CN111849966B - Constant temperature detection method for identifying lactobacillus brevis and special primer and kit thereof - Google Patents
Constant temperature detection method for identifying lactobacillus brevis and special primer and kit thereof Download PDFInfo
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Abstract
The invention discloses a constant temperature detection method for identifying lactobacillus brevis, and a special primer and a kit thereof. The specific primer group provided by the invention consists of a primer F3, a primer B3, a primer FIP, a primer BIP, a primer LF and a primer LB, wherein the six primers are sequentially shown as sequences 2 to 7 in a sequence table. The specific primer group is used as follows (b1) or (b 2): (b1) identifying or assisting in identifying lactobacillus brevis; (b2) and detecting whether the sample to be detected contains the lactobacillus brevis or not. The invention utilizes the loop-mediated isothermal amplification technology to amplify the specific gene fragment of lactobacillus brevis, fills the blank of the LAMP detection method of lactobacillus brevis in beer, has the advantages of strong specificity, simple operation, rapidness, convenience and the like, can be used for beer quality monitoring, and is suitable for the primary application of a brewery and the rapid field detection.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a constant-temperature detection method for identifying lactobacillus brevis, and a special primer and a kit thereof.
Background
Lactobacillus brevis (Lactobacillus brevis) is widely distributed in nature, belongs to the order of Lactobacillales, the family of Lactobacillaceae, the genus Lactobacillus brevis, and is commonly found in milk, kefir, cheese, and sauerkraut. When Lactobacillus brevis exists in beer, the acidity and viscosity of the beer are increased, and further turbid and sediment of the beer are caused, so that the taste and quality of the beer are influenced. Beer quality events caused by lactobacillus brevis pollution are frequent, statistics shows that more than 50% of daily beer pollution events are caused by lactobacillus brevis, and the rapid detection of lactobacillus brevis becomes a great problem to be urgently solved for quality monitoring of breweries.
At present, the detection methods for lactobacillus brevis mainly comprise a culture identification method, a bioluminescence and immunology method and a molecular biology method. The culture identification method is to use NBB-A, NBB-B, MRS and other culture mediums to culture thalli, and determine the type of the thalli through staining microscopy, physiological and biochemical experiments and the like, and is the most common detection method in domestic breweries at present. The method has higher accuracy, low cost and low requirement on experimenters in all types of detection methods at present, can meet the basic application requirements, but no matter which culture medium is adopted for culture identification, the result detection needs more than 5-7 days, cannot reflect the beer pollution condition in time, and cannot adapt to the requirement of rapid quality monitoring. The bioluminescence and immunology methods mainly comprise an ATP bioluminescence detection technology and an antigen-antibody detection method; the ATP bioluminescence detection technology achieves the purpose of detecting microorganisms by detecting ATP generated in the microbial metabolism process, can quickly obtain a detection result and also can quantify the microorganisms when the ATP bioluminescence detection technology is used for detecting the microorganisms, but cannot qualitatively identify different microorganisms, is easy to be interfered by the outside and has unstable results; the antigen-antibody method is based on the principle that an antigen and a corresponding antibody are specifically combined to form a precipitate compound to carry out qualitative detection on microorganisms, including agglutination reaction, precipitation reaction, enzyme-linked immunosorbent and the like.
The molecular biology method is based on nucleic acid level detection, common molecular biology methods applied to microorganism detection include Fluorescence In Situ Hybridization (FISH), gene chip technology, polymerase chain reaction PCR and the like, and the methods have the advantages of strong specificity and short time consumption, but are difficult to be effectively developed in winery detection due to the problems of complex operation, high requirements on equipment and operators and the like.
Disclosure of Invention
The invention aims to provide a constant temperature detection method for identifying lactobacillus brevis, and a special primer and a kit thereof.
The specific primer group provided by the invention consists of a primer F3, a primer B3, a primer FIP, a primer BIP, a primer LF and a primer LB;
the primer F3 is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and have the same functions as the sequence 2;
the primer B3 is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown in sequence 3 of the sequence table;
(a4) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 3;
the primer FIP is (a5) or (a6) as follows:
(a5) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;
(a6) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same functions as the sequence 4;
the primer BIP is (a7) or (a8) as follows:
(a7) a single-stranded DNA molecule shown in sequence 5 of the sequence table;
(a8) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 5 and has the same function as the sequence 5;
primers LF are as follows (a9) or (a 10):
(a9) a single-stranded DNA molecule shown in sequence 6 of the sequence table;
(a10) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 6 and has the same function as the sequence 6.
The primers LB are as follows (a11) or (a 12):
(a11) a single-stranded DNA molecule shown in sequence 7 of the sequence table;
(a12) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 7 and has the same function as the sequence 7.
The specific primer group is a primer group based on loop-mediated isothermal amplification.
The specific primer group is used as follows (b1) or (b 2): (b1) identifying or assisting in identifying lactobacillus brevis; (b2) and detecting whether the sample to be detected contains the lactobacillus brevis or not.
In the specific primer group, the molar ratio of a primer F3, a primer B3, a primer FIP, a primer BIP, a primer LF and a primer LB is 1:1:8:8:4:4 in sequence.
The invention also protects the application of the specific primer group in the preparation of reagents or kits; the reagent or the kit has the following uses (b1) or (b 2): (b1) identifying or assisting in identifying lactobacillus brevis; (b2) and detecting whether the sample to be detected contains the lactobacillus brevis or not.
The invention also protects a specific reagent which comprises the following components: the specific primer group; the specific reagent is in a liquid state, and the content of each primer in each 11 mu L of reagent is as follows: primer F32.5 pmol, primer B32.5pmol, primer FIP 20pmol, primer BIP 20pmol, primer LF 10pmol, and primer LB 10 pmol. The specific reagent further comprises: isotermal Master Mix and water. Each 11. mu.L reagent contained 7.5. mu.L of Isotermal Master Mix.
The invention also protects a kit containing the specific primer group or the specific reagent; the use of the kit is as follows (b1) or (b 2): (b1) identifying or assisting in identifying lactobacillus brevis; (b2) and detecting whether the sample to be detected contains the lactobacillus brevis or not.
The invention also provides a method for identifying or assisting in identifying the lactobacillus brevis, which comprises the following steps:
(1) extracting the genome DNA of the microorganism to be detected;
(2) taking the genomic DNA extracted in the step (1) as a template, and performing loop-mediated isothermal amplification by adopting the specific primer group; if the specific primer group can realize specific amplification by taking the genome DNA as a template, the microorganism to be detected is or is selected as lactobacillus brevis; if the specific primer group can not realize specific amplification by taking the genome DNA as a template, the microorganism to be detected is or is selected as non-lactobacillus brevis.
The invention also provides a method for detecting whether the sample to be detected contains lactobacillus brevis, which comprises the following steps:
(1) extracting the total DNA of a sample to be detected;
(2) taking the total DNA extracted in the step (1) as a template, and adopting the specific primer group to perform loop-mediated isothermal amplification; if the primer group can realize specific amplification by taking the total DNA as a template, the sample to be detected contains or is suspected to contain lactobacillus brevis; if the specific primer group can not realize specific amplification by taking the total DNA as a template, the sample to be detected does not contain or is suspected to contain lactobacillus brevis.
The sample to be tested can be a beer sample.
The invention also provides a method for identifying or assisting in identifying the lactobacillus brevis, which comprises the following steps:
detecting whether the genome DNA of the microorganism to be detected has a specific DNA molecule; if the specific DNA molecule exists in the genome DNA of the microorganism to be detected, the microorganism to be detected is or is selected as lactobacillus brevis; if the specific DNA molecule is not contained in the genome DNA of the microorganism to be tested, the microorganism to be tested is or is selected as non-lactobacillus brevis; the specific DNA molecule is a target sequence of a specific primer pair in the genome DNA of the lactobacillus brevis; the specific primer pair consists of a single-stranded DNA molecule shown in a sequence 2 of the sequence table and a single-stranded DNA molecule shown in a sequence 3 of the sequence table.
The specific DNA molecule can be specifically shown as a sequence 1 in a sequence table.
The invention also provides a method for detecting whether the sample to be detected contains lactobacillus brevis, which comprises the following steps:
detecting whether the total DNA of a sample to be detected has specific DNA molecules; if the specific DNA molecules exist in the total DNA of the sample to be detected, the sample to be detected contains or is suspected to contain lactobacillus brevis; if the total DNA of the sample to be detected does not contain the specific DNA molecules, the sample to be detected does not contain or is suspected to contain lactobacillus brevis; the specific DNA molecule is a target sequence of a specific primer pair in the genome DNA of the lactobacillus brevis; the specific primer pair consists of a single-stranded DNA molecule shown in a sequence 2 of the sequence table and a single-stranded DNA molecule shown in a sequence 3 of the sequence table.
The specific DNA molecule can be specifically shown as a sequence 1 in a sequence table.
The sample to be tested can be a beer sample.
Reaction system for loop-mediated isothermal amplification (12.5 μ L): template solution 1.5. mu.L, Isotermal Master Mix 7.5. mu.L, primers of primer set, balance water. In the reaction system, the content of each primer is as follows: primer F32.5 pmol, primer B32.5pmol, primer FIP 20pmol, primer BIP 20pmol, primer LF 10pmol, and primer LB 10 pmol.
The reaction temperature for the loop-mediated isothermal amplification may be 65 ℃.
Reaction conditions for loop-mediated isothermal amplification: at 65 deg.C for 30 min.
Loop-mediated isothermal amplification (LAMP).
So far, no LAMP special primer and kit for detecting lactobacillus brevis are available in the market.
The invention has the beneficial effects that: (1) the specificity is stronger: 8 regions of the target gene are locked, and 2 inner primers, 2 outer primers and 2 loop primers are used for detection, so that the specificity is stronger; (2) the sensitivity is higher: the detection sensitivity is at least 1 order of magnitude higher than that of other molecular biological methods such as polymerase chain reaction and the like; (3) the result identification is simple and convenient: the amplification product is directly judged by observing the fluorescence amplification curve and the dissolution curve, so that the subsequent steps of gel electrophoresis and sequencing detection are avoided, the operation is simple, the cost is low, and the method is more suitable for field detection; (4) the time consumption is shorter: the amplification detection time only needs 30min, the detection period is shorter, and the speed is faster; (5) the matching equipment has high portability: lithium electricity supply does not need professional laboratory and any laboratory equipment, and the routine environment scene realizes "integral type" operations such as sample processing, detection.
The invention utilizes the loop-mediated isothermal amplification technology to amplify the specific gene fragment of lactobacillus brevis, fills the blank of the LAMP detection method of lactobacillus brevis in beer, has the advantages of strong specificity, simple operation, rapidness, convenience and the like, can be used for beer quality monitoring, and is suitable for the primary application of a brewery and the rapid field detection.
Drawings
FIG. 1 is a fluorescent amplification curve of LAMP reaction carried out using three primer sets, respectively, in example 2.
FIG. 2 is a melting curve of LAMP reaction using the P1 primer set in example 2.
FIG. 3 is a fluorescent amplification curve in example 3.
FIG. 4 is a fluorescent amplification curve in example 4.
FIG. 5 is a fluorescent amplification curve of LAMP in example 5.
FIG. 6 is a fluorescent amplification curve of real-time fluorescent PCR in example 5.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 primer design for LAMP detection of Lactobacillus brevis
A plurality of specific nucleic acid fragments of lactobacillus brevis are screened by a large number of sequence comparison, and an LAMP primer group with the optimal effect is finally obtained through LAMP primer design and pre-experimental effect verification and is named as a specific primer group. The target sequence of the specific primer group is shown as a sequence 1 in a sequence table.
The specific primer group consists of six primers, and the sequences are as follows:
primer F3 (SEQ ID NO: 2 of the sequence Listing): 5'-AAACCGCCAAGCCGATTG-3', respectively;
primer B3 (SEQ ID NO: 3 of the sequence Listing): 5'-GCGGTTTTGGCTTTTAAGGT-3', respectively;
primer FIP (SEQ ID NO: 4 of the sequence table): 5'-ACCAATTGCTGGTCCAGGTGTTAACGTTGGAACTCCCATCAC-3';
primer BIP (sequence 5 of the sequence table): 5'-AATACGCATACCAAGCCGCTCAGCCTGTTGCTGACGTTCAT-3';
primer LF (sequence 6 of the sequence listing): 5'-GTCCAGTAATCACGATGCAGG-3', respectively;
primer LB (SEQ ID NO: 7 of the sequence Listing): 5'-CAACCCGTGTTGATTCTCGAA-3' is added.
Example 2 comparison of Effect of primer set
When a specific primer set is obtained by screening, preliminary experiments are performed on a large number of primer sets to compare the effects. The present embodiment only shows a partial effect comparison by way of example.
The sequences of the primers of the three primer sets are shown in Table 1. Each primer was synthesized manually. The P1 primer set is the specific primer set of example 1. The P2 primer set and the P3 primer set are exemplary other primer sets in the screening process.
TABLE 1
Three primer sets were used for LAMP reactions, respectively, to compare the effects.
The test bacteria are: lactobacillus brevis CICC 24450.
1. Taking test bacteria liquid (with bacteria concentration of 10)5CFU/mL), treating at 99 ℃ for 5min, then terminating the reaction at 4 ℃, and collecting the supernatant to obtain a template solution containing the genomic DNA.
2. LAMP was carried out.
Reaction (12.5 μ L): template solution 1.5. mu.L, Isotermal Master Mix 7.5. mu.L, primers of primer set, balance water. In the reaction system, the content of each primer is as follows: primer F32.5 pmol, primer B32.5pmol, primer FIP 20pmol, primer BIP 20pmol, primer LF 10pmol, and primer LB 10 pmol. Isotermal Master Mix: beijing Frorron Biotechnology Ltd, the product number is ISO-004.
A negative control was set up with double distilled water instead of template solution.
Reaction conditions are as follows: at 65 deg.C for 30 min. The reaction was carried out in a Genie II real-time fluorescence detector.
The fluorescent amplification curves of LAMP reactions using three primer sets, respectively, are shown in FIG. 1. The primer group with the fluorescence value P1 is superior to other primer groups in both the amplification time and the fluorescence value. The annealing temperature of the P1 primer set was 88 deg.C (see FIG. 2).
Example 3 optimization of reaction temperature
The test bacteria are: lactobacillus brevis CICC 24450.
1. Taking test bacteria liquid (with bacteria concentration of 10)5CFU/mL), treating at 99 ℃ for 5min, then terminating the reaction at 4 ℃, and collecting the supernatant to obtain a template solution containing the genomic DNA.
2. LAMP was carried out.
The reaction system was the same as in example 2. The specific primer set of example 1 was used.
Reaction conditions are as follows: at a certain temperature (60 deg.C, 61 deg.C, 62 deg.C, 63 deg.C, 64 deg.C, 65 deg.C, 66 deg.C or 67 deg.C) for 30 min. The reaction was carried out in a Genie II real-time fluorescence detector.
The fluorescence amplification curve is shown in FIG. 3. The optimal amplification temperature is 65 ℃.
Example 4 specificity
The test bacteria are respectively as follows: lactobacillus brevis CICC24450(Lactobacillus brevis), Lactobacillus brevis CICC20014(Lactobacillus brevis), Lactobacillus casei CICC6117(Lactobacillus casei), Lactobacillus plantarum CICC20261(Lactobacillus plantarum), Lactobacillus acidophilus CICC6074(Lactobacillus acidophilus), Lactobacillus delbrueckii CICC6047(Lactobacillus delbrueckii), Bifidobacterium longum CICC6069(Bifidobacterium longum), Streptococcus thermophilus CICC6038(Streptococcus thermophilus), Lactobacillus lactis CICC6242 (Lactobacillus lactis), Leuconostoc mesenteroides CICC20714(Leuconostoc mesenteroides), Bacillus subtilis CICC 713(Bacillus subtilis), Pediococcus pentosaceus strain 62 (Streptococcus faecalis 21821821804), and Escherichia aeruginosa Escherichia coli (Escherichia coli) 10210003. CICC is China Industrial Culture Collection (China Center of Industrial Culture Collection), http:// sales.
1. Preparation of template solution
Taking test bacteria liquid (the bacteria concentration is 10)5CFU/mL), treating at 99 ℃ for 5min, then terminating the reaction at 4 ℃, and collecting the supernatant to obtain a template solution containing the genomic DNA.
2. LAMP was carried out.
The reaction system was the same as in example 2. The specific primer set of example 1 was used.
The reaction conditions are as follows: at 65 deg.C for 30 min. The reaction was carried out in a Genie II real-time fluorescence detector.
The fluorescence amplification curve is shown in FIG. 4. Lactobacillus brevis 1 and Lactobacillus brevis 2, both positive. The other test bacteria were negative. The results show that the specific primer group has high specificity.
Example 5 sensitivity
The test bacteria are: lactobacillus brevis CICC 24450.
1. Preparation of template solution
The bacterial concentration in the bacterial suspension of the test bacteria is determined by plate counting, and then the bacterial suspension is diluted by sterile physiological saline to obtain bacterial suspensions with the bacterial concentrations of 1, 10, 100, 1000, 10000 and 100000CFU/mL respectively.
Taking the bacterial suspension, treating for 5min at 99 ℃, then terminating the reaction at 4 ℃, and collecting the supernatant to obtain the template solution containing the genome DNA.
2. LAMP was carried out.
The reaction system was the same as in example 2. The specific primer set of example 1 was used.
Reaction conditions are as follows: at 65 deg.C for 30 min. The reaction was carried out in a Genie II real-time fluorescence detector.
The fluorescence amplification curve is shown in FIG. 5. A100-100000 CFU/mL bacterial suspension can generate a fluorescence amplification curve. The result shows that the detection sensitivity of the specific primer group to the lactobacillus brevis is 100CFU/mL through simple nucleic acid extraction.
3. Performing real-time fluorescent PCR
Reaction system (20 μ L): 2 μ L of template solution, 10 μ L of TB Green Fast qPCR Mix (2X), 0.4 μ L of ROX Reference Dye (50X), primer F3 and primer B3, and the balance of water. In the reaction system, the content of each primer is as follows: primer F38 pmol, primer B38 pmol.
Primer F3 (SEQ ID NO: 2 of the sequence Listing): 5'-AAACCGCCAAGCCGATTG-3', respectively;
primer B3 (SEQ ID NO: 3 of the sequence Listing): 5'-GCGGTTTTGGCTTTTAAGGT-3' are provided.
The reaction conditions are as follows: 2min at 50 ℃; 10min at 95 ℃; at 95 ℃ for 15s, 60 ℃ for 1min, 45 cycles. The reaction is carried out in an ABI7900 real-time fluorescence detector, and the amplification result with the Ct value less than or equal to 40 is judged to be positive.
The fluorescence amplification curve is shown in FIG. 6. The bacterial suspension with the concentration of 1000-100000CFU/mL can generate a fluorescence amplification curve, and the detection sensitivity is 1000 CFU/mL.
The results show that the LAMP detection method of the invention has 10 times higher sensitivity than real-time fluorescent PCR by the same nucleic acid extraction steps.
Example 6 preparation of LAMP detection reagent for detecting Lactobacillus brevis
Detection reagent: isotermal Master Mix 7.5. mu.L, each primer of the specific primer set of example 1, balance water. In each 11 mu L of detection reagent, the content of each primer is as follows: primer F32.5 pmol, primer B32.5pmol, primer FIP 20pmol, primer BIP 20pmol, primer LF 10pmol, and primer LB 10 pmol.
The use method of the detection reagent comprises the following steps: taking 11 mu L of detection reagent, adding 1.5 mu L of genome DNA or total DNA of a sample to be detected, and then carrying out LAMP reaction. Reaction conditions are as follows: at 65 deg.C for 30 min. And (5) observing a fluorescence amplification curve.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.
Sequence listing
<110> Beijing Yanjing beer GmbH
China food fermentation industry research institute Co., Ltd
<120> constant temperature detection method for identifying lactobacillus brevis, and special primer and kit thereof
<130> GNCYX201560
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 202
<212> DNA
<213> Lactobacillus breris
<400> 1
aaaccgccaa gccgattgcc ttagatggca acaaattaac gttggaactc ccatcacccc 60
tgcatcgtga ttactggacc catcaacacc tggaccagca attggtagaa tacgcatacc 120
aagccgctca cgaagatatt caacccgtgt tgattctcga aaatgaacgt cagcaacagg 180
ccaccttaaa agccaaaacc gc 202
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aaaccgccaa gccgattg 18
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
<210> 4
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
accaattgct ggtccaggtg ttaacgttgg aactcccatc ac 42
<210> 5
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aatacgcata ccaagccgct cagcctgttg ctgacgttca t 41
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gtccagtaat cacgatgcag g 21
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
caacccgtgt tgattctcga a 21
Claims (7)
1. The specific primer group consists of a primer F3, a primer B3, a primer FIP, a primer BIP, a primer LF and a primer LB;
the primer F3 is a single-stranded DNA molecule shown in a sequence 2 in a sequence table;
the primer B3 is a single-stranded DNA molecule shown in a sequence 3 in a sequence table;
the primer FIP is a single-stranded DNA molecule shown in a sequence 4 of a sequence table;
the primer BIP is a single-stranded DNA molecule shown in a sequence 5 of a sequence table;
the primer LF is a single-stranded DNA molecule shown in a sequence 6 in a sequence table;
the primer LB is a single-stranded DNA molecule shown in a sequence 7 of the sequence table.
2. The use of the specific primer set of claim 1 in the preparation of a reagent or a kit; the reagent or the kit has the following uses (b1) or (b 2): (b1) identifying or assisting in identifying lactobacillus brevis; (b2) and detecting whether the sample to be detected contains the lactobacillus brevis or not.
3. The specific reagent comprises the following components: the specific primer set of claim 1; the specific reagent is in a liquid state, and the content of each primer in each 11 mu L of detection reagent is as follows: primer F32.5pmol, primer B32.5pmol, primer FIP 20pmol, primer BIP 20pmol, primer LF 10pmol, primer LB 10 pmol.
4. A kit comprising the specific primer set according to claim 1 or the specific reagent according to claim 3; the use of the kit is as follows (b1) or (b 2): (b1) identifying or assisting in identifying lactobacillus brevis; (b2) and detecting whether the sample to be detected contains the lactobacillus brevis or not.
5. A method for identifying or assisting in identifying Lactobacillus brevis comprises the following steps:
(1) extracting the genome DNA of the microorganism to be detected;
(2) taking the genomic DNA extracted in the step (1) as a template, and carrying out loop-mediated isothermal amplification by using the specific primer group of claim 1; if the specific primer set of claim 1 can realize specific amplification using the genomic DNA as a template, the microorganism to be tested is or is selected from Lactobacillus brevis; if the specific primer set according to claim 1 cannot achieve specific amplification using the genomic DNA as a template, the microorganism to be tested is or is candidate for non-Lactobacillus brevis.
6. A method for detecting whether a sample to be detected contains lactobacillus brevis or not comprises the following steps:
(1) extracting the total DNA of a sample to be detected;
(2) taking the total DNA extracted in the step (1) as a template, and carrying out loop-mediated isothermal amplification by adopting the specific primer group in claim 1; if the primer group of claim 1 can realize specific amplification by using the total DNA as a template, the sample to be tested contains or is suspected to contain lactobacillus brevis; if the specific primer set of claim 1 cannot achieve specific amplification using the total DNA as a template, the sample to be tested does not contain or is suspected to contain Lactobacillus brevis.
7. The method of claim 6, wherein: the sample to be detected is a beer sample.
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| CN101553566A (en) * | 2006-11-30 | 2009-10-07 | 札幌啤酒株式会社 | Method for detection of hop latent virus, primer set for the detection, and kit for the detection |
| CN104513867A (en) * | 2014-12-29 | 2015-04-15 | 张成标 | Primer set, reagent kit and method for detecting hop stunt viroid based on loop-mediated isothermal amplification technology |
| EP3187595A1 (en) * | 2015-12-30 | 2017-07-05 | Fundacion Gaiker | Method for the detection of legionella spp. strains in environmental samples based on loop-mediated isothermal amplification (lamp), detection reagent, set of primers, device for dosing and preparing the sample for analysis, support, and system for dosing and preparing the sample for analysis |
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| CN101553566A (en) * | 2006-11-30 | 2009-10-07 | 札幌啤酒株式会社 | Method for detection of hop latent virus, primer set for the detection, and kit for the detection |
| CN104513867A (en) * | 2014-12-29 | 2015-04-15 | 张成标 | Primer set, reagent kit and method for detecting hop stunt viroid based on loop-mediated isothermal amplification technology |
| EP3187595A1 (en) * | 2015-12-30 | 2017-07-05 | Fundacion Gaiker | Method for the detection of legionella spp. strains in environmental samples based on loop-mediated isothermal amplification (lamp), detection reagent, set of primers, device for dosing and preparing the sample for analysis, support, and system for dosing and preparing the sample for analysis |
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