CN112924429B - A probe and kit for absolute quantification of Lactobacillus jinshani - Google Patents

Abstract

The invention discloses a Lactobacillus jin Shini absolute quantitative probe and a kit, belonging to the fields of biology, fermentation and detection. The Lactobacillus jin shinshi quantitative probe and the kit can realize the total amount detection of Lactobacillus jin shinshi, do not need expensive instruments when used for detection and quantification of Lactobacillus jin shinshi, and can quickly complete the quantitative work within 2.5 h. 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 jin Shini, and have the characteristics of rapidness, convenience, cheapness and accuracy.

Description

A probe and kit for absolute quantification of Lactobacillus jinshani

technical field

The invention relates to a probe and a kit for absolute quantification of Lactobacillus jinshani, belonging to the fields of biology, fermentation and detection.

Background technique

Lactobacillus jinshani is widely distributed in traditional fermented food brewing systems. For example, in the liquor brewing system, the abundance of Lactobacillus jinshani increased sharply after 5 days of fermentation, and became the dominant microorganism in the middle and late fermentation. It has a significant correlation and plays an important role in the fermentation process; it is also reported that it is also an important functional microorganism in the vinegar fermentation system. The biomass and succession of the bacteria in the process of food fermentation are important microorganisms to characterize whether the food is fermented normally or not. Therefore, real-time tracking of the biomass of Lactobacillus jinshani has important guiding significance for judging the stability of fermentation batches and regulating fermentation parameters. However, at present, most of the traditional fermented food systems are multi-strain co-fermentation systems, and the content of Lactobacillus jinshani in the sample cannot be determined by simple OD colorimetry. Quantification of Lactobacillus jinshani, however, requires expensive equipment and a demanding operating environment. Therefore, in order to track the growth trend of Lactobacillus jinshani in samples conveniently, quickly and accurately, it is necessary to develop corresponding quantitative methods and kits of Lactobacillus jinshani.

The principle of G-quadruplex/heme-mimicking enzyme activity detection is that G-quadruplex can form a DNA-mimicking enzyme with catalase activity with heme, which can catalyze the oxidation of hydrogen peroxide to ABTS 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 critical to the entire detection process. If the G-quadruplex sequence is improperly designed, when the G-quadruplex sequence forms a dimer with other bases, the G-quadruplex sequence cannot form a G-quadruplex. The use of quantitative methods based on this principle can lead to underestimation of the target gene content in the sample, reducing the sensitivity and accuracy of the detection method.

At present, the principle of G-quadruplex/heme-mimicking enzyme activity detection has been reported to be used for the specific detection of microorganisms; for example, Wang Y, Li X, Xi D, Wang X. Visual detection of Fusarium proliferatum based on asymmetric recombinase polymerase amplification and hemin/G-quadruplex DNAzyme. Rsc Advances 2019; 9:37144-37147. Asymmetric specific primers are used (upstream primers add reverse sequence modification of G-quadruplex, downstream is not modified), this method only It can be applied to the detection of specific bacteria Fusarium proliferatum in the sample, but cannot achieve the total amount detection of Lactobacillus jinshani; in addition, when using the asymmetric specific primers for detection in this document, different concentrations of upstream and downstream primers (upstream and downstream primers) are added to the PCR system. The primer concentration is low and the downstream primer concentration is high), which is amplified by recombinant polymerase amplification (RPA) to form a double-stranded product. As the PCR reaction proceeds, the upstream primer is exhausted, and the downstream primer uses newly synthesized double-stranded DNA as The template is amplified to form single-stranded DNA with G-quadruplex ends to detect Fusarium proliferatum in samples using the G-quadruplex/heme-mimetic enzyme activity assay. However, this quantitative method still requires a PCR step to generate G-quadruplexes, and the PCR process still requires expensive PCR equipment and a strict operating environment.

SUMMARY OF THE INVENTION

A probe, kit and application for absolute quantification of Lactobacillus jinshani of the present invention solves at least one of the following technical problems: (1) the existing method cannot realize the total amount detection of all Lactobacillus jinshani; (2) the current Some quantitative methods have low species resolution and/or insufficient detection accuracy; (3) Existing quantitative methods require expensive equipment and/or strict operating environment, and are not suitable for timely detection after production sampling; (4) ) The 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 signal probe sequence is shown in SEQ ID NO. 1 (GGGTGGGTGGGTGGGTAAATAATCTACGGGAGTGCG).

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

A second object of the present invention is to provide a method for the quantification of Lactobacillus jinshani comprising the use of the probe of the present invention.

The method includes: melting the DNA in the sample to be tested; adding an excess signal probe (sequence such as SEQ ID NO. 1), combining with the target nucleotide fragment of the sample to be tested to form a double strand, so that the G quadruplex is naked and leaked Outside the sequence; add enough quenching probe (sequence such as SEQ ID NO. 2) to form a double strand with the unbound signal probe, destroy the G quadruplex structure; use 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 Lactobacillus jinshani.

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

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

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

In one embodiment, the sample is a fermented food or a sample taken from the fermentation process of the 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 beverages, alcoholic beverages, yogurt, cheese, fruit vinegar , fermented fermented rice, tempeh, fermented bean curd, fermented rice noodles, etc.; the environmental samples are selected from the intestinal tract, soil, water and the like.

In one embodiment, the melting of the DNA in the sample to be tested is performed in a high temperature manner. Optionally, the sample to be tested is treated at a temperature higher than 90°C. It can be any kind of environment that can provide the corresponding temperature, such as metal bath, water bath, oven, thermostat, etc.

In one embodiment, the melting is performed in a buffer. Optionally, the buffer may be a Tris-HCl buffer, which further contains 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 when all the target nucleotide fragments of the test sample can be combined to form double strands. The specific dosage can be determined by those skilled in the art in combination with common knowledge in the art 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 signal probe binds to the target nucleotide fragment of the sample to be tested to form a double strand, which is performed at a temperature range of 50-60°C.

In one embodiment, the sufficient amount refers to the amount of quencher probe that is added in an amount sufficient to form duplexes with all unbound signaling probes. The specific dosage can be determined by those skilled in the art in combination with common knowledge in the art or a specific sample to be tested, or through preliminary experiments.

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

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

In one embodiment, the G-quadruplex/heme mimetic enzyme with catalase activity is formed by reacting with the naked G-quadruplex with heme, and combined with the activity of catalase to characterize Lactobacillus jinshani Biomass refers to adding ABTS and H ₂ O ₂ after adding heme to the system for reaction, and then characterizing the catalase activity by the absorbance 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, specifically:

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

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

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

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

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

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

(7) Quantify Lactobacillus jinshani in the sample by combining the absorbance value.

In one embodiment, the quantitative method further includes: configuring samples with different known contents of Lactobacillus jinshani, and measuring the absorbance values obtained by different samples after being processed by the above method; drawing a standard curve between the absorbance values and different contents of Lactobacillus jinshani; The absorbance value obtained after the sample to be tested is processed by the above method is substituted into the standard curve, that is, the content of Lactobacillus jinshani in the sample to be tested is obtained.

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

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

In one embodiment, the detection kit further contains any one or more of the following: heme, buffer, 2,2-azino-bis-(3-ethylbenzodihydrothiazoline- ₆ -sulfonic _acid ) diammonium salt (ABTS), H2O2. These reagents may not be included, and the operator must prepare separately when using the kit.

In one embodiment, in the detection kit, the buffer may be Tris-HCl buffer, and further 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 Lactobacillus jinshani absolute quantification kit, and the kit includes four reagents (Reagent 1, Reagent 2, Reagent 3, Reagent 4) and a set of Lactobacillus jinshani quantitative probes ( Signal probe, quenching probe); the reagent 1 includes heme; the reagent 2 includes a 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 reagents or probes may be in a liquid state or a solid state, and those skilled in the art can routinely adjust the concentration to a suitable concentration during use.

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

In one embodiment, the method of using comprises: adding excess signal probe to the sample to be tested in which the DNA is melted for a period of time, so that the signal probe binds to the target fragment in the sample to be tested; Quenching the probe to make it form 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 Lactobacillus jinshani in samples.

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

(1) DNA melting of the sample to be tested; (2) Add signal probe, react at 55°C for 30min; (3) Add quench probe, react at 55°C for 30min; (4) Add heme, react at 37°C ℃ react for 30min; (5) add 2,2-azino-bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt (ABTS) and H ₂ O ₂ , at 37 ℃ The temperature was reacted for 30 minutes; (6) the absorbance value of the reactant at a wavelength of 420 nm was detected; (7) Lactobacillus jinshani in the sample was quantified in combination with the absorbance value.

The fifth object of the present invention is to provide the application of the kit in the quantification of Lactobacillus jinshani.

In one embodiment, the application is for the technical field of fermented food; optionally, 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 beverages, alcoholic beverages, yogurt, cheese, fruit vinegar, fermented wine, tempeh, fermented bean curd, fermented rice noodles, etc.

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

Beneficial effects:

In the present invention, the G quadruplex is combined with a specific sequence to form a signal probe, the signal probe is combined with the target sequence so that the G quadruplex is naked outside the sequence, and a sufficient amount of quenching probe and unreacted signal probe are added. Forms a double strand, destroys the G-quadruplex structure, and forms a G-quadruplex/heme-mimicking enzyme by reacting with heme, exhibits catalase activity, and characterizes microbial biomass with catalase activity. The Lactobacillus jinshani quantitative probe of the present invention can realize the total amount detection of Lactobacillus jinshani; further, the signal probe is optimized, and the signal probe sequence is:

GGGTGGGTGGGTGGGTAAATAATCTACGGGAGTGCG (SEQ ID NO. 1), the quencher probe is CGCACTCCCGTAGATTATTTACCCA (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 additional spatial structure with the specific sequence (Fig. 1), and the detection accuracy is higher and the lowest detection rate is obtained. Limit improvement.

When the probe of the present invention is used for detection and quantification of Lactobacillus jinshani, 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 2.5h. In the present invention, in order to avoid the use of expensive equipment, such as a PCR instrument, the quantification of microorganisms is realized by a combination of signal probes and quenching probes. The present invention solves the problem that the current microbial quantification methods rely on relatively expensive instruments and are very limited in the actual application process.

Further, the present invention can realize the rapid detection of Lactobacillus jinshani, the sample does not have to be subjected to nucleic acid extraction, only the microorganisms in the sample need to be eluted in the buffer, and the subsequent experiments can be directly carried out. Meanwhile, compared with the quantitative results of fluorescence 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 Lactobacillus jinshani quantification.

Description of drawings

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

Figure 2: Specificity of Lactobacillus jinshani probes.

Figure 3: Standard curve of Lactobacillus jinshani quantitative probes based on genome extraction.

Figure 4: Standard curve of Lactobacillus jinshani quantification probe based on no sample genome extraction.

Figure 5: qPCR standard curve.

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

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

Detailed ways:

Example 1: Lactobacillus jinshani quantitative probe combination reagent

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

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

Example 2: Lactobacillus jinshani quantitative kit and its use

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

When the 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 O ₂ .

The usage method is:

(1) Solution configuration. Prepare 100nM heme solution (reagent 1); prepare Tris-HCL with a final concentration of 50mM, KCl with a final concentration of 50mM, and a final pH of 7.9 (reagent 2); 7mM of 2,2-azino-bis-( 3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt (ABTS) (reagent ₃ ) and a ₇ mM solution of H2O2 (reagent 4); all solvents were sterile water.

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

(3) The quenching probe forms a double strand with the unbound signal probe. The quencher probe forms a duplex with the unbound signaling probe, disrupting the G-quadruplex structure. 8 μL of 20 μM quenching probe was added to the system after the reaction in step (4), and the reaction was carried out at 55° C. for 30 min.

(4) The heme/G quadruplex structure is formed. Reagent 1 with a final concentration of 100 nM was added to the system after the reaction in step (5), and treated at 37°C for 30 min.

(5) Color reaction. Reagent (ABTS) with a final concentration of 7 mM and reagent 4 with a final concentration of 7 mM were added to the system after the reaction in (4), and treated 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 Lactobacillusjinshani in the sample.

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

Example 3: Lactobacillus jinshani quantitative kit

The Lactobacillus jinshani 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 .

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 in H2O2.

Example 4: Specificity of Lactobacillus jinshani Quantitative Kit

(1) Lactobacillus jinshani from fermented grains was selected as the positive control, and 36 bacterial species and 6 fungal species widely existed in the fermented food samples were selected as the negative control. The bacterial microorganisms were Lactobacillus buchneri, Lactobacillus dioilvorans, Lactobacillus brevis,Lactobacillus crustorum,Lactobacillus plantarum,Lactobacillus harbinensis,Lactobacillus acidiliscis,Pediococcus ethanolidurans,Pediococcusacidilactici,Pediococcus pentosaceus,Lactobacillus murinus,Lactobacilluscurvatus,Lactobacillus casei,Lactobacillus reuteri,Lactobacillus panis,Lactobacillus fermentum,Lactobacillus johnsonii,Lactobacillus delbrueckii,Lactococcus lactis,Weissella confusa , Weissella paramesenteroides, Weissellaviridescens, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Enterococcus italicus, Enterococcus lactis, Enterococcus faecalis, Bacillus coagulans, Bacillus licheniformis, Bacillustoc equilensis, Bacillus subtilis, Bacillus velecius, Enterococcus pasteur. The fungal microorganisms were Aspergillus tubingensis, Mucor rouxianus, Schizosaccharomyces pombe, Zygosaccharomyces bailii, Pichia kudriavzevii, Saccharomycopsis fibuligera.

(2) The above microorganisms are cultured in different mediums, among which Lactobacillus jinshani, Lactobacillus buchneri, Lactobacillus dioilvorans, Lactobacillus brevis, Lactobacillus crustorum, Lactobacillus plantarum, Lactobacillus harbinensis, Lactobacillus acidiliscis, Pediococcus ethanolidurans, Pediococcus pentosacidilactici, Pediococcus pentosaceus, Lactobacillus mucurva , Lactobacillus casei, Lactobacillus reuteri, Lactobacillus panis, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus delbrueckii, Lactococcus lactis, Weissella confusa, Weissella paramesenteroides, Weissellaviridescens, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc mesenteroides medium using MRSoconostoc pseudomesenteroides medium Tryptone 10.0g/L, beef extract 8.0g/L, yeast extract 4.0g/L, glucose 18.0g/L, sorbitan oleate 0.8mL/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 conditions were 30°C for 48h. Enterococcus italicus, Enterococcus lactis, Enterococcusfaecalis, Bacillus coagulans, Bacillus licheniformis, Bacillus tequilensis, Bacillus subtilis, Bacillus velezensis, Acetobacter pasteurianus, Enterococcusfaecium use LB medium, the medium formula is peptone 10.0g/L, yeast powder 5g/L, chlorinated Sodium 10g/L. The culture condition was 37°C for 24h. Aspergillus tubingensis, Mucor rouxianus, Schizosaccharomyces pombe, Zygosaccharomyces bailii, Pichia kudriavzevii, Saccharomycopsis fibuligera use YPD medium, the medium formula is yeast extract 10g/L, peptone 20g/L, glucose 20g/L. The culture conditions were as follows: mold was cultured at 30°C for 5 days, and yeast was cultured at 30°C for 2 days.

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

(4) The probe was selected as a Lactobacillus jinshani-specific probe, and the sequence of the signal probe was

GGGTGGGTGGGTGGGTAAATAATCTACGGGAGTGCG (SEQ ID NO. 1), the sequence of the quenching probe is CGCACTCCCGTAGATTATTTACCCA (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), 4 μL of genomic DNAs of different microorganisms were added, and treated in a water bath at 90° C. for 10 min. After adding 4 μL of 20 μM signal probe, the reaction was performed at 55° C. for 30 min.

(5) The quenching probe forms a double strand with the unbound signal probe, which destroys the G-quadruplex structure. 8 μL of 20 μM quenching probe was added to the system after the reaction in step (4), and the reaction was carried out at 55° C. for 30 min.

(6) The heme/G quadruplex structure is formed. Reagent 1 (heme) with a final concentration of 100 nM was added to the system after the reaction in step (5), and treated at 37° C. for 30 min.

(7) 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 reaction system in (6), and treated at 37° C. for 30 min. The results are shown in Figure 2. The experimental group added with the Lactobacillus jinshani genome showed a color reaction, while the experimental group added with non-Lactobacillus jinshani and the blank control group did not have a color reaction, which proves the specificity of the kit for detecting Lactobacillus jinshani.

Example 5: Quantitative method accuracy assessment

(1) Lactobacillus jinshani bacterial liquid was obtained according to the culture method in Example 4, the microbial concentration was determined by the plate counting method, and the extraction of the genome was the same as that in Example 4.

(2) Lactobacillus jinshani genomic DNA was diluted by 10-fold gradient.

(3) Using the Lactobacillus jinshani probe, the color reaction was carried out with different concentrations of Lactobacillus jinshani genomic DNA. The sequence of the signal probe is GGGTGGGTGGGTGGGTAAATAATCTACGGGAGTGCG (SEQ ID NO. 1), and the sequence of the quencher probe is CGCACTCCCGTAGATTATTTACCCA (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), 4 μL of different dilutions of genomic DNA were added (no sample DNA was added as a blank control). Treated in a water bath at 90 °C for 10 min. After adding 4 μL of 20 μM signal probe, the reaction was performed at 55° C. for 30 min.

(5) The quenching probe forms a double strand with the unbound signal probe, which destroys the G-quadruplex structure. 8 μL of 20 μM quenching probe was added to the system after the reaction in step (4), and the reaction was carried out at 55° C. for 30 min.

(6) The heme/G quadruplex structure is formed. Reagent 1 (heme) with a final concentration of 100 nM was added to the system after the reaction in step (5), and treated at 37° C. for 30 min.

(7) 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 reaction system in (6), 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.

(8) A standard curve is constructed 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 to 10 ⁷ ). The accuracy of the quantitative method of the kit provided by the present invention is proved.

Example 6: Quantitative experiment of Lactobacillus jinshani in wine samples

(1) Refer to the Materials and methods method of 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. The samples were collected in A well-known wine manufacturer in Yantai, Shandong. The genome concentration was 658.39ng/μL. (2) Use the probe of Lactobacillus jinshani to perform color reaction. The sequence of the signal probe is

GGGTGGGTGGGTGGGTAAATAATCTACGGGAGTGCG (SEQ ID NO. 1), the sequence of the quenching probe is CGCACTCCCGTAGATTATTTACCCA (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) was added 4 μL of wine metagenomic DNA (no sample DNA was added as a blank control). Treated in a water bath at 90 °C for 10 min. After adding 4 μL of 20 μM signal probe, the reaction was performed at 55° C. for 30 min.

(5) The quenching probe forms a double strand with the unbound signal probe, which destroys the G-quadruplex structure. 8 μL of 20 μM quenching probe was added to the system after the reaction in step (4), and the reaction was carried out at 55° C. for 30 min.

(6) The heme/G quadruplex structure is formed. Reagent 1 (heme) with a final concentration of 100 nM was added to the system after the reaction in step (5), and treated at 37° C. for 30 min.

(7) Color reaction. 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 were added to the reaction system in (6), 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, and the absorbance value was 0.

(8) According to the standard curve obtained in Example 5, the total amount of Lactobacillus jinshani in the sample was calculated to be 0 log ₁₀ CFU/mL.

(9) Quantify Lactobacillus jinshani in the same sample above by fluorescence quantitative PCR method (quantitative steps and materials are the same as in Example 11 (6)), the result shows that the total amount of Lactobacillus jinshani is 0 log ₁₀ CFU/mL, which is the same as that determined by the above method. The quantitative results were consistent4.

Example 7: Absolute quantification of Lactobacillus jinshani in wine grain samples

(1) Refer to the method in MATERIALSAND METHODS of SongZW, Du H, ZhangY, XuY. Unraveling core functional microbiotain traditional solid-state fermentation by high-throughput amplicons and metatranscriptomics sequencing. Frontiers in microbiology 2017; 8:1294, extract wine from Shandong Province Metagenomes in fermented grain samples with a genome concentration of 100.02ng/μL.

(2) Use the probe of Lactobacillus jinshani to perform color reaction. The sequence of the signal probe is

GGGTGGGTGGGTGGGTAAATAATCTACGGGAGTGCG (SEQ ID NO. 1), the sequence of the quenching probe is CGCACTCCCGTAGATTATTTACCCA (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 with a final concentration of 50 mM, KCl with a final concentration of 50 mM, and a final pH of 7.9), 4 μL of fermented grains metagenomic DNA was added (no sample DNA was added as a blank control). Treated in a water bath at 90 °C for 10 min. After adding 4 μL of 20 μM signal probe, the reaction was performed at 55° C. for 30 min.

(4) The quenching probe forms a double strand with the unbound signal probe, which destroys 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) The heme/G quadruplex structure is formed. Reagent 1 (heme) with a final concentration of 100 nM was added to the system after the reaction in step (4), and treated at 37°C for 30 min.

(6) Color reaction. To the reaction system in (5), 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, 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 0.602.

(7) According to the standard curve obtained in Example 2, the total amount of microorganisms of Lactobacillus jinshani in the sample was calculated to be 7.33 log ₁₀ CFU/mL.

(8) Quantify Lactobacillus jinshani in the same fermented grains sample by fluorescence quantitative method (quantitative steps and materials are the same as in Example 11(6)), the result shows that the total amount of microorganisms of Lactobacillus jinshani is 7.42 log ₁₀ CFU/mL, which is the same as that of Lactobacillus jinshani. The quantitative results determined by the above methods were basically consistent (coefficient of variation, CV=0.009).

Example 8: Absolute quantification method of Lactobacillus jinshani based on non-extraction of sample genome

(1) Lactobacillus jinshani bacterial liquid was obtained according to the culture method in Example 4, and the microbial concentration was determined by the plate counting method.

(2) Dilute the Lactobacillus jinshani bacterial solution in (1) through a 10-fold gradient

(3) Use the probe of Lactobacillus jinshani to perform color reaction. The sequence of the signal probe is

GGGTGGGTGGGTGGGTAAATAATCTACGGGAGTGCG (SEQ ID NO. 1), the sequence of the quenching probe is CGCACTCCCGTAGATTATTTACCCA (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), 10 μL of bacterial solutions of different dilutions were added (no sample bacterial solution was added as a blank control). Treated in a boiling water bath for 20 min. After adding 4 μL of 20 μM signal probe, the reaction was performed at 55° C. for 30 min. (5) The quenching probe forms a double strand with the unbound signal probe, which destroys the G-quadruplex structure. 8 μL of 20 μM quenching probe was added to the system after the reaction in step (4), and the reaction was carried out at 55° C. for 30 min.

(6) The heme/G quadruplex structure is formed. Reagent 1 (heme) with a final concentration of 100 nM was added to the system after the reaction in step (5), and treated at 37° C. for 30 min.

(7) 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 reaction system in (6), 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.

(8) A standard curve is constructed 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 to 10 ⁷ ). Prove the accuracy of the kit quantitative method provided by the present invention

Example 9: Determination of the content of Lactobacillus jinshani in wine samples based on the absolute quantification method of microorganisms without extracting the sample genome

(1) The sample was collected from a well-known wine manufacturer in Yantai, Shandong Province. 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. 5 mL of phosphate buffer was added to the cells obtained in (1), and the cells were collected by centrifugation at 12,000 × g for 2 min, and the procedure was repeated once.

(3) Resuspend the bacteria, add 1 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) to the bacterial cells obtained in (2), and mix by pipetting. uniform.

(4) Use the probe of Lactobacillus jinshani to perform color reaction. The sequence of the signal probe is

GGGTGGGTGGGTGGGTAAATAATCTACGGGAGTGCG (SEQ ID NO. 1), the sequence of the quenching probe is CGCACTCCCGTAGATTATTTACCCA (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 with a final concentration of 50 mM, KCl with a final concentration of 50 mM, and a final pH of 7.9), 10 μL of grape fermentation bacteria solution was added (no sample bacteria solution was added as a blank control). Treated in boiling water bath for 20min. After adding 4 μL of 20 μM signal probe, the reaction was performed at 55° C. for 30 min.

(6) The quenching probe forms a double strand with the unbound signal probe, which destroys the G-quadruplex structure. 8 μL of 20 μM quenching probe was added to the system after the reaction in step (5), and the reaction was carried out at 55° C. for 30 min.

(7) The heme/G quadruplex structure is formed. Reagent 1 (heme) with a final concentration of 100 nM was added to the system after the reaction in step (6), and treated 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 reaction system 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, and the absorbance value was 0.

(9) According to the standard curve obtained in Example 8, the total amount of Lactobacillus jinshani in the sample was calculated to be 4.98 log ₁₀ CFU/mL.

(10) Quantify Lactobacillus jinshani in the same sample above by fluorescence quantitative PCR (quantitative steps and materials are the same as in Example 11(6)), the results show that the total amount of Lactobacillus jinshani is 0 log ₁₀ CFU/mL, which is similar to the quantitative determination of the above method. The results are consistent.

Example 10: Determination of the content of Lactobacillus jinshani in wine grain samples based on absolute quantitative method without extracting the sample genome

(1) The sample comes from fermented fermented grains of a winery in Shandong. The sample processing method is as follows: add 5 mL of phosphate buffer to 1 g of the sample, and centrifuge at 3000 × g for 10 min to collect the bacteria.

(2) Washing. 5 mL of phosphate buffer was added to the cells obtained in (1), and the cells were collected by centrifugation at 12,000 × g for 2 min, and the procedure was repeated once.

(3) Resuspend the bacteria, add 1 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) to the bacterial cells obtained in (2), and mix by pipetting. uniform.

(4) Use the probe of Lactobacillus jinshani to perform color reaction. The sequence of the signal probe is

GGGTGGGTGGGTGGGTAAATAATCTACGGGAGTGCG (SEQ ID NO. 1), the sequence of the quenching probe is CGCACTCCCGTAGATTATTTACCCA (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 with a final concentration of 50 mM, KCl with a final concentration of 50 mM, and a final pH of 7.9), 10 μL of fermented fermented fermented bacteria was added (no sample bacterial solution was added as a blank control). Treated in boiling water bath for 20min. After adding 4 μL of 20 μM signal probe, the reaction was performed at 55° C. for 30 min.

(6) The quenching probe forms a double strand with the unbound signal probe, which destroys the G-quadruplex structure. 8 μL of 20 μM quenching probe was added to the system after the reaction in step (5), and the reaction was carried out at 55° C. for 30 min.

(7) The heme/G quadruplex structure is formed. Reagent 1 (heme) with a final concentration of 100 nM was added to the system after the reaction in step (6), and treated 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 reaction system 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, and the absorbance value was 0.612.

(9) according to the standard curve obtained in Example 8, the total amount of Lactobacillus jinshani calculated in the sample is 7.45log ₁₀ CFU/mL,

(10) Quantify Lactobacillus jinshani in the same sample above by fluorescent quantitative PCR (quantitative steps and materials are the same as in Example 11 (6)), the results show that Lactobacillus jinshani

The total amount was 7.42 log ₁₀ CFU/mL, which was basically consistent with the two sets of data determined by the above method (coefficient of variation, CV=0.002). Example 11: Comparison of Quantitative Microbial Detection Kit and Fluorescence Quantitative PCR Detection Results

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

(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) 5 mL of phosphate buffer was added to 1 g of the sample, and the cells were collected by centrifugation at 3000 × g for 10 min. 5 mL of phosphate buffer was added to the obtained cells, and the cells were collected by centrifugation at 12,000 × g for 2 min, and repeated once. The bacteria were resuspended, 1 mL of reagent 2 buffer was added to the obtained bacteria, and the mixture was mixed by pipetting.

(3) Use the probe of Lactobacillus jinshani to perform color reaction. The sequence of the signal probe is

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

(4) Determination based on a kit quantitative method without genome extraction.

(i) The signal probe forms double strands 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), 10 μL of fermented fermented fermented bacteria was added (no sample bacterial solution was added as a blank control). Treated in a boiling water bath for 20 min. After adding 4 μL of 20 μM signal probe, the reaction was performed at 55° C. for 30 min.

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

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

(iv) 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 reaction system (iii), 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, and the absorbance values were 0.635, 0.642, and 0.633.

(v) According to the standard curve obtained in Example 8, the total amount of Lactobacillus jinshani in the sample was calculated to be 7.70±0.048log ₁₀ CFU/mL.

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

(i) The signal probe forms double strands 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), 4 μL of fermented grains metagenomic DNA was added (no sample DNA was added as a blank control). Treated in a water bath at 90 °C for 10 min. After adding 4 μL of 20 μM signal probe, the reaction was performed at 55° C. for 30 min.

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

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

(iv) Color reaction. To the reaction system in (5), 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, 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 0.635, 0.654, and 0.623.

(v) According to the standard curve obtained in Example 5, the total amount of Lactobacillus jinshani in the sample is calculated to be 7.67±0.15log ₁₀ CFU/mL.

(6) Quantification of Lactobacillus jinshani content in samples by qPCR

(i) Lactobacillus jinshani bacterial liquid was obtained according to the culture method in Example 4, the microbial concentration was determined by the plate counting method, and the extraction of the genome was the same as that in Example 4.

(ii) Serial dilution of Lactobacillus jinshani genomic DNA by 10-fold.

(iii) The qPCR system was 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) qPCR reaction program: pre-denaturation at 95°C for 5min, cycle stage: 95°C for 5s, 60°C for 20s; the number of cycles is 40, and the melting curve increases from 65°C to 95°C, increasing by 0.5°C every 5s.

(v) qPCR was performed on the extracted genome using Lactobacillus jinshani specific primers, the downstream sequence of the primer sequence was AAATAATCTACGGGAGTGCG (SEQ ID NO. 5), and the downstream sequence was

GTAGGAAATGGCTTGGTAGTGA (SEQ ID NO. 6).

(vi) The standard curve of CT value and Lactobacillus jinshani bacterial concentration was established by 10-fold serial dilution of genomic DNA, as shown in FIG. 4 , R ² =0.99.

(vii) qPCR system and reaction conditions are the same as (iii), (iv). According to the CT value at the end of the reaction, the concentration of Lactobacillus jinshani in the sample calculated by the established standard curve was 7.72±0.09log ₁₀ CFU/g.

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

Example 12: Detection Limits Using Two Different Sequence Signal Probes

(1) Lactobacillus jinshani bacterial liquid was obtained according to the culture method in Example 4, and the concentration of microorganisms was determined by plate counting method, and the concentration was 7.49log10 CFU/mL. The extraction of the genome was the same as that in Example 4.

(2) DNA template of 2.25log10 CFU/mL was obtained by 10-fold gradient dilution of Lactobacillus jinshani genomic DNA.

(3) The sequence of the Lactobacillus jinshani signal probe provided by the present invention is:

GGGTGGGTGGGTGGGTAAATAATCTACGGGAGTGCG (SEQ ID NO. 1), the sequence of the quenching probe is CGCACTCCCGTAGATTATTTACCCA (SEQ ID NO. 2). 3.2 log ₁₀ CFU/mL actobacillus jinshani genomic DNA obtained in (2) was added to carry out a color reaction.

(4) The Lactobacillus jinshani signal probe sequence is (SEQ ID NO.3)

GGGATTGGGATTGGGATTGGGAAATAATCTACGGGAGTGCG, the quenching probe sequence is CGCACTCCCGTAGATTATTTCCCAA (SEQ ID NO. 4). The 3.2log10 CFU/mLLactobacillus jinshani genomic DNA obtained in (2) was added to carry out a color reaction.

(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) was added 4 μL of Actobacillus jinshani genomic DNA (no sample DNA was added as a blank control). Treated in a water bath at 90 °C for 10 min. 4 μL of 20 μM of different signal probes were added respectively and reacted at 55° C. for 30 min.

(6) The quenching probe forms a double strand with the unbound signal probe, which destroys the G-quadruplex structure. 8 μL of 20 μM quenching probes were added to the system after the reaction in step (5), and the reaction was carried out at 55° C. for 30 min.

(7) The heme/G quadruplex structure is formed. Reagent 1 (heme) with a final concentration of 100 nM was added to the system after the reaction in step (6), and treated 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 respectively added to the reaction system 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) Steps (5) (6) (7) (8) are repeated 9 times, and the stability of the detection results is compared, as shown in FIG. 7 . The coefficient of variation (CV) of the quantitative result of the signal sequence based on SEQ ID NO.3 was 1.11; the coefficient of variation of the quantitative result of the signal sequence based on SEQ ID NO.1 was 0.071, and the detection effect was stable.

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

SEQUENCE LISTING

<110> Jiangnan University

<120> A probe and kit for absolute quantification of Lactobacillus jinshani

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 36

<212> DNA

<213> Synthetic

<400> 1

gggtgggtgg gtgggtaaat aatctacggg agtgcg 36

<210> 2

<211> 25

<212> DNA

<213> Synthetic

<400> 2

cgcactcccg tagattattt accca 25

<210> 3

<211> 41

<212> DNA

<213> Synthetic

<400> 3

gggattggga ttgggattgg gaaataatct acgggagtgc g 41

<210> 4

<211> 25

<212> DNA

<213> Synthetic

<400> 4

cgcactcccg tagattattt cccaa 25

Claims (11)

1. a group of probes, is characterized in that, comprises signal probe and quenching probe; 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 . 2. A detection kit, comprising the signal probe and quencher probe according to claim 1. 3. The detection kit according to claim 2, wherein the detection kit also contains any one or more of the following: heme, buffer, 2,2-azinyl-bis-(3 - ethylbenzodihydrothiazoline- ₆ -sulfonic _acid ) diammonium salt, H2O2. 4. A Lactobacillus jinshani quantitative method, characterized in that the method uses the probe described in claim 1, or the detection kit described in any one of claims 2-3. 5 . The quantitative method according to claim 4 , wherein the method comprises: melting of DNA in the sample to be tested; adding excess signal probes to combine with the target nucleotide fragments of the sample to be tested to form double strands. 6 . , make the G quadruplex leak out of the sequence; add a sufficient amount of quenching probe and unbound signal probe to form a double strand, destroy 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 characterizes the biomass of Lactobacillus jinshani . 6. The quantitative method according to claim 4, wherein the method is absolute quantification or relative quantification; when the method is absolute quantification, further comprising: establishing catalase activity or combining with catalase activity The indicators that are correlated are the standard curves of the biomass of Lactobacillus jinshani ; when the samples to be tested are detected, the detected catalase activity or the indicators that are correlated with the catalase activity are substituted into the standard curve, that is, to obtain the standard curve. The biomass of Lactobacillus jinshani in the samples was measured. 7. The quantitative method according to any one of claims 5-6, wherein the sample to be tested is a sample containing bacterial cells, genomes or metagenomes. 8 . The quantitative method according to claim 7 , wherein the sample is a fermented food or a sample or an environmental sample taken from a fermentation process of the fermented food. 9 . 9. quantitative method according to claim 8 is characterized in that, described fermented food is more than any one of following: liquor, rice wine, soy sauce, beer, wine, vinegar, fermented tea, traditional fermented vegetables, fermented beverage, Alcoholic beverages, yogurt, cheese, fruit vinegar, fermented wine, tempeh, fermented bean curd, and fermented rice noodles; the environmental samples are selected from the intestinal tract, soil, and water bodies. 10. The method of use of any one of the kits of claims 2-3, wherein the method of use comprises: adding an excess of signal probes to the DNA melting sample to be tested and reacting for a period of time to make the signal probes Combine with the target fragment in the sample to be tested; then add a sufficient amount of 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, the absorbance value of the reactant was detected, and Lactobacillus jinshani in the sample was quantified in combination with the absorbance value. 11. A method for detecting Lactobacillus jinshani content in fermented food or environmental samples, characterized in that, using the probe described in claim 1, or the test kit described in any one of claims 2-3; 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 beverages, alcoholic beverages, yogurt, cheese, fruit vinegar, fermented wine, tempeh, fermented bean curd, and fermented rice noodles; The environmental samples were selected from the intestinal tract, soil, and water bodies.

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