CN112899381A

CN112899381A - Absolute quantitative probe, method and application thereof

Info

Publication number: CN112899381A
Application number: CN202110163271.9A
Authority: CN
Inventors: 吴群; 徐岩; 杜如冰
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-02-05
Filing date: 2021-02-05
Publication date: 2021-06-04
Anticipated expiration: 2041-02-05
Also published as: CN112899381B

Abstract

The invention discloses an absolute quantitative probe, a method and application thereof, belonging to the fields of biology, fermentation and detection. The acid-resistant Lactobacillus acetoterorans quantitative probe and the kit can realize the total amount detection of Lactobacillus acetoterorans, do not need expensive instruments when used for detection and Lactobacillus acetoterorans quantification, and can quickly complete the quantification within 2.5 hours. Meanwhile, the sample used in the present invention does not have to be subjected to nucleic acid extraction. The probe and the detection kit based on the invention are used for the quantification of Lactobacillus acetoterrans, and have the characteristics of rapidness, convenience, cheapness and accuracy.

Description

Absolute quantitative probe, method and application thereof

Technical Field

The invention relates to an absolute quantitative probe, a method and application thereof, belonging to the fields of biology, fermentation and detection.

Background

Acid-resistant Lactobacillus (Lactobacillus acetolerans) is a lactic acid bacterium widely distributed in a traditional fermented food fermentation system, and research shows that Lactobacillus acetolerans is an absolutely dominant microorganism in the middle and later periods of the white spirit fermentation process, correlation analysis shows that Lactobacillus acetolerans has correlation with the formation of various flavor compounds, the change of biomass is closely related to the quality of final products, and the biomass and succession of the Lactobacillus acetolerans in the food fermentation process are important marker microorganisms for representing normal food fermentation. Therefore, the real-time tracking of the biomass of Lactobacillus acetoterrans has important guiding significance for judging the stability of the fermentation batch and regulating and controlling the fermentation parameters. However, most of the conventional fermented food systems are multi-strain co-fermentation systems, the content of Lactobacillus acetoerrors in a sample cannot be judged by a simple OD colorimetric method, and although the quantification of Lactobacillus acetoerrors in a mixed bacteria system can be realized by combining a fluorescent quantitative PCR method with a specific primer or probe, high-volume equipment and high-requirement operating environment are required. Therefore, in order to conveniently, rapidly and accurately track the growth change trend of Lactobacillus acetoerrors in a sample, a corresponding Lactobacillus acetoerrors quantitative method and a kit need to be developed.

The principle of enzyme activity detection of the G quadruplex/heme analogue is that the G quadruplex can form DNA mimic enzyme with catalase activity with heme, can catalyze hydrogen peroxide to oxidize ABTS to generate ABTS +, presents green color development reaction, and can detect characteristic light absorption value under the wavelength of 420 nm. The stability of the structure of the G quadruplex is crucial to the whole detection process, if the design is not proper, when the G quadruplex sequence and other bases form a dimer, the G quadruplex sequence cannot form the G quadruplex, and a quantitative method based on the principle can underestimate the content of a target gene in a sample in use and reduce the sensitivity and the accuracy of the detection method.

At present, the principle of enzyme activity detection based on G quadruplex/heme simulation has been reported to be used for specific detection of microorganisms; for example, the documents WangY, Li X, Xi D, Wang X.visual detection of Fusarium proliferatum based on enzymmetric cloning enzyme amplification and hemin/G-quadruplex DNAzyme. Rsc Advances 2019; 37144-37147. in the method, an asymmetric specific primer (an upstream primer is modified by adding a reverse sequence of a G quadruplex, and the downstream is not modified) is used, so that the method is only suitable for detecting specific bacteria Fusarium proliferatum in a sample, and cannot realize the total amount detection of Lactobacillus acetogerans; in the case of detection using the asymmetric specific primer, the upstream primer and the downstream primer are added to a PCR system at different concentrations (the upstream primer is low in concentration and the downstream primer is high in concentration), a double-stranded product is formed by amplification using Recombinant Polymerase Amplification (RPA), the upstream primer is depleted as the PCR reaction proceeds, the downstream primer is amplified using newly synthesized double-stranded DNA as a template, and a single-stranded DNA having a G quadruplex end is formed, and Fusarium proliferatum in a detection sample is detected using a G quadruplex/heme mimic enzyme activity. However, this quantitative method still requires a PCR step to generate G quadruplexes, and the PCR process still requires high-volume PCR equipment and a strict operating environment.

Disclosure of Invention

The probe, the kit and the application for the absolute quantification of Lactobacillus acetotropirans of the invention solve at least one technical problem as follows: (1) the existing method can not realize the total amount detection of all Lactobacillus acetoterorans; (2) the existing quantitative method has low species resolution and/or insufficient detection accuracy; (3) the existing quantitative method needs high-volume instruments and equipment and/or strict operation environment, and is not suitable for timely detection after production sampling; (4) the existing quantitative method has complicated operation and the like.

The first purpose of the invention is to provide a group of probes, which comprise a signaling probe and a quenching probe; the sequence of the signal probe is shown as SEQ ID NO.1 (GGGTGGGTGGGTGGGTAAAAAGCAGAGTGGAGAAAATACT).

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

It is a second object of the present invention to provide a method for quantification of Lactobacillus acetomolecules, which comprises using the probe of the present invention.

The method comprises the following steps: melting DNA in a sample to be detected; adding excessive signal probe (sequence shown as SEQ ID NO.1), and combining with target nucleotide fragment of the sample to be detected to form double chains, so that the G quadruplex is exposed outside the sequence; adding sufficient quenching probe (with sequence shown as SEQ ID NO.2) to form double chains with the unbound signal probe, and destroying the structure of the G quadruplex; the G quadruplex with the naked leakage outside reacts with the heme to form G quadruplex/heme mimic enzyme with catalase activity, and the biomass of Lactobacillus acetotropirans is characterized by combining the activity of the catalase.

In one embodiment, the method is an absolute quantitative method, further comprising: establishing a standard curve of catalase activity (or an index which is related to the catalase activity, such as an absorbance value of a solution after ABTS is catalyzed and oxidized by hydrogen peroxide to generate ABTS + at a wavelength of 420 nm) and biomass of Lactobacillus acetogens; and when a sample to be detected is detected, substituting the detected catalase activity into the standard curve to obtain the biomass of the Lactobacillus acetotropirans in the sample to be detected.

In one embodiment, the method is a relative quantification method, further comprising: detecting a plurality of samples, and determining the relative value of the biomass of the Lactobacillus acetomolecules in the plurality of different samples according to the relative ratio of the catalase activities detected by the different samples.

In one embodiment, the sample to be tested is a sample containing bacteria, genome, metagenome, or the like. Optionally, the sample to be detected is a finished fermented food product or a sample obtained in a fermentation process of fermented food; optionally, the sample to be tested is subjected to pretreatment such as centrifugation and collection of bacteria, and then subsequent measurement is performed. Preferably, the cells in the sample are collected and subjected to DNA melting directly without genome extraction.

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

In one embodiment, the fermented food is any one or more of: white spirit, yellow wine, soy sauce, beer, wine, table vinegar, fermented tea, traditional fermented vegetables, fermented beverages, alcoholic drinks, yogurt, cheese, fruit vinegar, fermented glutinous rice, fermented soya beans, fermented bean curd, fermented rice and flour foods and the like; the environmental sample is selected from intestinal tract, soil, water body and the like.

In one embodiment, the melting of the DNA in the sample to be tested is performed at a high temperature. Optionally, the sample to be tested is treated at a temperature above 90 ℃. Can be any one of metal bath, water bath, oven, heat preservation instrument and the like which can provide the environment with corresponding temperature.

In one embodiment, the melting is performed in a buffer. Optionally, the buffer solution can be Tris-HCl buffer solution, and also contains KCl and NH₄Any one or more of Cl and NaCl. Optionally, the buffer is Tris-HCl, KCl, pH 7.9.

In one embodiment, the excess is an amount that is greater than the amount of signaling probe required to form a duplex with all of the target nucleotide fragments of the sample to be tested. The specific amount can be determined by one skilled in the art by combining with common knowledge in the art or a specific sample to be tested, or by pre-experiment.

In one embodiment, the excess is in excess of 10¹⁰Copies of the signal probe.

In one embodiment, the binding of the signaling probe to the target nucleotide fragment of the sample to be tested to form a duplex is performed at a temperature in the range of 50-60 ℃.

In one embodiment, the sufficient amount is an amount of quenching probe that is required to form a double strand with all unbound signaling probes. The specific amount can be determined by one skilled in the art by combining with common knowledge in the art or by a specific sample to be tested, or by preliminary experiments.

In one embodiment, the sufficient amount is a double amount of signaling probe.

In one embodiment, the addition of a sufficient amount of quenching probe to form a duplex with the unbound signaling probe is at a temperature such that the quenching probe forms a duplex with the unbound signaling probe; one skilled in the art can determine or determine the specific sample to be tested in combination with common knowledge in the art.

In one embodiment, the use of the bare leak in the outer G quadruplex with bloodThe heme reaction forms G quadruplex/heme mimic enzyme with catalase activity, and the characterization of the biomass of Lactobacillus acetoterorans by combining the activity of catalase means that ABTS and H are added after heme reaction is added into a system₂O₂The catalase activity was then characterized by the absorbance of the reaction.

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

In one embodiment, the quantification method is, in particular:

(1) carrying out DNA unzipping treatment on a sample to be detected;

(2) adding a signal probe, and reacting at 55 ℃ for 30 min;

(3) adding a quenching probe, and reacting at 55 ℃ for 30 min;

(4) adding heme, and reacting at 37 deg.C for 30 min;

(5) adding 2, 2-azino-bis- (3-ethylbenzodihydropyrazoline-6-sulfonic acid) diammonium salt (ABTS) and H₂O₂Reacting at 37 ℃ for 30 min;

(6) detecting the light absorption value of the reactant at the wavelength of 420 nm;

(7) and (4) quantifying Lactobacillus acetomolecules in the sample by combining the absorbance value.

In one embodiment, the quantification method further comprises: preparing samples with different known Lactobacillus acetoterorans contents, and measuring the light absorption values of the different samples after the different samples are treated by the method; drawing a standard curve of the light absorption value and the contents of different Lactobacillus acetomolecules; substituting the light absorption value obtained by processing the sample to be detected by the method into the standard curve to obtain the content of Lactobacillus acetoterorans in the sample to be detected.

The third purpose of the invention is to provide a detection kit for the absolute quantification of Lactobacillus acetotropirans, which contains a signal probe with the sequence shown as SEQ ID NO. 1.

In one embodiment, the detection kit further comprises a quenching probe with a sequence shown in SEQ ID NO. 2.

In one embodiment, the method comprisesThe detection kit also comprises any one or more of the following components: heme, buffer solution, 2-azino-bis- (3-ethylbenzodihydropyrazoline-6-sulfonic acid) diammonium salt (ABTS), and H₂O₂. These reagents may not be contained, and an operator may prepare the reagent kit separately when the reagent kit is used.

In one embodiment, in the detection kit, the buffer solution can be Tris-HCl buffer solution, and also contains KCl and NH₄Any one or more of Cl and NaCl. Optionally, the buffer is Tris-HCl, KCl, pH 7.9.

In one embodiment, the detection kit is a Lactobacillus acetoerrors absolute quantification kit, which simultaneously comprises four reagents (reagent 1, reagent 2, reagent 3, reagent 4) and a set of Lactobacillus acetoerrors quantitative probes (signal probes, quenching probes); the reagent 1 comprises heme; the reagent 2 comprises a buffer solution (Tris-HCl, KCl, pH 7.9; wherein KCl can be replaced by NH)₄Cl, NaCl); the reagent 3 comprises 2, 2-azino-bis- (3-ethylbenzodihydropyrazoline-6-sulfonic acid) diammonium salt (ABTS); the reagent 4 comprises H₂O₂。

In one embodiment, the reagent or probe in the test kit may be in a liquid state or a solid state, and may be adjusted to a suitable concentration by those skilled in the art in use.

The fourth purpose of the invention is to provide a using method of the kit.

In one embodiment, the method of use comprises: adding excessive signal probes into a sample to be detected after the DNA is unzipped, and reacting for a period of time to enable the signal probes to be combined with target fragments in the sample to be detected; then adding a sufficient amount of quenching probe to form a double strand with the unbound signaling probe; adding heme, reacting for a while, adding ABTS and H₂O₂Reacting for a period of time, detecting the light absorption value of the reactant, and quantifying Lactobacillus acetomolecules in the sample by combining the light absorption value.

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

(1) Carrying out DNA unzipping treatment on a sample to be detected; (2) adding a signal probe, and reacting at 55 ℃ for 30 min; (3) adding a quenching probe, and reacting at 55 ℃ for 30 min; (4) adding heme, and reacting at 37 deg.C for 30 min; (5) adding 2, 2-azino-bis- (3-ethylbenzodihydropyrazoline-6-sulfonic acid) diammonium salt (ABTS) and H₂O₂Reacting at 37 ℃ for 30 min; (6) detecting the light absorption value of the reactant at the wavelength of 420 nm; (7) and (4) quantifying Lactobacillus acetomolecules in the sample by combining the absorbance value.

The fifth purpose of the invention is to provide the application of the kit in the quantification of Lactobacillus acetotropirans.

In one embodiment, the use is in the technical or environmental field of fermented food; optionally, the fermented food is any one or more of the following: white spirit, yellow wine, soy sauce, beer, wine, table vinegar, fermented tea, traditional fermented vegetables, fermented beverages, alcoholic drinks, yogurt, cheese, fruit vinegar, fermented glutinous rice, fermented soya beans, fermented bean curd, fermented rice and flour foods and the like; the environmental sample is selected from intestinal tract, soil, water body and the like.

In one embodiment, the sample to be tested may be a sample containing bacteria, genome, metagenome, or the like. Optionally, the sample to be detected is a finished fermented food product or a sample obtained in a fermentation process of fermented food; optionally, the sample to be tested is subjected to pretreatment such as centrifugation and collection of bacteria, and then subsequent measurement is performed. Preferably, the cells in the sample are collected and subjected to DNA melting directly without genome extraction.

Has the advantages that:

the invention combines the G quadruplex with a specific sequence to form a signal probe, the signal probe is combined with a target sequence to ensure that the G quadruplex is barely leaked outside the sequence, a sufficient amount of quenching probe is added to form a double chain with an unreacted signal probe, the structure of the G quadruplex is damaged, a G quadruplex/heme mimic enzyme is formed by reaction with heme, catalase activity is shown, and the biomass of microorganisms is represented by the catalase activity. The quantitative probe of Lactobacillus acetoerrors can realize the total amount detection of the Lactobacillus acetoerrors; further, the signaling probe was optimized with the sequence of the signaling probe GGGTGGGTGGGTGGGTAAAAAGCAGAGTGGAGAAAATACT (SEQ ID NO.1) and the quenching probe AGTATTTTCTCCACTCTGCTTTTTACCCA (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 generate an additional spatial structure with a specific sequence (FIG. 1), the detection accuracy is higher, and the minimum detection limit is improved.

When the probe is used for detection and quantification of Lactobacillus acetomolecules, a detection process of an expensive instrument is not needed. The kit for absolutely quantifying the microorganisms is also provided for the first time, and the quantification work can be completed within 2.5 hours. The invention aims to avoid using high-cost equipment such as a PCR instrument and realize the quantification of microorganisms by combining a signal probe and a quenching probe. The invention solves the problem that the prior microorganism quantitative means all depend on expensive instruments and are very limited in the practical application process.

Furthermore, the rapid detection method can realize rapid detection of Lactobacillus acetocolumns, and a sample does not need to be subjected to nucleic acid extraction, and only the microorganisms in the sample need to be eluted into a buffer solution for directly carrying out subsequent experiments. Meanwhile, compared with the fluorescent quantitative PCR quantitative result, the quantitative result obtained by the method has no significant difference.

In conclusion, the probe and the detection kit provided by the invention are used for quantifying Lactobacillus acetotropirans, and have the characteristics of rapidness, cheapness and accuracy.

Drawings

FIG. 1: a dimeric structure of a signaling probe. (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 is used for microbial quantification.

FIG. 2: specificity of Lactobacillus acetolerans probes.

FIG. 3: a standard curve of quantitative probes based on genome extracted Lactobacillus acetomolecules.

FIG. 4: standard curve based on Lactobacillus acetoerrors quantitative probe without sample genome extraction.

FIG. 5: qPCR standard curve.

FIG. 6: comparing a quantitative experiment of a Lactobacillus acetoolorans probe based on genome extraction, a quantitative experiment of a Lactobacillus acetoolorans probe based on not extracting a sample genome and a quantitative experiment of qPCRLactobacillus acetoolorans; the quantitative analysis method comprises the following steps of (A) a quantitative experiment of a Lactobacillus acetoerrors probe based on non-extracted sample genome, (B) a quantitative experiment of a Lactobacillus acetoerrors probe based on genome extraction, and (C) a quantitative experiment of a qPCRLactulobacter acetoerrors probe.

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

The specific implementation mode is as follows:

example 1: lactobacillus acetolerans quantitative probe combined reagent

A probe combination reagent; comprises a signaling probe reagent and a quenching probe reagent which are packaged separately; wherein, the sequence of the signal probe is shown as SEQ ID NO.1, and the sequence of the quenching probe is shown as SEQ ID NO. 2.

The signal probe reagent and the quenching probe reagent are in a dry powder or liquid state; in the case of dry powder, it may be diluted to an appropriate concentration prior to the experiment, for example, to a concentration of 20. mu.M using sterile water or buffer; in the case of liquid, the concentration may be 20 to 200. mu.M, and the reagent may be diluted before use or may be used as it is.

Example 2: lactobacillus acetolerans quantitative kit and use thereof

The Lactobacillus acetotropirans quantitative kit comprises a signal probe reagent and a quenching probe reagent which are independently packaged; wherein, the sequence of the signal probe is shown as SEQ ID NO.1, and the sequence of the quenching probe is shown as SEQ ID NO. 2.

When the kit is used, the kit can be mixed with heme, buffer solution, 2-azino-bis- (3-ethylbenzodihydro-thiazoline-6-sulfonic acid) diammonium salt (ABTS), and H₂O₂Can be used in combination.

The using method comprises the following steps:

(1) and (4) solution preparation. Prepare 100nM heme solution (reagent 1); Tris-HCl at a final concentration of 50mM, KCl at a final concentration of 50mM, and a final pH of 7.9 (reagent 2); 7mM of 2, 2-azino-bis- (3-ethylbenzodithiazoline-6-sulfonic acid) diammonium salt (ABTS) (reagent 3) and 7mM of H₂O₂Solution (reagent 4); the solvent is sterile water.

(2) The signal probe forms a double strand with the sample DNA. To 2mL of

reagent

2, 4. mu.L of the sample genomic DNA was added, and the mixture was treated with a water bath at 90 ℃ for 10 min. After addition of 4. mu.L of 20. mu.M signal probe, the reaction was carried out at 55 ℃ for 30 min.

(3) The quenching probe forms a double strand with the unbound signaling probe. The quenching probe forms a double chain with the unbound signal probe, and the G quadruplex structure is damaged. To the system after the reaction in step (4), 8. mu.L of 20. mu.M quenching probe was added and the reaction was carried out at 55 ℃ for 30 min.

(4) Forming a heme/G quadruplex structure. To the system after the reaction in step (5), a reagent 1 was added to a final concentration of 100nM and treated at 37 ℃ for 30 min.

(5) And (4) color reaction. To the reaction-terminated system of (4), a reagent (ABTS) having a final concentration of 7mM and a reagent 4 having a final concentration of 7mM were added, and the mixture was treated at 37 ℃ for 30min to effect a revealing reaction (green).

Detecting the light absorption value of the reactant at the wavelength of 420 nm; and (4) quantifying Lactobacillus acetomolecules in the sample by combining the absorbance value.

Certainly, when absolute quantification is carried out, a standard curve of the light absorption value and the biomass of the Lactobacillus acetoerrors can be automatically drawn, or the biomass of the Lactobacillus acetoerrors can be directly converted according to a using method recommended by the kit and the standard curve.

Example 3: lactobacillus acetolerans quantitative kit

The kit also contains 100nM heme solution (reagent 1), Tris-HCL buffer solution and 7mM 2, 2-azino-bis- (3-ethylbenzodihydropyrazoline-6-sulfonic acid) diammonium salt (ABTS), 7mM H₂O₂And (3) solution.

Example 4: specificity of Lactobacillus acetolerans quantitative kit

(1) Lactobacillus Acetobacter derived from fermented cereals was selected as a positive control, 36 microorganisms of bacterial species and 7 microorganisms of fungal species widely present in fermented food samples were selected as negative controls, and the bacterial microorganisms were Lactobacillus buchneri, Lactobacillus diovorans, Lactobacillus brevis, Lactobacillus crusorus, Lactobacillus plantarum, Lactobacillus harbinensis, Lactobacillus acidophilus, Pediococcus Acetobacter, Pediococcus pentosaceus, Lactobacillus murinus, Lactobacillus curius, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus acidophilus, Bacillus strain, Lactobacillus strain, Lestrain, Bacillus strain, Lactobacillus strain, Lestrain, Bacillus strain, Lestrain strain, strain, Lestrain strain, Lestrain strain, strain, strain, strain, strain, strain, strain, strain, strain, strain, strain, strain, strain. The fungal microorganisms are Aspergillus tubingensis, Mucor rouxianus, Schizosaccharomyces pombe, Zygosaccharomyces bailii, Pichia kudriavzevii, Saccharomyces fibuligera, Saccharomyces cerevisiae, respectively.

(2) The above microorganisms are cultured by selecting different media, among which Lactobacillus acetogens, Lactobacillus buchneri, Lactobacillus diovulorans, Lactobacillus brevis, Lactobacillus crustorum, Lactobacillus plantarum, Lactobacillus harbinensis, Lactobacillus acicularis, Pediococcus acidilibacter, Pediococcus acicularis, Pediococcus acidilibacter, Pediococcus pendacesus, Lactobacillus acidophilus, Lactobacillus murinus, Lactobacillus curvatus, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus panis, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus acidophildelbrueckii, Lactococcus lactis, Weissella convusa, Weissella paramertes, Weissella virescens, Leucosoc citrum, Leucosoc lactis, Leucosoc mesenteroides, Leucosoc seroteroides, Leucosoc pseudosenoides using MRS medium formulated with tryptone 10.0g/L, beef extract 8.0g/L, yeast extract 4.0g/L, glucose 18.0g/L, anhydrous sorbitol oleate 0.8mL/L, K₂HPO₄2.5 g/L, 6.0g/L of sodium acetate trihydrate, 2.0g/L of ammonium citrate tribasic, MgSO₄·7H₂O 0.3g/L，MnSO₄·4H₂O is 0.08 g/L. The culture conditions were 30 ℃ for 48 h. Enterococcus italicus, Enterococcus lactis, Enterococcus faecalis, Bacillus coagulons, Bacillus licheniformis, Bacillus tequilensis, Bacillus subtilis, Bacillus velezensis, Acetobacter passaturinus and Enterococcus faecalis by using LB culture medium, the culture medium formula is 10.0g/L of peptone, 5g/L of yeast powder and 10g/L of sodium chloride. The culture conditions were 37 ℃ for 24 h. Aspergillus tubinensis, Mucor rouxianus, Schizosaccharomyces pombe, Zygosaccharomyces bailii, Pichia kudriavzevii, Saccharomyces fibuligera, Saccharomyces cerevisiae, YPD medium was used, and yeast extract was 10g/L, peptone was 20g/L, and glucose was 20 g/L. The culture conditions were: the mold is cultured for 5 days at 30 ℃, and the yeast is cultured for 2 days at 30 ℃.

(3) Extracting the genome of a single bacterium. The bacterial liquid is treated for 2min under the condition of 12000rpm, and precipitates are collected. The genomes of the pure cultures of 43 microorganisms were extracted using the DNeasy Tissue Kit.

(4) The probe is selected from a Lactobacillus acetomolecules specific probe, the sequence of the signal probe is GGGTGGGTGGGTGGGTAAAAAGCAGAGTGGAGAAAATACT (SEQ ID NO.1), and the sequence of the quenching probe is AGTATTTTCTCCACTCTGCTTTTTACCCA (SEQ ID NO. 2).

(4) The signal probe forms a double strand with the sample DNA. To 2mL of each of the reagents 2 (including Tris-HCl of 50mM final concentration, KCl of 50mM final concentration, final pH 7.9) was added 4. mu.L of genomic DNA of different microorganisms, and treated in a water bath at 90 ℃ for 10 min. After addition of 4. mu.L of 20. mu.M signal probe, the reaction was carried out at 55 ℃ for 30 min.

(5) The quenching probe forms a double chain with the unbound signal probe, and the G quadruplex structure is damaged. To the system after the reaction in step (4), 8. mu.L of 20. mu.M quenching probe was added and the reaction was carried out at 55 ℃ for 30 min.

(6) Forming a heme/G quadruplex structure. To the system after the reaction in step (5), reagent 1 (heme) was added to a final concentration of 100nM and treated at 37 ℃ for 30 min.

(7) And (4) color reaction. To the reaction-terminated system of (6), reagent 3(ABTS) was added at a final concentration of 7mM, and reagent 4 (H) was added at a final concentration of 7mM₂O₂) And treating at 37 ℃ for 30 min. The result is shown in fig. 2, the experimental group added with the genome of Lactobacillus acetoerrors has a color reaction, and the experimental group added with the genome of non-Lactobacillus acetoerrors and the blank control group have no color reaction, so that the specificity of detecting Lactobacillus acetoerrors in the kit is proved.

Example 5: quantitative method accuracy assessment

(1) The Lactobacillus acetotropians bacterial liquid is obtained according to the culture method in the embodiment 4, the microbial concentration is measured by a plate counting method, and the genome is extracted as in the embodiment 4.

(2) The genomic DNA of Lactobacillus acetoerrors was diluted by a 10-fold gradient.

(3) The chromogenic reaction was performed with genomic DNA of Lactobacillus acetolensans at different concentrations using probes of Lactobacillus acetolensans. The sequence of the signal probe is

GGGTGGGTGGGTGGGTAAAAAGCAGAGTGGAGAAAATACT (SEQ ID NO.1), and the sequence of the quenching probe is AGTATTTTCTCCACTCTGCTTTTTACCCA (SEQ ID NO. 2).

(4) The signal probe forms a double strand with the sample DNA. To 2mL of reagent 2 (including Tris-HCl at a final concentration of 50mM, KCl at a final concentration of 50mM, final pH 7.9) was added 4. mu.L of genomic DNA at different dilutions (blank control without sample DNA). Treating with water bath at 90 deg.C for 10 min. After addition of 4. mu.L of 20. mu.M signal probe, the reaction was carried out at 55 ℃ for 30 min.

(6) Forming a heme/G quadruplex structure. To the system after the reaction in step (5), reagent 1 (heme) was added at a final concentration of 100nM and treated at 37 ℃ for 30 min.

(7) And (4) color reaction. To the reaction-terminated system of (6), reagent 3(ABTS) was added at a final concentration of 7mM, and reagent 4 (H) was added at a final concentration of 7mM₂O₂) And treating at 37 ℃ for 30 min. The absorbance at a wavelength of 420nm was measured using an ultraviolet spectrophotometer, and the experimental group without sample DNA was used as a blank.

(8) A standard curve is constructed by calculating the linear relationship between the absorbance and the concentration of the bacteria liquid, as shown in FIG. 3, R²0.99(x is log10CFU/mL, y is OD₄₂₀Linear range of 10³～10⁷). The accuracy of the quantitative method of the kit provided by the invention is proved.

Example 6: quantitative experiment of Lactobacillus acetoterorans in wine samples

(1) Reference is made to the Materials and methods of Gayevshiy, V., & Goddard, M. (2012), GeogrAN _ SNhic deletions of yeast communities and publications with videos and in New Zealand. ISME J,6(7),1281 and 1290, samples were collected from a known wine manufacturer at the Shandong tobacco station. The genome concentration was 658.39 ng/. mu.L.

(2) The chromogenic reaction was performed using a probe from Lactobacillus acetolerans. The sequence of the signaling probe is GGGTGGGTGGGTGGGTAAAAAGCAGAGTGGAGAAAATACT (SEQ ID NO.1), and the sequence of the quenching probe is AGTATTTTCTCCACTCTGCTTTTTACCCA (SEQ ID NO. 2).

(4) The signal probe forms a double strand with the sample DNA. To 2mL of reagent 2 (including Tris-HCl at a final concentration of 50mM, KCl at a final concentration of 50mM, final pH 7.9) was added 4. mu.L of sample metagenomic DNA (blank control without sample DNA). Treating with water bath at 90 deg.C for 10 min. After addition of 4. mu.L of 20. mu.M signal probe, the reaction was carried out at 55 ℃ for 30 min.

(7) And (4) color reaction. To the reaction-terminated system of (6), reagent 3(ABTS) was added at a final concentration of 7mM and reagent 4(7mM H) was added at a final concentration of 7mM₂O₂) And treating at 37 ℃ for 30 min. The absorbance at a wavelength of 420nm was measured using an ultraviolet spectrophotometer, and the absorbance was 0 as a blank control using the test group without the sample DNA.

(8) According to the standard curve obtained in example 5, the total amount of Lactobacillus acetotropians in the sample was calculated to be 0log₁₀CFU/mL。

(9) The amount of Lactobacillus acetoerrors in the same sample was determined by quantitative PCR (the determination procedure and materials were as in example 11 and 6), and the total amount of Lactobacillus acetoerrors was found to be 0log₁₀CFU/mL, consistent with the quantitative results determined by the methods described above.

Example 7: absolute quantification of Lactobacillus acetotropirans in fermented grain samples

(1) Reference is made to Song Z W, Du H, Zhang Y, Xu Y. innovative core functional microbiological in a catalytic solid-state compensation by high-throughput amplification and formatting strategies in microbiology 2017; 8:1294, extracting metagenome from fermented grains samples from Shandong province, wherein the genome concentration is 100.02 ng/mu L.

(3) The signal probe forms a double strand with the sample DNA. To 2mL of reagent 2 (containing Tris-HCl at a final concentration of 50mM, KCl at a final concentration of 50mM, and final pH 7.9), 4. mu.L of fermented grain metagenomic DNA was added (blank control without sample DNA). Treating with water bath at 90 deg.C for 10 min. After addition of 4. mu.L of 20. mu.M signal probe, the reaction was carried out at 55 ℃ for 30 min.

(4) The quenching probe forms a double chain with the unbound signal probe, and the G quadruplex structure is damaged. To the system after the reaction in step (3), 8. mu.L of 20. mu.M quenching probe was added and the reaction was carried out at 55 ℃ for 30 min.

(5) Forming a heme/G quadruplex structure. To the system after the reaction in step (4), reagent 1 (heme) was added to a final concentration of 100nM, and treated at 37 ℃ for 30 min.

(6) And (4) color reaction. To the reaction-terminated system of (5), reagent 3(ABTS) was added at a final concentration of 7mM, and reagent 4 (H) was added at a final concentration of 7mM₂O₂) And treating at 37 ℃ for 30 min. The absorbance at a wavelength of 420nm was measured using an ultraviolet spectrophotometer, and the absorbance was 0.723 as a blank control using an experimental group without sample DNA.

(7) The total amount of microorganisms of Lactobacillus acetotropians in the sample was calculated to be 8.58log according to the standard curve obtained in example 2₁₀CFU/mL。

(8) The amount of Lactobacillus acetolerans in the same fermented grain sample was determined by the quantitative method (the determination steps and materials were the same as those in example 11 and 6), and the results showed that the total microbial amount of Lactobacillus acetolerans was 8.56log₁₀CFU/mL substantially matches the quantitative results measured by the above-described method (coefficient of variation, CV ═ 0.002).

Example 8: lactobacillus acetotropirans absolute quantification method based on no extraction of sample genome

(1) The Lactobacillus acetotropians bacterial solution was obtained according to the cultivation method in example 4, and the microbial concentration was determined by plate counting.

(2) Diluting the bacterial liquid of Lactobacillus acetoolorans in the step (1) by 10 times of gradient

(3) The chromogenic reaction was performed using a probe from Lactobacillus acetolerans. The sequence of the signaling probe is GGGTGGGTGGGTGGGTAAAAAGCAGAGTGGAGAAAATACT (SEQ ID NO.1), and the sequence of the quenching probe is AGTATTTTCTCCACTCTGCTTTTTACCCA (SEQ ID NO. 2).

(4) The signal probe forms a double strand with the sample DNA. To 2mL of reagent 2 (containing 50mM Tris-HCl, 50mM KCl, and 7.9 final pH) was added 10. mu.L of each of the dilutions (blank samples without addition of the sample). Treating in boiling water bath for 20 min. After addition of 4. mu.L of 20. mu.M signal probe, the reaction was carried out at 55 ℃ for 30 min. (5) The quenching probe forms a double chain with the unbound signal probe, and the G quadruplex structure is damaged. To the system after the reaction in step (4), 8. mu.L of 20. mu.M quenching probe was added and the reaction was carried out at 55 ℃ for 30 min.

(8) A standard curve is constructed by calculating the linear relationship between the absorbance and the concentration of the bacteria liquid, as shown in FIG. 4, R²0.99(x is log10CFU/mL, y is OD₄₂₀Linear range of 10³～10⁷). The accuracy of the quantitative method of the kit provided by the invention is proved

Example 9: method for determining content of Lactobacillus acetotropins in wine sample based on absolute quantification method of microorganisms without extracting sample genome

(1) The sample is collected from a certain famous grape wine manufacturer of Shandong tobacco Taiwan, and the sample treatment method comprises the following steps: 5mL of phosphate buffer was added to 1mL of the sample, and the mixture was centrifuged at 3000 Xg for 10min to collect the cells.

(2) And (6) washing. 5mL of phosphate buffer was added to the cells obtained in (1), and the cells were collected by centrifugation at 12000 Xg for 2min and repeated once.

(3) Resuspend the cells, add 1mL of reagent 2 (containing 50mM Tris-HCl, 50mM KCl, final pH 7.9) to the cells obtained in (2), aspirate and mix well.

(4) The chromogenic reaction was performed using a probe from Lactobacillus acetolerans. The sequence of the signaling probe is GGGTGGGTGGGTGGGTAAAAAGCAGAGTGGAGAAAATACT (SEQ ID NO.1), and the sequence of the quenching probe is AGTATTTTCTCCACTCTGCTTTTTACCCA (SEQ ID NO. 2).

(5) The signal probe forms a double strand with the sample DNA. To 2mL of reagent 2 (containing 50mM Tris-HCl, 50mM KCl, and 7.9 final pH), 10. mu.L of the fermented grape liquid (blank control without addition of this liquid) was added. Treating in boiling water bath for 20 min. After addition of 4. mu.L of 20. mu.M signal probe, the reaction was carried out at 55 ℃ for 30 min.

(6) The quenching probe forms a double chain with the unbound signal probe, and the G quadruplex structure is damaged. To the system after the reaction in step (5), 8. mu.L of 20. mu.M quenching probe was added and the reaction was carried out at 55 ℃ for 30 min.

(7) Forming a heme/G quadruplex structure. To the system after the reaction in step (6), reagent 1 (heme) was added at a final concentration of 100nM and treated at 37 ℃ for 30 min.

(8) And (4) color reaction. To the reaction-terminated system of (7), reagent 3(ABTS) was added at a final concentration of 7mM, and reagent 4 (H) was added at a final concentration of 7mM₂O₂) And treating at 37 ℃ for 30 min. The absorbance at a wavelength of 420nm was measured using an ultraviolet spectrophotometer, and the absorbance was 0 as a blank control using the test group without the sample DNA.

(9) According to the standard curve obtained in example 8, the total amount of Lactobacillus acetotropians in the sample was calculated to be 0log₁₀CFU/mL。

(10) The amount of Lactobacillus acetoerrors in the same sample was determined by quantitative PCR (the quantitative procedure and materials were as in example 11 and 6), and the total amount of Lactobacillus acetoerrors was found to be 0log₁₀CFU/mL, consistent with the quantitative results determined by the methods described above.

Example 10: absolute quantitative method for determining content of Lactobacillus acetoerolerans in fermented grain sample based on non-extracted sample genome

(1) The sample is from fermented grains of a certain brewery in Shandong, and the sample treatment method comprises the following steps: 5mL of phosphate buffer was added to 1g of the sample, and the mixture was centrifuged at 3000 Xg for 10min to collect the cells.

(5) The signal probe forms a double strand with the sample DNA. To 2mL of reagent 2 (containing 50mM Tris-HCl, 50mM KCl, and 7.9 final pH), 10. mu.L of fermented grain suspension was added (blank control without addition of this suspension). Treating in boiling water bath for 20 min. After addition of 4. mu.L of 20. mu.M signal probe, the reaction was carried out at 55 ℃ for 30 min.

(8) And (4) color reaction. To the reaction-terminated system of (7), reagent 3(ABTS) was added at a final concentration of 7mM, and reagent 4 (H) was added at a final concentration of 7mM₂O₂) And treating at 37 ℃ for 30 min. The absorbance at a wavelength of 420nm was measured using an ultraviolet spectrophotometer, and the blank was prepared from the test group without the sample DNA, and it was found that the absorbance was 0.672.

(9) According to the standard curve obtained in example 8, the total amount of Lactobacillus acetotropians in the sample was calculated to be 8.47log₁₀CFU/mL，

(10) The amount of Lactobacillus acetoerrors in the same sample was determined by quantitative PCR (the determination procedure and materials were as in example 11 and 6), and the total amount of Lactobacillus acetoerrors was 8.56log₁₀CFU/mL was substantially identical to the two sets of data measured by the above method (coefficient of variation, CV 0.007).

Example 11: comparison of results of quantitative detection kit and fluorescent quantitative PCR (polymerase chain reaction) detection for microorganisms

(1) The sample is three fermented white spirit samples from the fermentation end point of a certain brewery in Shandong.

(2) Sample treatment:

(i) total genome concentrations of 369 ng/. mu.L, 590 ng/. mu.L and 321.89 ng/. mu.L were extracted from the three samples.

(ii) 5mL of phosphate buffer was added to 1g of the sample, and the mixture was centrifuged at 3000 Xg for 10min to collect the cells. To the obtained cells, 5mL of phosphate buffer was added, and the cells were collected by centrifugation at 12000 Xg for 2min and repeated once. Resuspend the cells, add 1mL of reagent 2 buffer to the obtained cells, aspirate and mix them well.

(4) The quantitative method is determined based on a kit without extracting genome.

(i) The signal probe forms a double strand with the sample DNA. To 2mL of reagent 2 (containing 50mM Tris-HCl, 50mM KCl, and 7.9 final pH), 10. mu.L of fermented grain suspension was added (blank control without addition of this suspension). Treating in boiling water bath for 20 min. After addition of 4. mu.L of 20. mu.M signal probe, the reaction was carried out at 55 ℃ for 30 min.

(ii) The quenching probe forms a double chain with the unbound signal probe, and the G quadruplex structure is damaged. To the system after the reaction in step (i), 8. mu.L of 20. mu.M quenching probe was added and the reaction was carried out at 55 ℃ for 30 min.

(iii) Forming a heme/G quadruplex structure. To the system after the reaction in step (ii), reagent 1 (heme) was added at a final concentration of 100mM, and the mixture was treated at 37 ℃ for 30 min.

(iv) And (4) color reaction. (iv) to the reaction-terminated system of (iii), reagent 3(ABTS) was added at a final concentration of 7mM, and reagent 4 (H) was added at a final concentration of 7mM₂O₂) And treating at 37 ℃ for 30 min. Measuring absorbance at 420nm with ultraviolet spectrophotometer to obtain sample DNA free sampleThe test group served as a blank and showed absorbance values of 0.678, 0.695, 0.70.

(v) According to the standard curve obtained in example 8, the total amount of Lactobacillus acetotropians in the sample was calculated to be 8.68. + -. 0.12log₁₀CFU/mL。

(5) Kit quantitative method determination based on genome extraction

(i) The signal probe forms a double strand with the sample DNA. To 2mL of reagent 2 (containing Tris-HCl at a final concentration of 50mM, KCl at a final concentration of 50mM, and final pH 7.9), 4. mu.L of fermented grain metagenomic DNA was added (blank control without sample DNA). Treating with water bath at 90 deg.C for 10 min. After addition of 4. mu.L of 20. mu.M signal probe, the reaction was carried out at 55 ℃ for 30 min.

(iii) Forming a heme/G quadruplex structure. To the system after the reaction in step (ii), reagent 1 (heme) was added at a final concentration of 100nM and treated at 37 ℃ for 30 min.

(iv) And (4) color reaction. To the reaction-terminated system of (5), reagent 3(ABTS) was added at a final concentration of 7mM, and reagent 4 (H) was added at a final concentration of 7mM₂O₂) And treating at 37 ℃ for 30 min. The absorbance values at a wavelength of 420nm were measured using an ultraviolet spectrophotometer, and the blank control was an experimental group without sample DNA, showing absorbance values of 0.753, 0.745, and 0.732.

(v) The total amount of Lactobacillus acetotropins in the samples was calculated to be 8.77. + -. 0.10log according to the standard curve obtained in example 5₁₀CFU/mL。

(6) quantitative qPCR (quantitative polymerase chain reaction) on the content of Lactobacillus acetoterons in a sample

(i) The Lactobacillus acetotropians bacterial liquid is obtained according to the culture method in the embodiment 4, the microbial concentration is measured by a plate counting method, and the genome is extracted as in the embodiment 4.

(ii) The genomic DNA of Lactobacillus acetoerrors was diluted by a 10-fold gradient.

(iii) The qPCR system was SYBR Green 10. mu.L, upstream and downstream primers 20. mu.M, template DNA 0.5. mu.L, and sterile water supplemented 20. mu.L.

(iv) Reaction procedure for qPCR: pre-denaturation 95 ℃ for 5min, cycle phase: 5s at 95 ℃ and 20s at 60 ℃; the number of cycles was 40, the dissolution curve was raised from 65 ℃ to 95 ℃ by 0.5 ℃ every 5 s.

(v) The extracted genome was subjected to qPCR using Lactobacillus acetolerans specific primers with a sequence downstream of the primer sequence of AAAAAGCAGAGTGGAGAAAATACT (SEQ ID No.5) and a sequence downstream of CCAATAAAAAGAGCAACAGCA (SEQ ID No. 6).

(vi) The genome DNA was diluted by 10-fold gradient to establish a standard curve of CT value and Lactobacillus acetoserons bacterial concentration, as shown in FIG. 4, R²＝0.99。

(vii) The qPCR system and reaction conditions were as in (iii), (iv). According to the CT value of the reaction end, the concentration of Lactobacillus acetotropians in the sample is calculated to be 8.71 +/-0.12 log through the established standard curve₁₀ CFU/g。

(7) By significant difference analysis, the results are shown in FIG. 6, there is no significant difference between the three quantitative methods (P <0.05)

Example 12: detection limit for detection by using two different sequence signal probes

(1) The bacterial solution of Lactobacillus acetotropians was obtained according to the cultivation method of example 4, the microbial concentration was determined by plate counting method, and the genome was extracted at 7.49log10 CFU/mL as in example 4.

(2) The genomic DNA of Lactobacillus acetomolecules was diluted by a 10-fold gradient to give a 3.62log10 CFU/mL DNA template.

(3) The sequence of the Lactobacillus acetoolorans signal probe provided by the invention is

GGGTGGGTGGGTGGGTAAAAAGCAGAGTGGAGAAAATACT (SEQ ID NO.1), and the sequence of the quenching probe is AGTATTTTCTCCACTCTGCTTTTTACCCA (SEQ ID NO. 2). Adding the 3.2log obtained in (2)₁₀CFU/mL Lactobacillus acetomolecules genomic DNA for color reaction.

(4) The sequence of the signal probe utilizing Lactobacillus acetolerans is (SEQ ID NO.3)

GGGATTGGGATTGGGATTGGGAAAAAGCAGAGTGGAGAAAATACT, and the sequence of the quenching probe is AGTATTTTCTCCACTCTGCTTTTTCCCAA (SEQ ID NO. 4). 3.2log10CFU/mL of the genomic DNA of Lactobacillus acetomolecules obtained in (2) was added to conduct a color development reaction.

(5) The signal probe forms a double strand with the sample DNA. To 2mL of reagent 2 (including Tris-HCl at a final concentration of 50mM, KCl at a final concentration of 50mM, final pH 7.9) was added 4. mu.L of Lactobacillus acetoerrors genomic DNA (blank control without sample DNA). Treating with water bath at 90 deg.C for 10 min. After adding 4. mu.L of each 20. mu.M signal probe, the reaction was carried out at 55 ℃ for 30 min.

(6) The quenching probe forms a double chain with the unbound signal probe, and the G quadruplex structure is damaged. To the system after the reaction in step (5), 8. mu.L of 20. mu.M quenching probes were added, and the reaction was carried out at 55 ℃ for 30 min.

(8) And (4) color reaction. To the reaction-terminated system of (7), reagent 3(ABTS) was added at a final concentration of 7mM and reagent 4 (H) was added at a final concentration of 7mM₂O₂) And treating at 37 ℃ for 30 min. The absorbance at a wavelength of 420nm was measured using an ultraviolet spectrophotometer, and the experimental group without sample DNA was used as a blank.

(9) Repeating the steps (5), (6), (7) and (8) 9 times, and comparing the stability of the detection results, as shown in fig. 7. The Coefficient of Variation (CV) based on the quantitative results of the signal sequence of SEQ ID NO.3 was 0.83; the coefficient of variation of the quantitative result based on the signal sequence of SEQ ID NO.1 is 0.03, and the detection effect is stable.

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

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> absolute quantitative probe, method and use thereof

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 40

<212> DNA

<213> Artificial Synthesis

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gggtgggtgg gtgggtaaaa agcagagtgg agaaaatact 40

<210> 2

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<212> DNA

<213> Artificial Synthesis

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agtattttct ccactctgct ttttaccca 29

<210> 3

<211> 45

<212> DNA

<213> Artificial Synthesis

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gggattggga ttgggattgg gaaaaagcag agtggagaaa atact 45

<210> 4

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<212> DNA

<213> Artificial Synthesis

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agtattttct ccactctgct ttttcccaa 29

Claims

1. A set of probes, comprising a signaling probe and a quenching probe; the signal probe sequence comprises a sequence shown in SEQ ID NO. 1; the quenching probe sequence comprises a sequence shown in SEQ ID NO. 2.

2. A detection kit comprising the signaling probe of claim 1 and a quenching probe.

3. The detection kit according to claim 2, further comprising any one or more of: heme, buffer solution and 2, 2-linkageAzo-bis- (3-ethylchromazoline-6-sulfonic acid) diammonium salt, H₂O₂。

4. A method for quantifying Lactobacillus acetotropians, wherein the probe of claim 1 or the detection kit of any one of claims 2 to 3 is used.

5. A quantification method according to claim 4, characterized in that the method comprises: melting DNA in a sample to be detected; adding an excessive signal probe, and combining with a target nucleotide fragment of a sample to be detected to form a double chain so that a G quadruplex is exposed outside a sequence; adding sufficient quenching probe to form double chains with the unbound signal probe, and destroying the G quadruplex structure; the G quadruplex with the naked leakage outside reacts with the heme to form G quadruplex/heme mimic enzyme with catalase activity, and the biomass of Lactobacillus acetotropirans is characterized by combining the activity of the catalase.

6. A quantification method according to claim 4, wherein the method is absolute quantification or relative quantification; optionally, when the method is absolute quantification, the method further comprises: establishing a standard curve of catalase activity or an index which is correlated with the catalase activity and the biomass of Lactobacillus acetotropirans; when a sample to be detected is detected, substituting the detected catalase activity or indexes which are correlated with the catalase activity into the standard curve to obtain the biomass of the Lactobacillus acetoerolerans in the sample to be detected.

7. The method according to any one of claims 4 to 6, wherein the sample to be tested is a sample containing a cell, a genome, a metagenome, or the like; optionally, the sample is a fermented food or a sample taken from a fermentation process of a fermented food, or an environmental sample.

8. The method of claim 7, wherein the fermented food is any one or more of: white spirit, yellow wine, soy sauce, beer, wine, table vinegar, fermented tea, traditional fermented vegetables, fermented beverages, alcoholic drinks, yogurt, cheese, fruit vinegar, fermented glutinous rice, fermented soya beans, fermented bean curd and fermented rice-flour foods; the environmental sample is selected from intestinal tract, soil, water body and the like.

9. A method of using the kit of any one of claims 2 to 3, wherein the method of use comprises: adding excessive signal probes into a sample to be detected after the DNA is unzipped, and reacting for a period of time to enable the signal probes to be combined with target fragments in the sample to be detected; then adding a sufficient amount of quenching probe to form a double strand with the unbound signaling probe; adding heme, reacting for a while, adding ABTS and H₂O₂Reacting for a period of time, detecting the light absorption value of the reactant, and quantifying Lactobacillus acetomolecules in the sample by combining the light absorption value.

10. A method for detecting the amount of Lactobacillus acetoterorans in a fermented food product or an environmental sample, comprising using the probe of claim 1, or the kit of claims 2-3; the fermented food is any one or more of the following: white spirit, yellow wine, soy sauce, beer, wine, table vinegar, fermented tea, traditional fermented vegetables, fermented beverages, alcoholic drinks, yogurt, cheese, fruit vinegar, fermented glutinous rice, fermented soya beans, fermented bean curd, fermented rice and flour foods and the like; the environment sample is selected from intestinal tract, soil and water body.