0.5min, extension at 70 deg.C for 1min for 40 cycles, and annealing at 70 deg.C for 10 min.

The CT value measured by qPCR is used as the abscissa, and the cell value corresponding to the dilution concentration is used as the ordinate, and a standard curve is drawn (see FIG. 6 for the standard curve). Substituting the obtained CT value of 19.02 into the standard curve of FIG. 6 to finally obtain the yeast for making Daqu with the content of Saccharomyces cerevisiae: 10⁷cells/g. Since the copy number of the gene in the Saccharomyces cerevisiae cell is not unique, the content of Saccharomyces cerevisiae in the sample should be higher than 10⁷copies/g。

The patent application publication No. CN111172256A shows that the fungi in the high-temperature yeast powder are absolutely quantified, and the absolute content of Saccharomyces cerevisiae in the detection result is 10⁶About copies/g. The results of example 2 show that the absolute content of the Saccharomyces cerevisiae in the high temperature Daqu obtained by the method is 5.89X 10⁷copies/g. Therefore, the absolute content of the saccharomyces cerevisiae obtained by the method is closer to the true value.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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cttcgggttt ggtattgagt gatactctgt caagggttaa cttgaaatat tgacttagca 180

agagtgtact aataagcagt ctttctgaaa taatgtatta ggttcttcca actcgttata 240

tcagctaggc aggtttagaa gtattttagg ctcggcttaa caacaataaa ctaaaagttt 300

gacctcaaat caggtaggac tacccgctga acttaa 336

<210> 7

<211> 339

<212> DNA

<213> Saccharomycopsis fibuligera

<400> 7

cgaattgcga taagtaatgt gaattgcaga ttttcgtgaa tcatcgaatc tttgaacgca 60

tattgcgctc tatagtattc tatagagcat gcctgtttga gcgtcatttc tctcttaaac 120

ctttgggttt agtattgaag gttgtgttag cttctgctaa ctcctttgaa atgacttggc 180

aattgattga gttttccata tatttgctta aggatttaat attaggttct accaacttat 240

taaataccct tttgcgaagg acttactcgt gtatcaaggc cttataactt tgtcattaat 300

tttgacctca aatcaggtaa ggatacccgc tgaacttaa 339

<210> 8

<211> 406

<212> DNA

<213> Torulaspora delbrueckii

<400> 8

cgaaatgcga tacgtaatgt gaattgcaga attccgtgaa tcatcgaatc tttgaacgca 60

cattgcgccc cttggtattc cagggggcat gcctgtttga gcgtcatttc cttctcaaac 120

aatcatgttt ggtagtgagt gatactctgt caagggttaa cttgaaattg ctagcctgtt 180

atttggttgt gattttgctg gcttggatga ctttgtccag tctagctaat accgaattgt 240

cgtattaggt tttaccaact tcggcagact gtgtgttggc tcgggcgctt taaagacttt 300

gtcgtaaacg atttatcgtt tgtttgagct tttcgcatac gcaatccggc gaacaatact 360

ctcaaagttt gacctcaaat caggtaggaa tacccgctga acttaa 406

<210> 9

<211> 348

<212> DNA

<213> Rhizopus microsporus

<400> 9

aaagtgcgat aactagtgtg aattgcatat tcgtgaatca tcgagtcttt gaacgcagct 60

tgcactctat ggatcttcta tagagtacgc ttgcttcagt atcataacca acccacacat 120

aaaatttatt ttatgtggtg atggacaagc tcggttaaat ttaattatta taccgattgt 180

ctaaaataca gcctctttgt aattttcatt aaattacgaa ctacctagcc atcgtgcttt 240

tttggtccaa ccaaaaaaca tataatctag gggttctgct agccagcaga tattttaatg 300

atctttaact atgatctgaa gtcaagtggg actacccgct gaacttaa 348

<210> 10

<211> 382

<212> DNA

<213> Saccharomyces cerevisiae

<400> 10

gaaatgcgat acgtaatgtg aattgcagaa ttccgtgaat catcgaatct ttgaacgcac 60

attgcgcccc ttggtattcc agggggcatg cctgtttgag cgtcatttcc ttctcaaaca 120

ttcatgtttg gtagtgagtg atactctttg gagttaactt gaaattgctg gccttttcat 180

tggatgtttt ttttttccaa agagaggttt ctctgcgtgc ttgaggtata atgcaagtac 240

ggtcgtttta ggttttacca actgcggcta atctttttta tactgagcgt attggaacgt 300

tatcgataag aagagagcgt ctaggcgaac aatgttctta aagtttgacc tcaaatcagg 360

taggagtacc cgctgaactt aa 382

<210> 11

<211> 311

<212> DNA

<213> Paecilomyces variotii

<400> 11

gaaatgcgat aagtaatgtg aattgcagaa ttcagtgaat catcgagtct ttgaacgcac 60

attgcgcccc ctggtattcc ggggggcatg cctgtccgag cgtcatttct gccctcaagc 120

acggcttgtg tgttgggccc cgtcctccga tcccggggga cgggcccgaa aggcagcggc 180

ggcaccgcgt ccggtcctcg agcgtatggg gctttgtcac ccgctctgta ggcccggccg 240

gcgcttgccg atcaacccaa atttttatcc aggttgacct cggatcaggt agggataccc 300

gctgaactta a 311

<210> 12

<211> 422

<212> DNA

<213> Schizosaccharomyces pombe

<400> 12

gaaatgcgat acgtaatgtg aattgcagaa ttccgtgaat catcgaatct ttgaacgcac 60

attgcgcctt tgggttctac caaaggcatg cctgtttgag tgtcattaca atcttctcac 120

aaaaatgttt ttgatgaggt gttgaacgaa aatttgtttt ttttttaaaa taaatttagt 180

ttgaaatcga ttggtgaaaa caaaaggaag attgaaatta tttttctata ccttttttca 240

ttttttttct attgaacgta ataggtttta ccactttgtt tgatagaaaa agaaattagg 300

aaagaaaaat aactaaagtt ttaatctctt ttatatttga accttaacga aaaaaaatat 360

atttttttca cagcactctt ttatttgacc tcaaatcagg taggactacg cgctgaactt 420

aa 422

Claims

1. A method for absolute quantification of fungi in a sample, comprising the steps of:

s1, extracting the genome DNA of the sample to be detected;

s4, respectively introducing the ITS2 of each fungus, which is detected in the step S2, in the sample to be detected into plasmids, and constructing recombinant plasmids carrying the ITS2 genes of the fungi;

s6, detection:

2. The method of claim 1, wherein in step S2, high throughput sequencing is performed;

preferably, the high throughput sequencing primer hybridizes to SEQ ID No.: 2 and 3.

3. The method of claim 1, wherein an internal reference is added to the sample to be tested in step S3 until the concentration of the internal reference is 1 x10⁶-1×10⁸copies/mL；

Preferably, the internal reference is added to a concentration of 1 × 10⁷copies/mL。

4. The method of claim 1, wherein the fungus is selected from at least one of Pichia kudriavzevii (Pichia kudriavzevii), Rhodotorula mucilaginosa (Rhodotorula mulilaginosa), Saccharomyces wakei (Wickerhamomyces anomalus), Saccharomyces cerevisiae (Saccharomyces fibuigera), Torulaspora delbrueckii (Torulaspora delbrueckii), Rhizopus microsporus (Rhizopus), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Paecilomyces variotii (Paecilomyces variotiii), and Schizosaccharomyces pombe (Schizosaccharomyces pombe);

preferably, the IT2 in the step S4 is as shown in at least one of SEQ ID No. 4-12.

5. The method of claim 1, wherein each recombinant plasmid in step S5 is mixed with an internal reference in equal proportion.

6. The method of any one of claims 1 to 5, wherein the sample is at least one selected from the group consisting of Daqu and fermented grains;

preferably, the sample is selected from daqu.

7. The method of any one of claims 1 to 5, wherein the vector of the recombinant plasmid is selected from at least one of the group consisting of pET-28a (+) plasmid, pET-21a (+), and PGEX-6P plasmid;

preferably, the vector of the recombinant plasmid is pET-28a (+) plasmid.

8. The method of any one of claims 1 to 5, wherein the recombinant feed-transformed host cell is selected from the group consisting of fungi or bacteria;

preferably, the host cell is selected from bacteria;

preferably, the host cell is selected from the group consisting of E.coli.

9. The method of any one of claims 1-5, wherein the reads number is determined by high throughput sequencing.