CN113637788B

CN113637788B - Method for constructing recombinant plasmid to absolutely quantify fungi in sample

Info

Publication number: CN113637788B
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: 2024-09-20
Anticipated expiration: 2041-07-30
Also published as: CN113637788A

Abstract

The application belongs to the field of bioengineering, and particularly relates to a method for constructing recombinant plasmids and absolute quantifying 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 ITS2 genes of the fungi, finally mixing the recombinant plasmids, directly calculating the concentration of the fungi in the sample to be detected by taking the numbers of reads of the recombinant plasmids in the mixed plasmids as the basis, avoiding the step of counting microorganisms, having simpler operation and higher accuracy and having stronger purpose of absolute quantification of the fungi in the sample by using the method.

Description

Method for constructing recombinant plasmid to absolutely quantify fungi in sample

Technical Field

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

Background

The Chinese white spirit brewing history is long, and the unique taste is deeply favored by people. Historically, the characteristic wine culture formed in connection with drinking has even become part of the traditional Chinese culture. Nowadays, white spirit has become a daily consumer product, and sales amount has been increased year by year. Daqu is an indispensable component in traditional Chinese brewing, and plays important roles in saccharification, liquefaction and flavor substance provision in the brewing production of white spirit. Daqu is also an important index for controlling the product in the process of brewing white spirit. The brewing ancestors draw conclusions from the brewing practice that "bones of the wine are the yeast and" have good wine must have good yeast ". Common materials for Daqu include wheat, barley and pea, which are obtained by mixing pulverized materials with a certain amount of water and then compression molding. The growth and metabolism of the microorganisms in the process of culturing Daqu provides rich and unique microorganism population, enzymes and fermentation precursor substances for the brewing production of the later-period products. The Daqu can be classified into low temperature Daqu (40-50deg.C), medium temperature Daqu (50-60deg.C) and high temperature Daqu (60-65deg.C) according to the maximum Qu Wen in solid state fermentation process.

In the traditional solid white spirit brewing process, microorganisms are throughout the white spirit production process. Brewing is, to some extent, said to be the process of culturing microorganisms to obtain metabolites. The type and proportion of the microorganisms of the wine-making process determine the type and content of the metabolites, and therefore the type and content of the aroma substances present in the wine, and therefore the taste and style of the wine. Daqu contains abundant microbial communities such as mold, yeast and acetic acid bacteria. Among them, the action of fungi cannot be neglected. For example, schizosaccharomyces pombe has good alcohol-producing ability as a core wine-producing flora, and in Maotai-flavor white spirit, the schizosaccharomyces pombe is added as an enhanced yeast to fermented grains, so that the yield of ethanol can be improved without affecting the production of other flavor metabolites. Therefore, the diversity of the daqu fungus microbial community is revealed, the microbial driving force behind the change of the flavor substances is analyzed, the white spirit brewing process is improved, and the white spirit with stable quality is produced, so that the daqu fungus microbial community has great practical significance.

In recent years, high-throughput sequencing technology has been widely used for studying 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 our understanding of the white spirit microbial community structure. We can characterize the relative abundance of each species in the microbial community based on the ratio of the number of reads measured by OTU to the total number of reads of a single sample. However, due to the technical limitations of high throughput sequencing, the microbial composition is characterized only by relative abundance, and the absolute content index of the microorganism is lacking in the sequencing results. We can use the relative abundance to determine how advantageous a species is, but cannot be used to compare how much a species is in different samples. The representation form of the relative abundance makes cross-sample and cross-domain comparison difficult, and when the total amount of microorganisms in different samples is inconsistent, erroneous conclusion can be caused if the relative abundance is misused for cross-sample comparison. Therefore, absolute content is a constant quantitative indicator, is not affected by total amount variation, and has an irreplaceable advantage when comparing across samples.

However, the existing method for constructing recombinant plasmid to absolutely quantify fungi in white spirit samples is few, and the phenomenon that the fungi in white spirit samples are overestimated or underestimated in relative abundance compared with absolute abundance is common. Therefore, there are many problems to be solved in quantifying fungi in white spirit samples. The method for absolute quantification of fungi in white spirit samples fills the blank in the field, improves the recognition depth of the Daqu microbial community structure, compensates the absolute content deficiency in the high-throughput sequencing technology, and further analyzes the Daqu microbial community structure, which is very necessary and has important significance.

Disclosure of Invention

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

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

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

S2, sequencing the genome DNA extracted in the step S1 to obtain fungus species in a sample;

S3, adding an internal reference into the sample to be tested, wherein the internal reference is shown in SEQ ID NO.:1 is shown in the specification;

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

S5, mixing each recombinant plasmid constructed in the step S4 with the internal reference in the step S3 to obtain a mixture;

S6, detecting:

S6.1, detecting the following parameters of the sample to be tested containing the internal reference, which is prepared in S3: the number of reads R1 of the fungus in the test sample containing the internal reference and the number of reads R2 of the internal reference in the test sample containing the internal reference;

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

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

concentration of a certain fungus in a sample= (R1/R2) x correction factor of a certain fungus;

Correction factor for a fungus = concentration c× (r 1/r 2) of recombinant plasmid carrying ITS2 gene for that fungus in the mixture.

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

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

In some embodiments, the step S3 is to add an internal reference to the sample to be tested until the concentration of the internal reference is 1X 10 ⁶-1×10⁸ copies/mL; in some embodiments, the internal reference is added to a concentration of 1X 10 ⁷ copies/mL.

In some embodiments, the fungus is selected from at least one of pichia kudriavzevii (Pichia kudriavzevii), rhodotorula mucilaginosa (Rhodotorula mucilaginosa), wilms anomala (Wickerhamomyces anomalus), saccharomyces pombe (Saccharomycopsis fibuligera), torula delbrueckii (Torulaspora delbrueckii), rhizopus microsporidianus (Rhizopus microsporus), saccharomyces cerevisiae (Saccharomyces cerevisiae), paecilomyces varioti (Paecilomyces variotii), and schizosaccharomyces pombe (Schizosaccharomyces pombe).

In some embodiments, IT2 in step S4 is as set forth in at least one of SEQ ID NOS.4-12.

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

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

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

In some embodiments, the recombinant material transformed host cell is selected from fungi or bacteria; in some embodiments, the host cell is selected from bacteria; in some embodiments, the host cell is selected from the group consisting of E.coli.

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

The method of the invention is to obtain the kind of fungi in the sample to be tested, then to construct the recombinant plasmids with the ITS2 genes of the fungi respectively, and finally to mix the recombinant plasmids and to directly calculate the concentration of the fungi in the sample to be tested based on the reads number of each recombinant plasmid in the mixed plasmids, thereby avoiding the step of microorganism counting, having simpler operation and stronger purpose.

Compared with the previous method for quantifying all fungi in a sample, the method has the advantages that a recombinant plasmid is constructed for each fungus, the correction factor concept is introduced, one fungus is quantified by using one recombinant plasmid, the absolute abundance of the target fungus can be accurately estimated, and higher accuracy is realized.

The method makes up that only the relative abundance of the sample can be obtained by utilizing the high-flux sequencing technology, and the absolute abundance of the fungi can be accurately judged, so that the phenomenon that the abundance value of the fungi is overestimated or underestimated is effectively avoided. The accurate evaluation of the fungus abundance is beneficial to improving the understanding depth of the microbial community structure and makes up for the lack of absolute content in the high-throughput sequencing technology.

Drawings

Fig. 1: the pET-28a (+) plasmid and the synthesized double enzyme digestion verification result of internal reference DNA fragments respectively used for detecting absolute contents of pichia kudriavzevii, rhodotorula mucilaginosa, wilkham's yeast, oocyst membrane covered yeast, torulopsis delbrueckii, rhizopus microsporidianus, saccharomyces cerevisiae, paecilomyces variotii and schizosaccharomyces pombe.

Fig. 2: the PCR verification results of the recombinant plasmids are respectively used for detecting the absolute contents of pichia kudriavzevii, rhodotorula mucilaginosa, wilkham's yeast, oocyst complex film yeast, torulopsis delbrueckii, rhizopus microsporidianus, saccharomyces cerevisiae, paecilomyces variotii and schizosaccharomyces pombe.

Fig. 3: respectively used for detecting the plasmid maps of recombinant plasmids of absolute contents of pichia kudriavzevii, rhodotorula mucilaginosa, wilkham's yeast, oocyst complex film yeast, torulopsis delbrueckii, rhizopus microsporidianus, saccharomyces cerevisiae, paecilomyces variotii and schizosaccharomyces pombe.

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

Fig. 5: absolute quantitative sequencing results.

Table 4: abundance differences.

Fig. 6: a standard curve.

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples, which do not represent limitations on the scope of the present invention. Some insubstantial modifications and adaptations of the invention based on the inventive concept by others remain within the scope of the invention.

The invention is further illustrated below in conjunction with specific examples.

Coli JM109 and E.coli BL21 (DE 3) referred to in the examples below were purchased from North Nagawa, and pET-28a (+) plasmid was purchased from Novagen (E.coli BL21 (DE 3) of the above strain was commercially available, and no preservation for the patent program was required); the Daqu related in the following examples was purchased from Maotai-flavor liquor company in Guizhou area; the Plant Genomic DNA Kit plant genomic DNA extraction kit referred to in the examples below was purchased from root Biochemical technologies (beijing) limited.

The following examples relate to the following media:

LB liquid medium: yeast powder 5.0g L ^-1, tryptone 10.0g L ^-1、NaCl 10.0g L^-1, ampicillin 100 μ g L ^-1.

LB solid medium: yeast powder 5.0g L ^-1, tryptone 10.0g L ^-1、NaCl 10.0g L^-1, agar powder 15g L ^-1, ampicillin 100 μ g L ^-1.

Example 1: construction of methods useful for detecting absolute fungal content in a sample

The method comprises the following specific steps:

Extracting genome DNA of a sample to be detected, and carrying out high-throughput sequencing on a mixture by taking 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 according to an upstream primer and a downstream primer provided by reference "Fungi Sailing the Arctic Ocean:Speciose Communities in North Atlantic Driftwood as Revealed by High-Throughput Amplicon Sequencing" to obtain the type of fungi in the sample to be detected; adding an internal reference into a sample to be detected until the concentration of the internal reference in the sample to be detected is 1X 10 ⁷ copies/mL, and obtaining the sample to be detected containing the internal reference; respectively introducing ITS2 genes of fungi in a sample to be tested into the constructed recombinant plasmids to obtain recombinant plasmids respectively carrying the ITS2 genes of the fungi; mixing the recombinant plasmids with internal references to obtain a mixture; detecting the numbers of reads of each recombinant plasmid and the numbers of reads of the internal references in the mixture, detecting the numbers of reads of each fungus and the numbers of reads of the internal references in a sample to be tested containing the internal references, and calculating the concentration of each fungus in the sample to be tested according to the concentration of each recombinant plasmid in the mixture by using a formula, wherein the formula is as follows:

Concentration of a certain fungus in a sample = (number of reads of the fungus in a sample to be measured containing an internal reference/number of reads of an internal reference in a sample to be measured containing an internal reference) ×correction factor of the fungus;

Correction factor for a fungus = concentration of recombinant plasmid carrying the fungal ITS2 gene in the mixture/(number of reads of recombinant plasmid carrying the fungal ITS2 gene in the mixture/number of reads of reference in the mixture).

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

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

The absolute content of fungi in Daqu was measured using the method of example 1, the specific procedure is as follows:

Extracting genome DNA of Daqu to be detected by using Plant Genomic DNA Kit plant genome DNA extraction kit, taking DNA fragment with nucleotide sequence shown as SEQ ID NO.2 as an upstream primer, taking DNA fragment with nucleotide sequence shown as SEQ ID NO.3 as a downstream primer, performing high-throughput sequencing (high-throughput sequencing is completed by Aoweisen gene technology Co., ltd.), selecting Pichia kudri (Pichia kudriavzevii), rhodotorula mucilaginosa (Rhodotorula mucilaginosa), wilkinsoni (Wickerhamomyces anomalus), saccharomyces cerevisiae (Saccharomycopsis fibuligera), torulaspora delbrueckii (Torulaspora delbrueckii), rhizopus oligosporus (Rhizopus microsporus), saccharomyces cerevisiae (Saccharomyces cerevisiae), paecilomyces varioti (Paecilomyces variotii) and Schizosaccharomyces pombe (Schizosaccharomyces pombe) according to the high-throughput sequencing result, and determining the nucleotide sequences of Pichia kudri, rhodotorula glabra, paecilomyces anopsilosis, trichosporon, saccharomyces cerevisiae, paecilomyces varioti and Schizosaccharomyces pombe as shown in SEQ ID NO. 12-12, respectively, and the nucleotide sequences of Table (+) in SEQ ID NO.12 are determined.

According to the patent application text with publication number CN111172256A, preparing an internal reference; adding the internal reference into the yeast to be tested until the concentration of the internal reference in the yeast to be tested is 1X 10 ⁷ copies/mL, and obtaining the yeast to be tested containing the internal reference.

The method comprises the steps of chemically synthesizing ITS2 genes of Pichia kudriavzevii, rhodotorula mucilaginosa, wilkham's yeast, saccule-covered yeast, trichosporon delbrueckii, rhizopus microsporidianus, saccharomyces cerevisiae, paecilomyces variotii and Schizosaccharomyces pombe, and adding enzyme cutting sites of BamH and SalI at two ends of the ITS2 genes to obtain internal reference DNA fragments for detecting absolute contents of Pichia kudriavzevii, rhodotorula mucilaginosa, wilkham's yeast, saccule-covered yeast, trichosporon delbrueckii, rhizopus microsporidanus, saccharomyces cerevisiae, paecilomyces variotii and Schizosaccharomyces pombe.

The double enzyme digestion is carried out by taking the internal reference DNA fragments of pET-28a (+) plasmid and synthesized for detecting absolute contents of Pichia kudriavzevii, rhodotorula mucilaginosa, wilkham's yeast, zaocys, torulopsis delbrueckii, rhizopus microsporidianus, saccharomyces cerevisiae, paecilomyces variotii and Schizosaccharomyces pombe as templates, and BamHI and SalI (the enzyme digestion system is shown in Table 1, after the double enzyme digestion, 5 mu L of the double enzyme digestion products of the internal reference DNA fragments are taken and identified by 2% agarose gel electrophoresis, and the identification result is shown in figure 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 4 times of CP buffer into the double enzyme digestion product, adding the mixture into an upper layer pipe, centrifuging for 2min at 12000r/min, and repeating sample loading once; adding 700 mu L DNA Wash Buffer, centrifuging at 12000r/min for 2min, pouring out liquid of the lower collecting pipe, and repeating for one time; carrying out air separation on the empty adsorption column once, centrifuging for 2min at 12000r/min, replacing a new centrifuge tube with 1.5mL, and loading the column; heating to volatilize alcohol after air-separation, and heating to 60 ℃ for 1-2 min; adsorbing 30-50 mu L ddH ₂ O (preheated at 60 ℃), centrifuging at 12000r/min for 2min, eluting DNA; the column purified product was ligated using T4 ligase at 16℃for 16h (ligation systems see Table 2);

table 2 connection system

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

E.coli JM109 was transformed with the ligation product, the transformation product was spread on LB solid medium, cultured at 37℃for 8 hours, transformants were picked up on LB solid medium, inoculated on LB liquid medium, cultured at 37℃for 10 hours, and plasmids were extracted and subjected to ITS2F:5'-GCATCGATGAAGAACGCAGC-3' (nucleotide sequence shown as SEQ ID NO. 2) is an upstream primer, ITS2R:5'-TCCTCCGCTTATTGATATGC-3' (nucleotide sequence shown as SEQ ID NO. 3) is used as a downstream primer for PCR verification and sequencing to obtain recombinant plasmids which are respectively used for detecting absolute contents of pichia kudriavzevii, rhodotorula mucilaginosa, wilkham's yeast, zygosaccharomyces marxianus, trichosporon delbrueckii, rhizopus microsporum, saccharomyces cerevisiae, paecilomyces variotii and Schizosaccharomyces pombe and are correct in verification, wherein PCR reaction parameters are as follows: after 2min pre-denaturation at 94 ℃, 30s denaturation at 94 ℃, 45s annealing at 55 ℃, 30s extension at 72 ℃,25 cycles, 30s extension at 72 ℃ the PCR products were identified by 2% agarose gel electrophoresis (validation system see Table 3, results see 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

Transforming the recombinant plasmid which is verified to be correct into escherichia coli E.coli BL21 (DE 3) to obtain recombinant escherichia coli which respectively contains the recombinant plasmids for detecting the absolute contents of pichia kudriavzevii, rhodotorula mucilaginosa, wilkinsonii, zygosaccharomyces curcas, torulaspora delbrueckii, rhizopus microsporidianus, saccharomyces cerevisiae, paecilomyces variotii and Schizosaccharomyces pombe; coating the obtained recombinant escherichia coli on an LB solid culture medium, and culturing for 8-10 hours at 37 ℃ to obtain single colony; selecting single colony, inoculating the single colony into LB liquid culture medium, and culturing for 12-14 h at 37 ℃ to obtain seed liquid; inoculating the seed solution into LB liquid culture medium according to the inoculum size of 4% (v/v), and culturing at 37 ℃ and 200rpm for 8 hours to obtain fermentation liquor; centrifuging the fermentation broth at 4deg.C and 1000rpm for 20min, and collecting thallus; the recombinant plasmids obtained by extraction and amplification from thalli are respectively used for detecting the absolute content of pichia kudriavzevii, rhodotorula mucilaginosa, wilkham's yeast, oocyst membrane-covered yeast, torula delbrueckii, rhizopus microsporidianus, saccharomyces cerevisiae, paecilomyces varioti and schizosaccharomyces pombe (plasmid map will be shown in figure 3).

Mixing the obtained recombinant plasmids for detecting the absolute contents of pichia kudriavzevii, rhodotorula mucilaginosa, wilkham's yeast, oocyst membrane-covered yeast, torula delbrueckii, rhizopus microsporidianus, saccharomyces cerevisiae, paecilomyces variotii and schizosaccharomyces pombe with internal references according to the equal quantitative ratio (1:1:1:1:1:1:1:1:1:1) to obtain a mixture, wherein the contents of each recombinant plasmid and the internal references in the mixture are 1 multiplied by 10 ⁷ copies/g; the mixture was treated with ITS2F:5'-GCATCGATGAAGAACGCAGC-3' (nucleotide sequence shown as SEQ ID NO. 2) is an upstream primer, ITS2R:5'-TCCTCCGCTTATTGATATGC-3' (nucleotide sequence shown as SEQ ID NO. 3) is used as a downstream primer for high-throughput sequencing (high-throughput sequencing is completed by Beijing Aoweisen Gene technologies Co., ltd.) to obtain the numbers of reads of each recombinant plasmid and the numbers of reads of internal references in the mixture, and the Daqu to be tested containing the internal references is subjected to ITS2F:5'-GCATCGATGAAGAACGCAGC-3' (nucleotide sequence shown as SEQ ID NO. 2) is an upstream primer, ITS2R:5'-TCCTCCGCTTATTGATATGC-3' (nucleotide sequence shown as SEQ ID NO. 3) is used as a downstream primer for high-throughput sequencing (the high-throughput sequencing is completed by Beijing Aoweisen gene technology Co., ltd.) to obtain the numbers of reads of each fungus in the yeast to be tested containing the internal reference and the numbers of reads of the internal reference, and the concentration of each fungus in the yeast to be tested is calculated according to the concentration of each recombinant plasmid in the mixture by using a formula, wherein the formula is as follows:

Concentration= (number of reads R1 of the fungus in the sample to be measured containing the reference/number of reads R2 of the reference in the sample to be measured containing the reference) ×correction factor of the fungus;

Correction factor for a fungus = concentration c of recombinant plasmid carrying ITS2 gene for the fungus in the mixture/(number r1 of reads of recombinant plasmid carrying ITS2 gene for the fungus in the mixture/number r2 of reads of reference in the mixture).

Sequencing shows that the Daqu contains Pichia kudriavzevii, saccharomycetes, rhizopus oligosporus, saccharomyces cerevisiae, rhodotorula mucilaginosa, wilkham's yeast, torulopsis delbrueckii, paecilomyces variotii, and Schizosaccharomyces pombe is not included in the Daqu sample.

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

TABLE 5 quantitative results of concentration of R1, R2 values in Daqu samples for each fungus

From absolute quantitative calculations, we considered the 8 fungi contained in the Daqu as a whole, we compared the differences between their relative and absolute abundances according to the following formula (abundance comparison see fig. 4).

Difference in abundance of a fungus = the ratio of the relative abundance of the fungus in 8 fungi-the ratio of the absolute abundance of the 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 kudriavzevii overestimated was 50.73% compared to absolute abundance, while the bursa-covered yeast was underestimated 13.42% and saccharomyces cerevisiae was underestimated 28.62% (see table 6 for detailed results).

TABLE 6

Meanwhile, the quantitative result shows that the absolute content of saccharomyces cerevisiae in the Daqu is 5.89 multiplied by 10 ⁷ copies/g, the absolute content of the saccule-covered yeast in the Daqu is 4.85 multiplied by 10 ⁷ copies/g, the absolute content of rhizopus microsporidianus in the Daqu is 1.93 multiplied by 10 ⁷ copies/g, and the absolute content of pichia kudriavzevii in the Daqu is 8.59 multiplied by 10 ⁶ copies/g (the quantitative result is shown in table 5 and figure 5).

Example 3: verification of methods useful for detecting absolute fungal content in a sample

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

The specific implementation method is as follows: saccharomyces cerevisiae is screened from a sample, the cell number is 1.0X10 ⁹ cells/g by adopting a blood cell counting plate method, the cells are diluted to the corresponding cell number of 10 ⁸、10⁷、10⁶、10⁵、10⁴ cells/g respectively, the genomic DNA of the Saccharomyces cerevisiae is extracted by using a Plant Genomic Kit plant genomic DNA extraction kit, and Saccharomyces cerevisiae specific primers ScAIRIF-CACAATGGGGCAAAGGCTTC, SCAIRIR-GCCAGAACTGAAGCACAAGC are designed, and then fluorescence quantitative PCR is carried out on the extracted genome by a StepOnePlus instrument (purchased from Simer). The reaction system is as follows: the total system was 20. Mu.L, 2X FAST QPCR MASTER Mix 10. Mu.L, each of the upstream and downstream primers was 0.4. Mu.L, genome 1. Mu.L, and ultrapure water 8.2. Mu.L. The reaction conditions are as follows: pre-denaturation at 95℃for 5min, denaturation at 95℃for 10s, annealing at 50℃for 0.5min, extension at 70℃for 1min, 40 cycles total, 70℃for 10min.

And drawing a standard curve by taking CT values measured by qPCR as an abscissa and cell values corresponding to dilution concentrations as an ordinate (the standard curve is shown in figure 6). Substituting the obtained CT value 19.02 into the standard curve of FIG. 6 to finally obtain the Saccharomyces cerevisiae with the content of: 10 ⁷ cells/g. Because the copy number of the gene in the saccharomyces cerevisiae cell is not unique, the content of the saccharomyces cerevisiae in the sample is higher than 10 ⁷ copies/g.

The patent application text of publication No. CN111172256A carries out absolute quantification on each fungus in finished product height Wen Qufen, and the absolute content of Saccharomyces cerevisiae in the detection result is about 10 ⁶ copies/g. The results of example 2 show that the absolute content of Saccharomyces cerevisiae in the high temperature Daqu obtained by applying the method is 5.89×10 ⁷ copies/g. Therefore, the absolute content of the saccharomyces cerevisiae obtained by the method is closer to the true value.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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.

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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 genome DNA of a sample to be detected;

S3, adding an internal reference into the sample to be detected, wherein the sequence of the internal reference is shown as SEQ ID NO.:1, adding an internal reference to the mixture, wherein the concentration of the internal reference is 1X 10 ⁷ copies/mL;

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

s5, mixing the recombinant plasmids constructed in the step S4 with the internal reference in the step S3 according to the quantitative ratio to obtain a mixture, wherein the content of the recombinant plasmids and the internal reference in the mixture is 1 multiplied by 10 ⁷ copies/g;

S6, detecting:

Correction factor of a fungus = concentration c× (r 1/r 2) of recombinant plasmid carrying ITS2 gene of the fungus in the mixture;

The fungi are selected from at least one of pichia kudriavzevii (Pichia kudriavzevii), rhodotorula mucilaginosa (Rhodotorula mucilaginosa), wilkham's yeast (Wickerhamomyces anomalus), oocyst membrane-covered yeast (Saccharomycopsis fibuligera), torula delbrueckii (Torulaspora delbrueckii), rhizopus microsporidianus (Rhizopus microsporus), saccharomyces cerevisiae (Saccharomyces cerevisiae), paecilomyces varioti (Paecilomyces variotii) and schizosaccharomyces pombe (Schizosaccharomyces pombe);

The sequence of IT2 in the step S4 is shown as at least one of SEQ ID NO. 4-12;

The sample is Daqu.

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

3. The method of claim 2, wherein the primer for high throughput sequencing has a sequence set forth in SEQ ID No.:2 and 3.

4. The method of any one of claims 1-3, wherein the recombinant plasmid vector is selected from at least one of the group consisting of pET-28a (+) plasmid, pET-21a (+) plasmid, and PGEX-6P plasmid.

5. The method of any one of claims 1-3, wherein the recombinant plasmid vector is a pET-28a (+) plasmid.

6. A method according to any one of claims 1 to 3, wherein the host cell transformed with the recombinant plasmid is selected from fungi or bacteria.

7. The method of claim 6, wherein the host cell is selected from the group consisting of bacteria.

8. The method of claim 6, wherein the host cell is selected from the group consisting of E.coli.