CN112924429B - Absolute quantitative probe and kit for Lactobacillus jin Shini - Google Patents
Absolute quantitative probe and kit for Lactobacillus jin Shini Download PDFInfo
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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
Technical Field
The invention relates to a Lactobacillus jin Shini absolute quantitative probe and a kit, belonging to the fields of biology, fermentation and detection.
Background
The Lactobacillus jin shinshi is widely distributed in a traditional fermented food brewing system, for example, in a white spirit brewing system, the abundance of the Lactobacillus jin shinshi rapidly rises after 5 days of fermentation and becomes a dominant microorganism in the middle and later stages of fermentation, and the Lactobacillus jin shinshi is found to show a significant correlation with other microorganisms through correlation analysis and plays an important role in the fermentation process; it is reported that the microorganism is also an important functional microorganism in vinegar fermentation systems. The biomass and succession of the strain in the food fermentation process are important microorganisms for representing whether food is normally fermented or not. Therefore, the real-time tracking of the biomass of the Lactobacillus jin Shini has important guiding significance for judging the stability of the fermentation batch and regulating and controlling the fermentation parameters. However, most of the traditional fermented food systems are multi-strain co-fermentation systems, the content of Lactobacillus jin Shini in a sample cannot be judged by a simple OD colorimetric method, and although the quantification of Lactobacillus jin Shini in a mixed bacteria system can be realized by combining a fluorescent quantitative PCR method with a specific primer or probe, high-cost equipment and a high-requirement operating environment are required. Therefore, in order to conveniently, quickly and accurately track the growth change trend of Lactobacillus jin shinshi in a sample, a corresponding Lactobacillus jin shinshi quantitative method and a corresponding 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 digestion polymerization 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 primer 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 jin shinshi; 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 jin Shini solve at least one technical problem as follows: (1) the existing method can not realize the total amount detection of all Lactobacillus jin Shini; (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 (GGGTGGGTGGGTGGGTAAATAATCTACGGGAGTGCG).
In one embodiment, the quenching probe has the sequence shown in SEQ ID NO.2 (CGCACTCCCGTAGATTATTTACCCA).
It is a second object of the present invention to provide a method for quantification of Lactobacillus jin shinani, said method comprising the use of a 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; and (3) reacting the G quadruplex with heme by using the naked leakage outside to form G quadruplex/heme mimic enzyme with catalase activity, and characterizing the biomass of Lactobacillus jinshani by combining the activity of 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 correlated with the catalase activity, such as an absorbance value of a solution after ABTS is generated by catalyzing hydrogen peroxide to generate ABTS + at a wavelength of 420 nm) and the biomass of Lactobacillus jin shinhai; 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 jin shinhai 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 jin shinhai in the different samples according to the relative value of the catalase activity 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 step of reacting the G quadruplex with heme by using a bare leakage outside to form a G quadruplex/heme mimic enzyme with catalase activity, and the step of characterizing the biomass of Lactobacillus jin Shini by combining the catalase activity 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 jin Shini in the sample by combining the absorbance value.
In one embodiment, the quantification method further comprises: preparing samples with different known Lactobacillus jin Shini 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 different Lactobacillus jin Shini contents; substituting the light absorption value obtained by processing the sample to be detected by the method into the standard curve to obtain the Lactobacillus jin shinani content in the sample to be detected.
The third purpose of the invention is to provide a detection kit for the absolute quantification of Lactobacillus jin Shini, 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 test kit further comprises any one or more of: 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 jin shinshi absolute quantification kit comprising simultaneously four reagents (reagent 1, reagent 2, reagent 3, reagent 4) and a set of Lactobacillus jin shinshi quantitative probes (signal probe, quenching probe); 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 H2O2And reacting for a period of time, detecting the light absorption value of the reactant, and quantifying Lactobacillus jin Shini 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 jin Shini 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 jin Shini.
In one embodiment, the use is in the field of fermented food technology; 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.
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 fermented food fermentation process or an environmental sample; 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 Lactobacillus jin shinshi quantitative probe can realize the total amount detection of the Lactobacillus jin shinshi; further, the signal probe is optimized, and the sequence of the signal probe is
GGGTGGGTGGGTGGGTAAATAATCTACGGGAGTGCG (SEQ ID NO.1), and CGCACTCCCGTAGATTATTTACCCA (SEQ ID NO.2) as a quenching probe. 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 Lactobacillus jin Shini quantification, the detection process of expensive instruments 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 jin Shini, and the sample does not need to be subjected to nucleic acid extraction, and only the microorganism in the sample needs to be eluted into the 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 the quantification of Lactobacillus jin Shini, and have the characteristics of rapidness, cheapness and accuracy.
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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 the Lactobacillus jin Shini probe.
FIG. 3: a standard curve of a quantitative probe of Lactobacillus jin Shini extracted based on genome.
FIG. 4: standard curve based on Lactobacillus jin shanni quantitative probe without sample genome extraction.
FIG. 5: qPCR standard curve.
FIG. 6: comparing a quantitative experiment of a Lactobacillus jin Shini probe based on genome extraction, a quantitative experiment of a Lactobacillus jin Shini probe based on non-extracted sample genome and a quantitative experiment of qPCRLactobacillus jin Shini; the quantitative analysis method comprises the following steps of (A) a quantitative experiment of a Lactobacillus jin shinshi probe based on non-extracted sample genome, (B) a quantitative experiment of a Lactobacillus jin shinshi probe based on genome extraction, and (C) a quantitative experiment of qPCRLactobacterium jin shinshi.
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 jin inshani 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 jin inshani quantitative kit and use thereof
The Lactobacillus jin shinshi 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 jin Shini 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 Lactobacillus jin shinshi can be automatically drawn, or the biomass of Lactobacillus jin shinshi is directly converted according to a use method recommended by the kit and the standard curve.
Example 3: lactobacillus jin inshani quantitative kit
The Lactobacillus jin shinshi 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.
The kit further comprises 100nM heme solution (reagent 1), Tris-HCl buffer, 7mM 2, 2-azino-bis- (3-ethylbenzodihydrophthaloline-6-sulfonic acid) diammonium salt (ABTS), and 7mM H2O2And (3) solution.
Example 4: specificity of Lactobacillus jin Shini quantitative kit
(1) Lactobacillus kinsonii from fermented cereals was selected as a positive control, 36 microorganisms of bacterial species and 6 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 crustis, Lactobacillus plantaris, Lactobacillus harbinensis, Lactobacillus acicularis, Pediococcus acidilibacter, Pediococcus pecticii, Pediococcus pectobacterium, Lactobacillus rinus, Lactobacillus curvatus, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus strain. The fungal microorganisms are Aspergillus tubingensis, Mucor rouxianus, Schizosaccharomyces pombe, Zygosaccharomyces bailii, Pichia kudriavzevii, Saccharomyces fibuligera, respectively.
(2) The above microorganisms were cultured by selecting different media, among which Lactobacillus kinsonii, Lactobacillus buchneri, Lactobacillus diovorans, Lactobacillus brevis, Lactobacillus crustorum, Lactobacillus plantarum, Lactobacillus harbinensis, Lactobacillus acidophilus, Lactobacillus ethanolidii, Lactobacillus acidophilus, Pediococcus acidilactici, Pediococcus pentasus, Lactobacillus acidophilus, Lactobacillus murinus, Lactobacillus curvatus, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus panicus, Lactobacillus paniculatus, Lactobacillus fermentum, Lactobacillus judai, Lactobacillus helenci, Lactobacillus judai, Lactobacillus helospirius, Lactobacillus judai, Lactobacillus casei2HPO42.5 g/L, 6.0g/L of sodium acetate trihydrate, 2.0g/L of ammonium citrate tribasic, MgSO4·7H2O 0.3g/L，MnSO4·4H2O0.08g/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 used YPD medium with a formulation of yeast extract 10g/L, peptone 20g/L, and glucose 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 jin inshani specific probe, and 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) 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 results are shown in fig. 2, the experimental group added with the Lactobacillus jin Shini genome shows a color reaction, and the experimental group added with the non-Lactobacillus jin Shini and the blank control group do not show a color reaction, so that the detection of the specificity of the Lactobacillus jin Shini in the kit is proved.
Example 5: quantitative method accuracy assessment
(1) The Lactobacillus jin shinshi bacterial liquid was obtained according to the cultivation method of example 4, the concentration of the microorganism was measured by plate counting method, and the genome was extracted as in example 4.
(2) The genomic DNA of Lactobacillus jin shani was diluted by a 10-fold gradient.
(3) The chromogenic reaction was performed with genomic DNA of Lactobacillus jin Shini at different concentrations using a probe of Lactobacillus jin Shini. The sequence of the signaling probe is GGGTGGGTGGGTGGGTAAATAATCTACGGGAGTGCG (SEQ ID NO.1), and 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 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.
(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 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 log10 CFU/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 jin Shini in wine sample
(1) Reference is made to the Materials and methods of Gayevshiy, V., & Goddard, M. (2012), GeogrAN _ SNhic deletions of yeast communities and publications associated with the channels 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 carried out using a probe from Lactobacillus jin Shini. 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) 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 wine 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.
(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(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 jin Shini in the sample was calculated to be 0log10CFU/mL。
(9) The amount of Lactobacillus jin Shini in the same sample was determined by quantitative PCR (the same procedure and materials as in example 11 and 6), and the total amount of Lactobacillus jin Shini was 0log10CFU/mL, consistent with the quantitative results determined by the above method, 4.
Example 7: absolute quantification of Lactobacillus jin Shini in fermented grain sample
(1) Reference is made to SongZW, Du H, Zhang Y, XuY. innovative core functional microbiological in a crystalline-state implementation by high-through amplified algorithms and microorganisms in a microbiological search 2017; 8:1294, extracting the metagenome from the fermented grain sample from Shandong province, wherein the genome concentration is 100.02 ng/mu L.
(2) The chromogenic reaction was carried out using a probe from Lactobacillus jin Shini. 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).
(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 0.602 as a blank control with no sample DNA added.
(7) The total amount of microorganisms of Lactobacillus jin Shini in the sample was calculated to be 7.33log according to the standard curve obtained in example 210CFU/mL。
(8) The amount of Lactobacillus jin Shini in the same fermented grain sample was determined by a quantitative fluorescence method (the determination procedure and materials were the same as those in example 11 and 6), and the results showed that the total microbial amount of Lactobacillus jin Shini was 7.42log10CFU/mL was substantially identical to the quantitative results measured by the above method (coefficient of variation, CV ═ 0.009).
Example 8: lactobacillus jin Shini absolute quantitative method based on non-extracted sample genome
(1) The Lactobacillus jin shinshi bacterial solution was obtained according to the cultivation method of example 4, and the concentration of the microorganism was measured by plate counting.
(2) Diluting the Lactobacillus jin Shini bacterial liquid in the step (1) by 10 times of gradient
(3) The chromogenic reaction was carried out using a probe from Lactobacillus jin Shini. 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) 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.
(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. 4, R20.99(x is log10 CFU/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 jin Shini in wine sample based on absolute quantification method of microorganism 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 carried out using a probe from Lactobacillus jin Shini. 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).
(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 jin Shini in the sample was calculated to be 4.98log10CFU/mL。
(10) The Lactobacillus in the same sample was subjected to quantitative PCR (quantitative procedure and materials as in example 11(6))Quantification is carried out on the Bacillus jin Shini, and the result shows that the total amount of the Lactobacillus jin Shini is 0log10CFU/mL, consistent with the quantitative results determined by the methods described above.
Example 10: absolute quantitative method for determining content of Lactobacillus jin Shini 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.
(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 carried out using a probe from Lactobacillus jin Shini. 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).
(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.
(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. Measurement with ultraviolet spectrophotometerThe absorbance at a wavelength of 420nm, blank with the test group without sample DNA, was 0.612.
(9) According to the standard curve obtained in example 8, the total amount of Lactobacillus jin Shini in the sample was calculated to be 7.45log10CFU/mL，
(10) The amount of Lactobacillus jin Shini in the same sample was determined by quantitative PCR (the same quantitative procedure and materials as in example 11 and 6), and the results showed that Lactobacillus jin Shini
The total amount is 7.42log10CFU/mL was substantially identical to the two sets of data measured by the above method (coefficient of variation, CV ═ 0.002). 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.
(3) The chromogenic reaction was carried out using a probe from Lactobacillus jin Shini. 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) 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. 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 showed absorbance values of 0.635, 0.642, and 0.633.
(v) The total amount of Lactobacillus jin Shini in the sample was calculated to be 7.70. + -. 0.048log according to the standard curve obtained in example 810CFU/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.
(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 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 with no sample DNA added showed absorbance values of 0.635, 0.654, 0.623.
(v) The total amount of Lactobacillus jin Shini in the sample was calculated to be 7.67. + -. 0.15log according to the standard curve obtained in example 510CFU/mL。
(6) quantitative qPCR (quantitative polymerase chain reaction) for determining the content of Lactobacillus jin Shini in sample
(i) The Lactobacillus jin shinshi bacterial liquid was obtained according to the cultivation method of example 4, the concentration of the microorganism was measured by plate counting method, and the genome was extracted as in example 4.
(ii) The genomic DNA of Lactobacillus jin shani 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) qPCR was performed on the extracted genome using a Lactobacillus jin Shini specific primer with a sequence downstream of AAATAATCTACGGGAGTGCG (SEQ ID NO.5) and a sequence downstream of
GTAGGAAATGGCTTGGTAGTGA(SEQ ID NO.6)。
(vi) The genomic DNA was diluted by 10-fold gradient to establish a standard curve of CT value and Lactobacillus jin Shini 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 end of the reaction, the concentration of Lactobacillus jin Shini in the sample is calculated to be 7.72 +/-0.09 log according to 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 liquid of Lactobacillus jinshani was obtained according to the cultivation method of example 4, and the concentration of the microorganism was measured by plate counting method, and the genome was extracted at a concentration of 7.49log10 CFU/mL as in example 4.
(2) The genomic DNA of Lactobacillus jin shinani was diluted by a 10-fold gradient to give a DNA template of 2.25log10 CFU/mL.
(3) The sequence of the Lactobacillus jin Shini signal probe provided by the invention is
GGGTGGGTGGGTGGGTAAATAATCTACGGGAGTGCG (SEQ ID NO.1), and the sequence of the quenching probe is CGCACTCCCGTAGATTATTTACCCA (SEQ ID NO. 2). Adding the 3.2log obtained in (2)10CFU/mL Lactobacillus jin Shini genomic DNA for color reaction.
(4) The sequence of the signal probe using Lactobacillus jin Shini is shown as (SEQ ID NO.3)
GGGATTGGGATTGGGATTGGGAAATAATCTACGGGAGTGCG, and the sequence of the quenching probe is CGCACTCCCGTAGATTATTTCCCAA (SEQ ID NO. 4). 3.2log10 CFU/mL Lactobacillus jin shinhai genomic DNA 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.LLactobacterium jin shinhai 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.
(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 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 1.11; the coefficient of variation of the quantitative result based on the signal sequence of SEQ ID NO.1 is 0.071, 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> Lactobacillus jin Shini absolute quantitative probe and kit
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 36
<212> DNA
<213> Artificial Synthesis
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gggtgggtgg gtgggtaaat aatctacggg agtgcg 36
<210> 2
<211> 25
<212> DNA
<213> Artificial Synthesis
<400> 2
cgcactcccg tagattattt accca 25
<210> 3
<211> 41
<212> DNA
<213> Artificial Synthesis
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gggattggga ttgggattgg gaaataatct acgggagtgc g 41
<210> 4
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<212> DNA
<213> Artificial Synthesis
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cgcactcccg tagattattt cccaa 25
Claims (11)
1. A set of probes, comprising a signaling probe and a quenching probe; the sequence of the signal probe is a sequence shown in SEQ ID NO. 1; the sequence of the quenching probe is shown as 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, 2-azino-bis- (3-ethylbenzodihydropyrazoline-6-sulfonic acid) diammonium salt, and H2O2。
4. A kind ofLactobacillus jinshaniA method of quantification, which comprises using the probe according to claim 1 or the detection kit according to any one of claims 2 to 3.
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; g quadruplex/heme mimic enzyme with catalase activity is formed by reacting naked leakage outside G quadruplex with heme, and activity characterization of catalase is combinedLactobacillus jinshaniThe biomass of (a).
6. A quantification method according to claim 4, wherein the method is absolute quantification or relative quantification; when the method is absolute quantification, the method further comprises the following steps: establishing an index of catalase activity or a correlation with catalase activity, anLactobacillus jinshaniA standard curve of biomass of (a); when detecting a sample to be detected, substituting the detected catalase activity or an index which is correlated with the catalase activity into the standardCurves, i.e. obtaining in the sample to be testedLactobacillus jinshaniThe biomass of (a).
7. The method according to any one of claims 5 to 6, wherein the sample to be tested is a sample containing a cell, a genome or a metagenome.
8. The quantification method according to claim 7, wherein the sample is a fermented food or a sample obtained from a fermentation process of a fermented food or an environmental sample.
9. The method of claim 8, 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 and water body.
10. 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 enough quenching probe to form double chains with the unbound signal probe; adding heme, reacting for a while, adding ABTS and H2O2Reacting for a period of time, detecting the absorbance of the reactant, and combining the absorbance with the absorbance in the sampleLactobacillus jinshaniQuantification was performed.
11. In detecting fermented food or environmental sampleLactobacillus jinshaniA method for assaying a nucleic acid molecule comprising the step of using the probe according to claim 1 or the kit according to any one of claims 2 to 3; the fermented food is any one or more of the following: chinese liquor, yellow wine, soy sauce, beer, wine, edible vinegar, fermented tea, traditional fermented vegetables, fermented beverage, and alcoholic beverageProduct, yogurt, cheese, fruit vinegar, fermented glutinous rice, fermented soybean, fermented bean curd, and fermented rice and flour food; the environmental sample is selected from intestinal tract, soil and water body.
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| 基于三步荧光定量PCR技术揭示不同产区白酒酿造系统中Lactobacillus sp.的分布特征;杜如冰等;《微生物学通报》;20190529(第01期);8-19 * |
| 沙门菌实时荧光定量PCR快速检测方法的建立;田赛等;《生物技术通讯》;20191130(第06期);79-83+90 * |
| 荧光定量PCR方法检测白酒发酵过程中Aspergillus tubingensis生物量;陈笔等;《微生物学通报》;20140606(第12期);160-167 * |
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