CN112877459B - Probes for Absolute Quantification of Pichia kuderi Azwiya - Google Patents

Abstract

The invention discloses a probe for absolute quantification of Pichia kudriazvii, belonging to the fields of biology, fermentation and detection. The Pichia kudriavzevii quantitative probe and kit of the present invention can realize the detection of the total amount of Pichia kudriavzevii, do not need to use expensive instruments for the detection and quantification of Pichia kudriavzevii, and can quickly complete the quantitative work within 2.5 hours. At the same time, the samples used in the present invention do not necessarily undergo nucleic acid extraction. The probe and detection kit based on the present invention are used for Pichia kudriavzevii quantification, and have the characteristics of fast, convenient, cheap and accurate.

Description

Probes for Absolute Quantification of Pichia kuderi Azwiya

technical field

The invention relates to a probe for absolute quantification of Pichia kudriazvii, belonging to the fields of biology, fermentation and detection.

Background technique

Pichia kudriavzevii is an important functional microorganism in the traditional fermented food brewing system. For example, in the liquor brewing system, Pichia kudriavzevii is a high-abundance fungal microorganism that can metabolize the main components of liquor- Alcohol, the bacteria can regulate the microbial interaction and the structure of the fermenting microbial flora, which plays an important role in the normal fermentation of food. Therefore, real-time tracking of the biomass of Pichia kudriavzevii has important guiding significance for judging the stability of fermentation batches, judging whether the fermentation process is normal or not, and regulating real-time fermentation parameters. However, at present, most traditional fermented food systems are multi-strain co-fermentation systems, and the content of Pichia kudriavzevii in samples cannot be judged by simple OD colorimetry, although fluorescent quantitative PCR combined with specific primers or probes can achieve The quantification of Pichia kudriavzevii requires expensive equipment and a demanding operating environment. Therefore, in order to track the growth trend of Pichia kudriavzevii in samples conveniently, quickly and accurately, it is necessary to develop corresponding quantitative methods and kits for Pichia kudriavzevii .

The principle of G quadruplex/heme mimic enzyme activity detection is that G quadruplex can form a DNA mimic enzyme with catalase activity with heme, which can catalyze the oxidation of ABTS by hydrogen peroxide to generate ABTS+, showing a green color reaction , the characteristic absorbance value can be detected at a wavelength of 420 nm. The stability of the G quadruplex structure is crucial to the entire detection process. If the design is improper, when the G quadruplex sequence forms a dimer with other bases, it will cause the G quadruplex sequence to fail to form a G quadruplex. The use of quantitative methods based on this principle will lead to underestimation of the content of the target gene in the sample, reducing the sensitivity and accuracy of the detection method.

At present, the principle of G-quadruplex/heme-mimetic enzyme activity detection has been reported to be used for the specific detection of microorganisms; for example, the literature Wang Y, Li X, Xi D, Wang X. Visual detection of Fusariumproliferatum based on asymmetric recombinase In polymerase amplification andhemin/G-quadruplex DNAzyme. Rsc Advances 2019;9:37144-37147., asymmetric specific primers are used (the upstream primer adds the reverse sequence modification of the G quadruplex, and the downstream does not modify), this method only It can be applied to the detection of the specific bacteria Fusarium proliferatum in the sample, and the total detection of Pichia kudriavzevii cannot be realized; in addition, when the literature uses the asymmetric specific primers for detection, it adds different concentrations of upstream and downstream primers (upstream and downstream primers) to the PCR system. Low primer concentration, high concentration of downstream primers), amplified by recombinant polymerase amplification (RPA) to form double-stranded products, as the PCR reaction proceeds, the upstream primers are exhausted, and the downstream primers use newly synthesized double-stranded DNA as The template is amplified to form single-stranded DNA with G-quadruplex ends for the detection of Fusarium proliferatum in samples using the G-quadruplex/heme mimic enzyme assay. However, this quantitative method still requires a PCR step to generate a G quadruplex, and the PCR process still requires high-cost PCR equipment and a strict operating environment.

Contents of the invention

A probe, kit and application for the absolute quantification of Pichia kudriavzevii of the present invention solve at least one of the following technical problems: (1) the existing method cannot realize the total amount detection of all Pichia kudriavzevii ; (2) the current Some quantitative methods have low species resolution and/or insufficient detection accuracy; (3) Existing quantitative methods require high-cost instruments and/or strict operating environments, and are not suitable for timely detection after production sampling; (4) ) Existing quantitative methods are cumbersome to operate, etc.

The first object of the present invention is to provide a set of probes, including a signal probe and a quencher probe; the sequence of the signal probe is shown in SEQ ID NO.1 (GGGTGGGTGGGTGGGTGTTTGAGCGTCGTTTCCATC).

In one embodiment, the quenching probe sequence is shown in SEQ ID NO.2 (GATGGAAACGACGCTCCAAACACCCA).

A second object of the present invention is to provide a Pichia kudriavzevii quantification method comprising the use of the probes of the present invention.

The method comprises: melting of DNA in the sample to be tested; adding an excess signal probe (sequence such as SEQ ID NO.1) to combine with the target nucleotide fragment of the sample to be tested to form a double strand, so that the G quadruplex is exposed Outside the sequence; adding a sufficient amount of quenching probe (sequence such as SEQ ID NO.2) to form a double strand with the unbound signal probe, destroying the G quadruplex structure; using the naked G quadruplex and heme The reaction forms a G-quadruplex/heme mimetic enzyme with catalase activity, which in combination with catalase activity characterizes the biomass of Pichia kudriavzevii .

In one embodiment, the method is an absolute quantitative method, further comprising: establishing catalase activity (or an index correlated with catalase activity, such as catalyzing hydrogen peroxide to oxidize ABTS to generate ABTS+ after the solution is The absorbance value at a wavelength of 420 nm) and the standard curve of the biomass of Pichia kudriavzevii ; when detecting the sample to be tested, the detected catalase activity was substituted into the standard curve to obtain the biomass of Pichia kudriavzevii in the sample to be tested.

In one embodiment, the method is a relative quantitative method, further comprising: detecting a plurality of samples, and determining the ratio of the biomass of Pichia kudriavzevii in the plurality of different samples according to the relative ratio of the catalase activity detected by different samples relative value.

In one embodiment, the sample to be tested is a sample containing bacteria, genome or metagenomics. Optionally, the sample to be tested is a finished product of fermented food or a sample taken from the fermentation process of fermented food; optionally, the sample to be tested is subjected to pretreatment such as centrifugation and collection of bacteria before subsequent determination. Preferably, after the bacteria in the sample are collected, the DNA melting process is directly performed without genome extraction.

In one embodiment, the sample is a fermented food or a sample taken from a fermentation process of a fermented food or an environmental sample.

In one embodiment, the fermented food is any one or more of the following: liquor, rice wine, soy sauce, beer, wine, vinegar, fermented tea, traditional fermented vegetables, fermented beverage, alcoholic beverage, yogurt, cheese, fruit vinegar , fermented glutinous rice, fermented soya bean, curd, fermented rice noodle food, etc.; the environmental sample is an environmental sample selected from intestinal tract, soil, and water body.

In one embodiment, the melting of the DNA in the sample to be tested is carried out in a high temperature manner. Optionally, the sample to be tested is processed at a temperature higher than 90°C. It can be any environment that can provide the corresponding temperature, such as a metal bath, a water bath, an oven, or an incubator.

In one embodiment, said melting is performed in a buffer. Optionally, the buffer may be a Tris-HCl buffer, further containing any one or more of KCl, NH ₄ Cl, and NaCl. Optionally, the buffer is Tris-HCl, KCl, pH=7.9.

In one embodiment, the excess refers to that the added amount is higher than the amount of the signal probe required to combine with all the target nucleotide fragments of the sample to be tested to form a double strand. The specific dosage can be determined by those skilled in the art in combination with common knowledge in the field or specific samples to be tested, or through preliminary experiments.

In one embodiment, the excess refers to more than 10 ¹⁰ copies of the signaling probe.

In one embodiment, the combination of the signal probe with the target nucleotide fragment of the sample to be tested to form a double strand is carried out at a temperature range of 50-60°C.

In one embodiment, the sufficient amount refers to the amount of the quenching probe required when the added amount is sufficient to form double strands with all unbound signal probes. The specific dosage can be determined by those skilled in the art in combination with common knowledge in the field or specific samples to be tested, or determined through preliminary experiments.

In one embodiment, the sufficient amount refers to double the amount of signaling probes.

In one embodiment, the addition of a sufficient amount of the quenching probe to form a double strand with the unbound signal probe is performed at a temperature that enables the quencher probe to form a double strand with the unbound signal probe; Those skilled in the art can determine in combination with common knowledge in the field or a specific sample to be tested.

In one embodiment, the use of naked G quadruplexes to react with heme to form G quadruplexes/heme mimetic enzymes with catalase activity, combined with the activity of catalase to characterize Pichia kudriavzevii Biomass refers to adding ABTS and H ₂ O ₂ to the system after adding heme for reaction, and then characterizing the catalase activity by the light absorbance value of the reactants.

In one embodiment, the absorbance value is the absorbance value at a wavelength of 420 nm.

In one embodiment, the quantitative method is specifically:

(1) The sample to be tested is subjected to DNA melting treatment;

(2) Add the signal probe and react at 55 °C for 30 min;

(3) Add a quenching probe and react at 55 °C for 30 min;

(4) Add heme and react at 37 °C for 30 min;

(5) Add 2,2-azino-bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt (ABTS) and H ₂ O ₂ , react at 37°C for 30 min ;

(6) Detect the absorbance value of the reactant at a wavelength of 420 nm;

(7) Quantify Pichia kudriavzevii in the sample by combining the absorbance value.

In one embodiment, the quantitative method further includes: configuring samples with different known Pichia kudriavzevii contents, and measuring the absorbance values of different samples after being processed by the above method; drawing a standard curve between absorbance values and different Pichia kudriavzevii contents; Substitute the absorbance value obtained after the sample to be tested through the above method into the standard curve to obtain the content of Pichia kudriavzevii in the sample to be tested.

The third object of the present invention is to provide a detection kit for absolute quantification of Pichia kudriavzevii , which contains a signal probe of the sequence of the present invention such as SEQ ID NO.1.

In one embodiment, the detection kit further contains a quencher probe with a sequence such as SEQ ID NO.2.

In one embodiment, the detection kit also contains any one or more of the following: heme, buffer, 2,2-azino-bis-(3-ethylbenzodihydrothiazoline- 6-sulfonic acid) diammonium salt (ABTS), H ₂ O ₂ . These reagents may not be included, and the operator must prepare them separately when using the kit.

In one embodiment, in the detection kit, the buffer may be Tris-HCl buffer, and may also contain any one or more of KCl, NH ₄ Cl, and NaCl. Optionally, the buffer is Tris-HCl, KCl, pH=7.9.

In one embodiment, the detection kit is a Pichia kudriavzevii absolute quantification kit, which includes four reagents (reagent 1, reagent 2, reagent 3, reagent 4) and a set of Pichia kudriavzevii quantitative probes (signal probe, quenching probe); the reagent 1 includes heme; the reagent 2 includes buffer (Tris-HCl, KCl, pH=7.9; KCl can be replaced by NH ₄ Cl, NaCl) ; The reagent 3 includes 2,2-azino-bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt (ABTS); the reagent 4 includes H ₂ O ₂ .

In one embodiment, in the detection kit, the reagent or probe may be in a liquid state or a solid state, and those skilled in the art can routinely adjust to an appropriate concentration during use.

The fourth object of the present invention is to provide a method for using the kit.

In one embodiment, the method of use comprises: adding an excess signal probe to the sample to be tested in which the DNA melts and reacting for a period of time, so that the signal probe binds to the target fragment in the sample to be tested; Quantitatively quench the probe to form a double strand with the unbound signal probe; then add heme, add ABTS and H ₂ O ₂ after a period of reaction, react for a period of time, detect the absorbance value of the reactant, and combine the absorbance value Quantification of Pichia kudriavzevii in samples.

In one embodiment, the method includes adjusting reagents and probes to concentrations suitable for use.

(1) The sample to be tested was subjected to DNA melting treatment; (2) Signal probe was added and reacted at 55 °C for 30 min; (3) Quenching probe was added and reacted at 55 °C for 30 min; (4) Heme was added, React at 37°C for 30 min; (5) Add 2,2-azino-bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt (ABTS) and H ₂ O ₂ , React at 37°C for 30 min; (6) Detect the absorbance of the reactants at a wavelength of 420 nm; (7) Quantify Pichia kudriavzevii in the sample based on the absorbance.

The fifth object of the present invention is to provide the application of said kit in Pichia kudriavzevii quantification.

In one embodiment, the application is in the field of fermented food technology or the environment; optionally, the fermented food is any one or more of the following: white wine, rice wine, soy sauce, beer, wine, vinegar, fermented food Tea, traditional fermented vegetables, fermented beverages, alcoholic beverages, yogurt, cheese, fruit vinegar, fermented rice, tempeh, curd, fermented rice noodles, etc.; the environmental samples are selected from the intestinal tract, soil, and water.

In one embodiment, during the application, the sample to be tested may be a sample containing bacteria, genome or metagenomics. Optionally, the sample to be tested is a finished product of fermented food or a sample taken from the fermentation process of fermented food or an environmental sample; optionally, the sample to be tested is subjected to pretreatment such as centrifugation and collection of bacteria before subsequent determination. Preferably, after the bacteria in the sample are collected, the DNA melting process is directly performed without genome extraction.

Beneficial effect:

In the present invention, the G quadruplex is combined with a specific sequence to form a signal probe, and the signal probe is combined with the target sequence so that the G quadruplex is exposed outside the sequence, and a sufficient amount of quenched probe and unreacted signal probe are added Form a double chain, destroy the G quadruplex structure, form a G quadruplex/heme mimic enzyme by reacting with heme, exhibit catalase activity, and use catalase activity to characterize the biomass of microorganisms. The Pichia kudriavzevii quantitative probe of the present invention can realize the total detection of Pichia kudriavzevii ; further, the signal probe is optimized, the signal probe sequence is GGGTGGGTGGGTGGGTGTTTGAGCGTCGTTTCCATC (SEQ ID NO.1), and the quenching probe is GATGGAAACGACGCTCCAAACACCCA (SEQ ID NO.2). Compared with the signal sequence of SEQ ID NO.3, the G quadruplex sequence in the signal probe of SEQ ID NO.1 does not produce an additional spatial structure with the specific sequence (Figure 1), and the detection accuracy is higher and the lowest The detection limit is improved.

When the probe of the present invention is used for detection and quantification of Pichia kudriavzevii , the detection process of expensive instruments is not required. It also provides a kit for absolute quantification of microorganisms for the first time, which can complete the quantitative work within 2.5 hours. In order to avoid the use of high-cost equipment, such as a PCR instrument, the present invention realizes microbial quantification by combining signal probes and quenching probes. The invention solves the problem that the current microbiological quantification means rely on relatively expensive instruments and are very limited in the actual application process.

Further, the present invention can realize rapid Pichia kudriavzevii detection, the sample does not need to be subjected to nucleic acid extraction, and only the microorganisms in the sample need to be eluted in the buffer, and subsequent experiments can be directly performed. At the same time, compared with the quantitative results of fluorescent quantitative PCR, the quantitative results obtained by the present invention have no significant difference.

In conclusion, the probe and detection kit provided by the present invention are fast, cheap and accurate for the quantification of Pichia kudriavzevii .

Description of drawings

Figure 1: Signaling probe dimer structure. (A) The G-quadruplex sequence of SEQ ID NO.1 does not form a loop with the specific sequence; (B) The reported G-quadruplex sequence of SEQ ID NO.3 used for microbial quantification forms a self-contained sequence with the specific sequence ring.

Figure 2: Specificity of Pichia kudriavzevii probes.

Figure 3: Standard curve of Pichia kudriavzevii quantitative probes based on genome extraction.

Figure 4: Standard curve based on Pichia kudriavzevii quantitative probes without extracting the sample genome.

Figure 5: qPCR standard curve.

Figure 6: Comparison of Pichia kudriavzevii probe quantification experiments based on genome extraction, Pichia kudriavzevii probe quantification experiments based on non-extracted sample genomes, and qPCR Pichia kudriavzevii quantitative experiments; among them, (A) Pichia kudriavzevii probe quantification experiments based on non-extracted sample genomes Experiment, (B) Pichia kudriavzevii probe quantitative experiment based on genome extraction, (C) qPCR Pichia kudriavzevii quantitative experiment.

Figure 7: Comparison of the stability of detection results based on the probe of SEQ ID NO.1/SEQ ID NO.2 (A) and the probe of SEQ ID NO.3/SEQ ID NO.4 (B).

Detailed ways:

Example 1: Pichia kudriavzevii quantitative probe combination reagent

Probe combination reagent; containing independently packaged signal probe reagent and quenching probe reagent; wherein, the signal probe sequence is as shown in SEQ ID NO.1, and the quenching probe sequence is as shown in SEQ ID NO.2 .

Signaling probe reagents and quenching probe reagents are in dry powder or liquid form; in dry powder form, they can be diluted to an appropriate concentration before the experiment, for example, diluted with sterile water or buffer solution to a concentration of 20 μM; in liquid form When used, the concentration can be 20-200 μM, and the reagent can be diluted before use, or used directly.

Embodiment 2: Pichia kudriavzevii quantitative kit and its use

The Pichia kudriavzevii quantitative kit contains independently packaged signal probe reagents and quenching probe reagents; wherein, the signal probe sequence is shown in SEQ ID NO.1, and the quenching probe sequence is shown in SEQ ID NO.2.

When this kit is used, it can be mixed with heme, buffer, 2,2-azino-bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt (ABTS), H ₂ Use with _O2 .

The method of use is:

(1) Solution configuration. Prepare 100 nM heme solution (Reagent 1); prepare Tris-HCL with a final concentration of 50 mM, KCl with a final concentration of 50 mM, and a final pH of 7.9 (Reagent 2); 7 mM 2,2-azino - Bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt (ABTS) (Reagent 3) and ₇ mM _H2O2 solution (Reagent 4); solvents are sterile water .

(2) The signal probe forms a double strand with the sample DNA. Add 4 μL of sample genomic DNA to 2 mL of reagent 2, and treat in a water bath at 90 °C for 10 min. After adding 4 μL of 20 μM signal probe, react at 55 °C for 30 min.

(3) The quencher probe forms a double strand with the unbound signal probe. The quencher probe doubles with the unbound signal probe, disrupting the G quadruplex structure. Add 8 μL of 20 μM quenching probe to the system after the reaction in step (2), and react at 55 °C for 30 min.

(4) Formation of heme/G quadruplex structure. Add reagent 1 at a final concentration of 100 nM to the system after the reaction in step (3), and treat at 37 °C for 30 min.

(5) Color reaction. Add reagent (ABTS) with a final concentration of 7 mM and reagent 4 with a final concentration of 7 mM to the system after the reaction in (4), and treat at 37°C for 30 min to perform a display reaction (green).

Detect the absorbance value of the reactant at a wavelength of 420 nm; combine the absorbance value to quantify Pichia kudriavzevii in the sample.

Of course, when performing absolute quantification, you can draw the standard curve between the absorbance value and the biomass of Pichia kudriavzevii by yourself, or directly convert the biomass of Pichia kudriavzevii according to the method recommended by the kit and the standard curve.

Example 3: Pichia kudriavzevii Quantification Kit

The Pichia kudriavzevii quantitative kit contains independently packaged signal probe reagents and quenching probe reagents; wherein, the signal probe sequence is as shown in SEQ ID NO.1, and the quenching probe sequence is as shown in SEQ ID NO.2 .

The kit also contains 100 nM heme solution (Reagent 1), Tris-HCL buffer, 7 mM 2,2-azino-bis-(3-ethylbenzodihydrothiazoline-6- sulfonic acid) diammonium salt (ABTS), 7 mM H ₂ O ₂ solution.

Example 4: Specificity of the Pichia kudriavzevii Quantification Kit

(1) Pichia kudriavzevii from fermented grains was selected as a positive control, and 36 bacterial species and 6 fungal microorganisms widely present in fermented food samples were selected as negative controls. The bacterial microorganisms were Lactobacillus buchneri , Lactobacillus dioilvorans , Lactobacillus brevis , Lactobacillus crustorum , Lactobacillus plantarum , Lactobacillus harbinensis , Lactobacillus acidiliscis , Pediococcus ethanolidurans , Pediococcus acidilactici , Pediococcus pentosaceus , Lactobacillus murinus , Lactobacillus curvatus , Lactobacillus casei , Lactobacillus reuteri , Lactobacillus panis , Lactobacillus fermentum , Lactobacillus johnsonii , Lactobacillus delbrueckii , Lactococcus lactis , Weissella confusa , Weissella paramesenteroides , Weissella viridescens , Leuconostoc citreum , Leuconostoc lactis , Leuconostoc mesenteroides , Leuconostoc pseudomesenteroides , Enterococcus italicus , Enterococcus lactis , Enterococcus faecal is , Bacillus coagulans , Bacillus licheniformis , Bacillus tequilensis , Bacillus subtilis , Bacillus velezensis , Acetobacter pasteurianus , Enterococcus faecium . The fungal microorganisms are Aspergillus tubingensis , Mucor rouxianus , Schizosaccharomyces pombe , Zygosaccharomyces bailii , Saccharomycopsis fibuligera , Saccharomyces cerevisiae .

(2) The above microorganisms were cultured in different media, among which Lactobacillus buchneri , Lactobacillus dioilvorans , Lactobacillus brevis , Lactobacillus crustorum , Lactobacillus plantarum , Lactobacillus harbinensis , Lactobacillus acidiliscis , Pediococcus ethanolidurans , Pediococ cus acidilactici , Pediococcus pentosaceus , Lactobacillus murinus , Lactobacillus curvatus , Lactobacillus casei , Lactobacillus reuteri , Lactobacillus panis , Lactobacillus fermentum , Lactobacillus johnsonii , Lactobacillus delbrueckii , Lactococcus lactis , Weissella confusa , Weissella paramesenteroides , Weissella viridescens , Leuconostoc citreum , Leuconostoc lactis , Leuconostoc mesenteroides , Leuconostoc pseudomesenteroides use MRS medium, the medium formula is Tryptone 10.0 g/L, beef extract 8.0 g/L, yeast extract 4.0 g/L, glucose 18.0 g/L, anhydrous sorbitan oleate 0.8 mL/L, K ₂ HPO ₄ 2.5 g/L, Sodium acetate trihydrate 6.0 g/L, triammonium citrate 2.0 g/L, MgSO ₄ ·7H ₂ O 0.3 g/L, MnSO ₄ ·4H ₂ O 0.08 g/L. The culture condition was 30°C for 48 h. Enterococcus italicus , Enterococcus lactis , Enterococcus faecalis , Bacillus coagulans , Bacillus licheniformis , Bacillus tequilensis , Bacillus subtilis , Bacillus velezensis , Acetobacter pasteurianus , Enterococcus faecium use LB medium, The medium formula is peptone 10.0 g/L, yeast powder 5 g/L , sodium chloride 10 g/L. The culture condition was 37°C for 24 h. Aspergillus tubingensis , Mucor rouxianus , Schizosaccharomyces pombe , Zygosaccharomyces bailii , Pichia kudriavzevii , Saccharomycopsis fibuligera , Saccharomyces cerevisiae use YPD medium, the medium formula is yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L L. The culture conditions were as follows: the mold was cultured at 30°C for 5 days, and the yeast was cultured at 30°C for 2 days.

(3) Single bacterial genome extraction. The above bacterial solution was treated at 12000 rpm for 2 min, and the precipitate was collected. The genomes of 43 microbial pure cultures were extracted using the gene extraction kit DNeasy Tissue Kit.

(4) The probe was selected as a Pichia kudriavzevii specific probe, the sequence of the signal probe was GGGTGGGTGGGTGGGTGTTTGAGCGTCGTTTCCATC (SEQ ID NO.1), and the sequence of the quenching probe was GATGGAAACGACGCTCAAACACCCA (SEQ ID NO.2).

(4) The signal probe forms a double strand with the sample DNA. Add 4 μL of genomic DNA of different microorganisms to 2 mL of reagent 2 (including Tris-HCL at a final concentration of 50 mM, KCl at a final concentration of 50 mM, and a final pH of 7.9), and treat in a water bath at 90 °C for 10 min. After adding 4 μL of 20 μM signal probe, react at 55 °C for 30 min.

(5) The quencher probe forms a double strand with the unbound signal probe, destroying the G quadruplex structure. Add 8 μL of 20 μM quenching probe to the system after the reaction in step (4), and react at 55 °C for 30 min.

(6) Formation of heme/G quadruplex structure. Add reagent 1 (heme) at a final concentration of 100 nM to the system after the reaction in step (5), and treat at 37 °C for 30 min.

(7) Color reaction. Add reagent 3 (ABTS) with a final concentration of 7 mM and reagent 4 (H ₂ O ₂ ) with a final concentration of 7 mM to the system after the reaction in (6), and treat at 37°C for 30 min. The results are shown in Figure 2, the experimental group added Pichia kudriavzevii genome showed a color reaction, and the experimental group added non- Pichia kudriavzevii and the blank control group did not appear color reaction, proving the specificity of detecting Pichia kudriavzevii in this kit.

Example 5: Quantitative method accuracy assessment

(1) The Pichia kudriavzevii bacterial liquid was obtained according to the cultivation method in Example 4, the microbial concentration was determined by plate counting method, and the genome extraction was the same as in Example 4.

(2) Pichia kudriavzevii genomic DNA was diluted by 10-fold gradient.

(3) Using the probe of Pichia kudriavzevii , perform color reaction with different concentrations of Pichia kudriavzevii genomic DNA. The sequence of the signal probe is GGGTGGGTGGGTGGGTGTTTGAGCGTCGTTTCCATC (SEQ ID NO.1), and the sequence of the quencher probe is GATGGAAACGACGCTCAAACACCCA (SEQ ID NO.2).

(4) The signal probe forms a double strand with the sample DNA. Add 4 μL of different dilutions of genomic DNA to 2 mL of reagent 2 (including Tris-HCL at a final concentration of 50 mM, KCl at a final concentration of 50 mM, and a final pH of 7.9) (no sample DNA is used as a blank control) . Incubate in a water bath at 90°C for 10 min. After adding 4 μL of 20 μM signal probe, react at 55 °C for 30 min.

(5) The quencher probe forms a double strand with the unbound signal probe, destroying the G quadruplex structure. Add 8 μL of 20 μM quenching probe to the system after the reaction in step (4), and react at 55 °C for 30 min.

(6) Formation of heme/G quadruplex structure. Add reagent 1 (heme) at a final concentration of 100 nM to the system after the reaction in step (5), and treat at 37 °C for 30 min.

(7) Color reaction. Add reagent 3 (ABTS) with a final concentration of 7 mM and reagent 4 (H ₂ O ₂ ) with a final concentration of 7 mM to the system after the reaction in (6), and treat at 37°C for 30 min. The absorbance value at a wavelength of 420 nm was measured by an ultraviolet spectrophotometer, and the experimental group without sample DNA was used as a blank control.

(8) Construct a standard curve by calculating the linear relationship between the absorbance value and the concentration of the bacterial solution, as shown in Figure 3, R ² =0.99 (the unit of x is log10 CFU/mL, the unit of y is OD ₄₂₀ , and the linear range is 10 ³ ~10 ⁷ ). Prove the accuracy of the kit quantitative method provided by the present invention.

Example 6: Quantitative experiment of Pichia kudriavzevii in wine samples

(1) Refer to Gayevskiy, V.,&Goddard, M.(2012). Geographic delineations of yeast communities and populations associated with vines and wines in New Zealand. ISME J, 6(7), 1281-1290. Materials and methods method, sample Collected from a well-known wine manufacturer in Yantai, Shandong. Genome concentration was 658.39 ng/μL.

(2) Color reaction using the probe of Pichia kudriavzevii . The sequence of the signal probe is GGGTGGGTGGGTGGGTGTTTGAGCGTCGTTTCCATC (SEQ ID NO.1), and the sequence of the quencher probe is GATGGAAACGACGCTCAAACACCCA (SEQ ID NO.2).

(4) The signal probe forms a double strand with the sample DNA. To 2 mL of Reagent 2 (including Tris-HCL at a final concentration of 50 mM, KCl at a final concentration of 50 mM, and a final pH of 7.9), add 4 μL of sample metagenomic DNA (without sample DNA as a blank control). Incubate in a water bath at 90°C for 10 min. After adding 4 μL of 20 μM signal probe, react at 55 °C for 30 min.

(5) The quencher probe forms a double strand with the unbound signal probe, destroying the G quadruplex structure. Add 8 μL of 20 μM quenching probe to the system after the reaction in step (4), and react at 55 °C for 30 min.

(6) Formation of heme/G quadruplex structure. Add reagent 1 (heme) at a final concentration of 100 nM to the system after the reaction in step (5), and treat at 37 °C for 30 min.

(7) Color reaction. Add reagent 3 (ABTS) with a final concentration of 7 mM and reagent 4 (7 mM H ₂ O ₂ ) with a final concentration of 7 mM to the system after the reaction in (6), and treat at 37°C for 30 min. The absorbance value at a wavelength of 420 nm was measured by an ultraviolet spectrophotometer, and the experimental group without sample DNA was used as a blank control, and the absorbance value was 0.44.

(8) According to the standard curve obtained in Example 5, the total amount of Pichia kudriavzevii in the sample was calculated to be 5.92 log ₁₀ CFU/mL.

(9) Quantify Pichia kudriavzevii in the same sample above by fluorescent quantitative PCR (quantification steps and materials are the same as in Example 11 (6)), and the results show that the total amount of Pichia kudriavzevii is 5.92 log ₁₀ CFU/mL, which is the same as that determined by the above method The quantitative results were basically consistent (coefficient of variation, CV=0.0005).

Example 7: Absolute quantification of Pichia kudriavzevii in a sample of fermented grains

(1) Refer to Song Z W, Du H, Zhang Y, Xu Y. Unraveling core functional microbiota in traditional solid-state fermentation by high-throughput amplicons and metatranscriptomics sequencing. Frontiers in microbiology 2017;8:1294 MATERIALS AND METHODS method, extract The metagenomics in the fermented grains samples from Shandong Province, the genome concentration was 100.02 ng/μL.

(2) Color reaction using the probe of Pichia kudriavzevii . The sequence of the signal probe is GGGTGGGTGGGTGGGTGTTTGAGCGTCGTTTCCATC (SEQ ID NO.1), and the sequence of the quencher probe is GATGGAAACGACGCTCAAACACCCA (SEQ ID NO.2).

(3) The signal probe forms a double strand with the sample DNA. To 2 mL of reagent 2 (including Tris-HCL at a final concentration of 50 mM, KCl at a final concentration of 50 mM, and a final pH of 7.9), add 4 μL of fermented grain metagenomic DNA (no sample DNA was added as a blank control). Incubate in a water bath at 90°C for 10 min. After adding 4 μL of 20 μM signal probe, react at 55 °C for 30 min.

(4) The quencher probe forms a double strand with the unbound signal probe, destroying the G quadruplex structure. Add 8 μL of 20 μM quenching probe to the system after the reaction in step (3), and react at 55 °C for 30 min.

(5) Formation of heme/G quadruplex structure. Add reagent 1 (heme) at a final concentration of 100 nM to the system after the reaction in step (4), and treat at 37 °C for 30 min.

(6) Color reaction. Add reagent 3 (ABTS) with a final concentration of 7 mM and reagent 4 (H ₂ O ₂ ) with a final concentration of 7 mM to the system after the reaction in (5), and treat at 37°C for 30 min. The absorbance value at a wavelength of 420 nm was measured by an ultraviolet spectrophotometer, and the experimental group without sample DNA was used as a blank control, and the absorbance value was 0.612.

(7) According to the standard curve obtained in Example 2, the total microbial count of Pichia kudriavzevii in the sample was calculated to be 6.78 log ₁₀ CFU/mL.

(8) Quantify Pichia kudriavzevii in the same fermented grain sample above by fluorescence quantification (quantification steps and materials are the same as in Example 11 (6)), and the results show that the total microbial load of Pichia kudriavzevii is 6.71log ₁₀ CFU/mL, which is the same as The quantitative results determined by the above methods were basically consistent (coefficient of variation, CV=0.008).

Embodiment 8: Absolute quantitative method based on Pichia kudriavzevii without extracting sample genome

(1) The Pichia kudriavzevii bacterial liquid was obtained according to the cultivation method in Example 4, and the microbial concentration was determined by the plate count method.

(2) Dilute the Pichia kudriavzevii bacterial solution in (1) by 10 times

(3) Color reaction using the probe of Pichia kudriavzevii . The sequence of the signal probe is GGGTGGGTGGGTGGGTGTTTGAGCGTCGTTTCCATC (SEQ ID NO.1), and the sequence of the quencher probe is GATGGAAACGACGCTCAAACACCCA (SEQ ID NO.2).

(4) The signal probe forms a double strand with the sample DNA. To 2 mL of reagent 2 (including Tris-HCL with a final concentration of 50 mM, KCl with a final concentration of 50 mM, and a final pH of 7.9), add 10 μL of bacterial solutions of different dilutions (no sample bacterial solution is used as a blank control) ). Treat in a boiling water bath for 20 min. After adding 4 μL of 20 μM signal probe, react at 55 °C for 30 min.

(5) The quencher probe forms a double strand with the unbound signal probe, destroying the G quadruplex structure. Add 8 μL of 20 μM quenching probe to the system after the reaction in step (4), and react at 55 °C for 30 min.

(6) Formation of heme/G quadruplex structure. Add reagent 1 (heme) at a final concentration of 100 nM to the system after the reaction in step (5), and treat at 37 °C for 30 min.

(7) Color reaction. Add reagent 3 (ABTS) with a final concentration of 7 mM and reagent 4 (H ₂ O ₂ ) with a final concentration of 7 mM to the system after the reaction in (6), and treat at 37°C for 30 min. The absorbance value at a wavelength of 420 nm was measured by an ultraviolet spectrophotometer, and the experimental group without sample DNA was used as a blank control.

(8) Construct a standard curve by calculating the linear relationship between the absorbance value and the concentration of the bacterial solution, as shown in Figure 4, R ² =0.99 (the unit of x is log10 CFU/mL, the unit of y is OD ₄₂₀ , and the linear range is 10 ³ ~10 ⁷ ). Prove the accuracy of the kit quantitative method provided by the present invention

Example 9: Determination of the content of Pichia kudriavzevii in wine samples based on the absolute quantitative method of microorganisms without extracting the sample genome

(1) The samples were collected from a well-known wine manufacturer in Yantai, Shandong. The sample processing method was as follows: 5 mL of phosphate buffer was added to 1 mL of the sample, and the bacteria were collected by centrifugation at 3000 × g for 10 min.

(2) Washing. Add 5 mL of phosphate buffer solution to the cells obtained in (1), centrifuge at 12,000 × g for 2 min to collect the cells, and repeat once.

(3) To resuspend the bacteria, add 1 mL of reagent 2 (including Tris-HCL at a final concentration of 50 mM, KCl at a final concentration of 50 mM, and a final pH of 7.9) to the bacterial cells obtained in (2), Mix by blowing and aspirating.

(4) Use the probe of Pichia kudriavzevii for color reaction. The sequence of the signal probe is GGGTGGGTGGGTGGGTGTTTGAGCGTCGTTTCCATC (SEQ ID NO.1), and the sequence of the quencher probe is GATGGAAACGACGCTCAAACACCCA (SEQ ID NO.2).

(5) The signal probe forms a double strand with the sample DNA. Add 10 μL of grape fermentation broth to 2 mL of Reagent 2 (including Tris-HCL at a final concentration of 50 mM, KCl at a final concentration of 50 mM, and a final pH of 7.9) (no sample broth is used as a blank control). Incubate in a boiling water bath for 20 min. After adding 4 μL of 20 μM signal probe, react at 55 °C for 30 min.

(6) The quencher probe forms a double strand with the unbound signal probe, destroying the G quadruplex structure. Add 8 μL of 20 μM quenching probe to the system after the reaction in step (5), and react at 55 °C for 30 min.

(7) Formation of heme/G quadruplex structure. Add reagent 1 (heme) at a final concentration of 100 nM to the system after the reaction in step (6), and treat at 37 °C for 30 min.

(8) Color reaction. Add reagent 3 (ABTS) with a final concentration of 7 mM and reagent 4 (H ₂ O ₂ ) with a final concentration of 7 mM to the system after the reaction in (7), and treat at 37°C for 30 min. The absorbance value at a wavelength of 420 nm was measured by an ultraviolet spectrophotometer, and the experimental group without sample DNA was used as a blank control, and the absorbance value was 0.512.

(9) According to the standard curve obtained in Example 8, the total amount of Pichia kudriavzevii in the sample was calculated to be 5.89 log ₁₀ CFU/mL.

(10) Quantify Pichia kudriavzevii in the same sample above by fluorescent quantitative PCR (quantification steps and materials are the same as in Example 11 (6)), the results show that the total amount of Pichia kudriavzevii is 5.92 log ₁₀ CFU/mL, which is the same as that determined by the above method The quantitative results were basically consistent (coefficient of variation, CV=0.003).

Example 10: Determination of the content of Pichia kudriavzevii in the fermented grains sample based on the absolute quantitative method without extracting the sample genome

(1) The samples came from the fermented grains of a winery in Shandong. The sample processing method was as follows: 5 mL of phosphate buffer was added to 1 g of the sample, and the bacteria were collected by centrifugation at 3000 × g for 10 min.

(2) Washing. Add 5 mL of phosphate buffer solution to the cells obtained in (1), centrifuge at 12,000 × g for 2 min to collect the cells, and repeat once.

(3) To resuspend the bacteria, add 1 mL of reagent 2 (including Tris-HCL at a final concentration of 50 mM, KCl at a final concentration of 50 mM, and a final pH of 7.9) to the bacterial cells obtained in (2), Mix by blowing and aspirating.

(4) Use the probe of Pichia kudriavzevii for color reaction. The sequence of the signal probe is GGGTGGGTGGGTGGGTGTTTGAGCGTCGTTTCCATC (SEQ ID NO.1), and the sequence of the quencher probe is GATGGAAACGACGCTCAAACACCCA (SEQ ID NO.2).

(5) The signal probe forms a double strand with the sample DNA. To 2 mL of Reagent 2 (including Tris-HCL at a final concentration of 50 mM, KCl at a final concentration of 50 mM, and a final pH of 7.9), add 10 μL of wine fermented grains (without sample bacteria as a blank control). Incubate in a boiling water bath for 20 min. After adding 4 μL of 20 μM signal probe, react at 55 °C for 30 min.

(6) The quencher probe forms a double strand with the unbound signal probe, destroying the G quadruplex structure. Add 8 μL of 20 μM quenching probe to the system after the reaction in step (5), and react at 55 °C for 30 min.

(7) Formation of heme/G quadruplex structure. Add reagent 1 (heme) at a final concentration of 100 nM to the system after the reaction in step (6), and treat at 37 °C for 30 min.

(8) Color reaction. Add reagent 3 (ABTS) with a final concentration of 7 mM and reagent 4 (H ₂ O ₂ ) with a final concentration of 7 mM to the system after the reaction in (7), and treat at 37°C for 30 min. The absorbance value at a wavelength of 420 nm was measured by an ultraviolet spectrophotometer, and the experimental group without sample DNA was used as a blank control, and the absorbance value was 0.592.

(9) According to the standard curve obtained in Example 8, the total amount of Pichia kudriavzevii in the sample was calculated to be 6.69 log ₁₀ CFU/mL,

(10) Quantify Pichia kudriavzevii in the same sample above by fluorescent quantitative PCR (quantification steps and materials are the same as in Example 11 (6)), the results show that the total amount of Pichia kudriavzevii is 6.71 log ₁₀ CFU/mL, which is the same as that determined by the above method The two groups of data were basically consistent (coefficient of variation, CV=0.0017).

Embodiment 11: Microbial Quantitative Detection Kit and Fluorescent Quantitative PCR Detection Result Comparison

(1) The samples were selected from three samples of liquor fermented grains from a winery in Shandong at the end of fermentation.

(2) Sample processing:

(i) The total genomes in the three samples were extracted, and the genome concentrations were 369 ng/μL, 590 ng/μL, and 321.89 ng/μL, respectively.

(ii) Add 5 mL of phosphate buffer to 1 g of sample, and centrifuge at 3000 × g for 10 min to collect the bacteria. Add 5 mL of phosphate buffer to the obtained cells, collect the cells by centrifugation at 12000 × g for 2 min, and repeat once. The bacteria were resuspended, and 1 mL of reagent 2 buffer was added to the obtained bacteria, and mixed by pipetting.

(3) Color reaction using the probe of Pichia kudriavzevii . The sequence of the signal probe is GGGTGGGTGGGTGGGTGTTTGAGCGTCGTTTCCATC (SEQ ID NO.1), and the sequence of the quencher probe is GATGGAAACGACGCTCAAACACCCA (SEQ ID NO.2).

(4) Determination based on the quantitative method of the kit without extracting the genome.

(i) The signal probe forms a double strand with the sample DNA. To 2 mL of Reagent 2 (including Tris-HCL at a final concentration of 50 mM, KCl at a final concentration of 50 mM, and a final pH of 7.9), add 10 μL of wine fermented grains (without sample bacteria as a blank control). Treat in a boiling water bath for 20 min. After adding 4 μL of 20 μM signal probe, react at 55 °C for 30 min.

(ii) The quencher probe forms a duplex with the unbound signal probe, disrupting the G quadruplex structure. Add 8 μL of 20 μM quenching probe to the system after the reaction in step (i), and react at 55 °C for 30 min.

(iii) Formation of a heme/G quadruplex structure. Add reagent 1 (heme) with a final concentration of 100 mM to the system after the reaction in step (ii), and treat at 37 °C for 30 min.

(iv) Color reaction. Add reagent 3 (ABTS) with a final concentration of 7 mM and reagent 4 (H ₂ O ₂ ) with a final concentration of 7 mM to the system after the reaction in (iii), and treat at 37°C for 30 min. The absorbance value at a wavelength of 420 nm was measured by an ultraviolet spectrophotometer, and the experimental group without sample DNA was used as a blank control, and the absorbance values were 0.598, 0.595, and 0.620.

(v) According to the standard curve obtained in Example 8, the total amount of Pichia kudriavzevii in the sample was calculated to be 6.82 ± 0.14 log ₁₀ CFU/mL.

(5) Determination of kit quantitative method based on genome extraction

(i) The signal probe forms a double strand with the sample DNA. To 2 mL of reagent 2 (including Tris-HCL at a final concentration of 50 mM, KCl at a final concentration of 50 mM, and a final pH of 7.9), add 4 μL of fermented grain metagenomic DNA (no sample DNA was added as a blank control). Incubate in a water bath at 90°C for 10 min. After adding 4 μL of 20 μM signal probe, react at 55 °C for 30 min.

(ii) The quencher probe forms a duplex with the unbound signal probe, disrupting the G quadruplex structure. Add 8 μL of 20 μM quenching probe to the system after the reaction in step (i), and react at 55 °C for 30 min.

(iii) Formation of a heme/G quadruplex structure. Add reagent 1 (heme) at a final concentration of 100 nM to the system after the reaction in step (ii), and treat at 37 °C for 30 min.

(iv) Color reaction. Add reagent 3 (ABTS) with a final concentration of 7 mM and reagent 4 (H ₂ O ₂ ) with a final concentration of 7 mM to the system after the reaction in (5), and treat at 37°C for 30 min. The absorbance value at a wavelength of 420 nm was measured by an ultraviolet spectrophotometer, and the experimental group without sample DNA was used as a blank control, and the absorbance values were 0.613, 0.625, and 0.612.

(v) According to the standard curve obtained in Example 5, the total amount of Pichia kudriavzevii in the sample was calculated to be 6.83 ± 0.071 log ₁₀ CFU/mL.

(6) Pichia kudriavzevii content in quantitative samples by qPCR

(i) The Pichia kudriavzevii bacterial liquid was obtained according to the cultivation method in Example 4, the microbial concentration was determined by the plate counting method, and the extraction of the genome was the same as in Example 4.

(ii) Pichia kudriavzevii genomic DNA was diluted by a 10-fold gradient.

(iii) The qPCR system is 10 μL of SYBR Green, 20 μM of upstream and downstream primers, 0.5 μL of template DNA, and 20 μL of sterile water.

(iv) Reaction program for qPCR: pre-denaturation at 95 °C for 5 min, cycle phase: 95 °C for 5 s, 60 °C for 20 s; cycle number 40, melting curve from 65 °C to 95 °C, every 5 s Increase by 0.5 °C.

(v) qPCR was performed on the extracted genome using Pichia kudriavzevii specific primers, the downstream sequence of the primer sequence was GTTTGAGCGTCGTTTCCATC (SEQ ID NO.5), and the downstream sequence was ATACCCTTCTAACACCTGGC (SEQ ID NO.6).

(vi) Genomic DNA was serially diluted 10 times to establish a standard curve between CT value and bacterial concentration of Pichia kudriavzevii , as shown in Figure 4, R ² =0.99.

(vii) The qPCR system and reaction conditions are the same as (iii) and (iv). According to the CT value at the end of the reaction, the concentration of Pichia kudriavzevii in the sample was calculated by the established standard curve to be 6.799 ± 0.122 log ₁₀ CFU/g.

(7) Through significant difference analysis, the results are shown in Figure 6, there is no significant difference among the three quantitative methods ( P <0.05)

Example 12: Detection limit of detection using two different sequence signal probes

(1) The Pichia kudriavzevii bacterial liquid was obtained according to the cultivation method in Example 4, and the microbial concentration was determined by plate counting method, and the concentration was 7.49 log10 CFU/mL. The extraction of the genome was the same as in Example 4.

(2) Genomic DNA of Pichia kudriavzevii was serially diluted 10 times to obtain 2.1 log10 CFU/mL DNA template.

(3) The sequence of the Pichia kudriavzevii signal probe provided by the present invention is GGGTGGGTGGGTGGGTGTTTGAGCGTCGTTTCCATC (SEQ ID NO.1), and the sequence of the quenching probe is GATGGAAACGACGCTCAAACACCCA (SEQ ID NO.2). Add 3.2 log ₁₀ CFU/mL Pichia kudriavzevii genomic DNA obtained in (2) for color reaction.

(4) The Pichia kudriavzevii signal probe sequence is (SEQ ID NO.3) GGGATTGGGATTGGGATTGGGGTTTGAGCGTCGTTTCCATC, and the quenching probe sequence is GATGGAAACGACGCTCAAACCCCAA (SEQ ID NO.4). Add 3.2 log10 CFU/mL Pichia kudriavzevii genomic DNA obtained in (2) for color reaction.

(5) The signal probe forms a double strand with the sample DNA. 4 μL of Pichia kudriavzevii genomic DNA will be added to 2 mL of Reagent 2 (including Tris-HCL at a final concentration of 50 mM, KCl at a final concentration of 50 mM, and a final pH of 7.9) (no sample DNA is added as a blank control). Incubate in a water bath at 90°C for 10 min. After adding 4 μL of 20 μM different signal probes, react at 55 °C for 30 min.

(6) The quencher probe forms a double strand with the unbound signal probe, destroying the G quadruplex structure. Add 8 μL of 20 μM quenching probes to the system after the reaction in step (5), and react at 55 °C for 30 min.

(7) Formation of heme/G quadruplex structure. Add reagent 1 (heme) at a final concentration of 100 nM to the system after the reaction in step (6), and treat at 37 °C for 30 min.

(8) Color reaction. Reagent 3 (ABTS) with a final concentration of 7 mM and reagent 4 (H ₂ O ₂ ) with a final concentration of 7 mM were added to the system after the reaction in (7), and treated at 37°C for 30 min. The absorbance value at a wavelength of 420 nm was measured by an ultraviolet spectrophotometer, and the experimental group without sample DNA was used as a blank control.

(9) Repeat steps (5) (6) (7) (8) 9 times to compare the stability of the test results, as shown in Figure 7. The coefficient of variation (CV) of the quantitative result of the signal sequence based on SEQ ID NO.3 is 1.04; the coefficient of variation of the quantitative result of the signal sequence based on SEQ ID NO.1 is 0.02, and the detection effect is stable.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

SEQUENCE LISTING

<110> Jiangnan University

<120> Probes for Absolute Quantification of Pichia Kudriazvii

<160> 4

<170> PatentIn version 3.3

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<212>DNA

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gggtgggtgg gtgggtgttt gagcgtcgtt tccatc 36

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<212>DNA

<213> Synthetic

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gatggaaacg acgctcaaac acccca 25

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<212>DNA

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gggattggga ttgggattgg ggtttgagcg tcgtttccat c 41

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<212> DNA

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gatggaaacg acgctcaaac cccaa 25

Claims (11)

1. A group of probes, characterized in that, consist of signal probes and quenching probes; the signal probe sequence is the sequence shown in SEQ ID NO.1; the quenching probe sequence is the sequence shown in SEQ ID NO.2 sequence. 2. A detection kit, characterized in that it contains the signaling probe and the quenching probe according to claim 1. 3. The detection kit according to claim 2, characterized in that, the detection kit also contains any one or more of the following: heme, buffer, 2,2-azino-bis-(3 -Ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt, H ₂ O ₂ . 4. A Pichia kudriavzevii quantitative method, characterized in that the method uses the probe according to claim 1, or the detection kit according to any one of claims 2-3. 5. The quantitative method according to claim 4, characterized in that, the method comprises: the DNA in the sample to be tested melts; adding an excess signal probe combines with the target nucleotide fragment of the sample to be tested to form a double strand , to make the G quadruplex naked out of the sequence; add enough quencher probe to form a double strand with the unbound signal probe, destroying the G quadruplex structure; use the naked G quadruplex to react with heme Formation of a G-quadruplex/heme mimetic enzyme with catalase activity combined with catalase activity to characterize the biomass of Pichia kudriavzevii . 6. quantitative method according to claim 4, is characterized in that, described method is absolute quantification or relative quantification; When described method is absolute quantification, also comprises: establish catalase activity or with catalase activity Correlation index, and the standard curve of the biomass of Pichia kudriavzevii ; When detecting the sample to be tested, the detected catalase activity or the index that is correlated with the catalase activity are substituted into the standard curve to obtain the standard curve to be tested. The biomass of Pichia kudriavzevii in the samples was measured. 7. The quantitative method according to claim 5 or 6, wherein the sample to be tested is a sample containing bacteria, genome or metagenomic group. 8. The quantitative method according to claim 7, wherein the sample is a fermented food or a sample taken from a fermented food fermentation process, or an environmental sample. 9. The quantitative method according to claim 8, wherein the fermented food is any one or more of the following: soy sauce, vinegar, cheese, tempeh, fermented bean curd, traditional fermented vegetables, fermented beverages, alcoholic beverages, Fermented rice noodle food; the environmental sample is an environmental sample selected from intestinal tract, soil, and water body. 10. The method for using the kit according to any one of claims 2-3, characterized in that, the method for using comprises: adding an excessive amount of signal probe to the sample to be tested in which the DNA melts and reacting for a period of time to make the signal probe Combine with the target fragment in the sample to be tested; then add enough quenching probe to form a double strand with the unbound signal probe; then add heme, add ABTS and H ₂ O ₂ after a period of reaction, After reacting for a period of time, detect the absorbance value of the reactant, and combine the absorbance value to quantify the Pichia kudriavzevii in the sample. 11. A method for detecting Pichia kudriavzevii content in fermented food or environmental samples, comprising utilizing the probe described in claim 1, or the kit described in any one of claims 2-3; said fermented food is any of the following One or more species: soy sauce, vinegar, cheese, tempeh, fermented bean curd, traditional fermented vegetables, fermented beverages, alcoholic beverages, fermented rice noodle foods; the environmental samples are environmental samples selected from intestinal tract, soil, and water bodies.