Primer probe, kit and method for rapid identification of Lactobacillus casei

Abstract

The present invention relates to the field of biotechnology, and in particular to a primer probe, a kit and a method for rapidly identifying lactobacillus casei. The primer probe is designed according to the nucleotide sequence shown in SEQ ID NO.1 or the nucleotide sequence shown in SEQ ID NO.2. The primer probe and the kit product of the present invention can rapidly detect whether a product contains lactobacillus casei, and the detection result has high accuracy and strong stability. The application of the primer probe and the kit product of the present invention in production, life, medicine and health care, etc. has broad market prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a primer probe, a kit and a method for rapidly identifying Lactobacillus casei. Background Art

[0002] Probiotics are a class of active microbial food raw materials that are beneficial to human health. Commonly used probiotics have two major categories: Lactobacillus acidophilus and Bifidobacterium, which are called "healthy three beneficial bacteria" together with Lactobacillus casei. Lactobacillus casei belongs to the genus Lactobacillus, is a Gram-positive bacterium, does not produce spores, has no flagella, does not move, and facultative heterofermentation of lactose. After entering the human body, Lactobacillus casei can survive in large quantities in the intestine, play a role in regulating the balance of intestinal flora and promoting human digestion and absorption. At the same time, Lactobacillus casei has high-efficiency blood pressure reduction, cholesterol reduction, lactose intolerance, enhancement of human immunity and prevention of cancer and other probiotic health care effects, and has received more and more attention in the field of functional food development. Therefore, increasing the research on Lactobacillus casei and developing functional dairy products with it is of great significance for human production and life.

[0003] ERIC sequence is a repetitive sequence existing in the genome of Enterobacter. The target gene is amplified by ERIC-PCR technology to reflect the characteristic bands of bacteria. Compared with traditional bacterial identification methods, ERIC-PCR technology is relatively simple and convenient, and can be widely used in microbiology. The existing ERIC-PCR technology has the technical defects of large detection error and can only distinguish the casei subspecies of Lactobacillus casei in the identification of Lactobacillus casei. At the same time, the DNA purity of the sample is high during the detection reaction, which limits the application scenarios of Lactobacillus casei detection and cannot achieve targeted detection of Lactobacillus casei in complex habitats.

[0004] However, no specific fragments that can effectively identify Lactobacillus casei have been screened yet. Summary of the invention

[0005] The main purpose of the present invention is to provide primers, a kit and a method for rapid identification of Lactobacillus casei. The present invention screened and obtained two gene fragments that can specifically identify Lactobacillus casei, as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides the use of the nucleotide sequence shown in SEQ ID NO.1 and / or the nucleotide sequence shown in SEQ ID NO.2 in identifying Lactobacillus casei. The present invention has found through a large number of screenings that all or part of the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2 can be used to identify Lactobacillus casei.

[0008] In a second aspect, the present invention provides a primer probe for rapid identification of Lactobacillus casei, wherein the primer probe is designed according to the nucleotide sequence shown in SEQ ID NO.1 or the nucleotide sequence shown in SEQ ID NO.2.

[0009] Preferably, the primers are capable of amplifying all or part of the sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0010] Preferably, the primer probe comprises any one or two of the following primer pairs;

[0011] 1142-aF: 5'-CTAAGGTAGCTGATCGGTGGCACGATC-3',

[0012] 1142-aR: 5'-CACTCTTGCCAATGGTCATCGCTG-3';

[0013] 1142-2-F: 5'-CAATCCATCAGTCAGAATGTGGAAGC-3',

[0014] 1142-2-R: 5'-CGAATGCACATGAGGATATCATTTCAGC-3';

[0015] The primer pair 1142-aF and 1142-aR is designed based on the nucleotide sequence shown in SEQ ID NO.1; the primer pair 1142-2-F and 1142-2-R is designed based on the nucleotide sequence shown in SEQ ID NO.2.

[0016] The third aspect of the present invention provides a kit for rapid identification of Lactobacillus casei, the kit comprising one or two of the following primer probes:

[0017] Design a primer probe according to the nucleotide sequence shown in SEQ ID NO.1;

[0018] The primer probe was designed according to the nucleotide sequence shown in SEQ ID NO.2.

[0019] Preferably, the kit includes any one or two of the following primers and probes:

[0020] 1142-aF: 5'-CTAAGGTAGCTGATCGGTGGCACGATC-3',

[0021] 1142-aR: 5'-CACTCTTGCCAATGGTCATCGCTG-3';

[0022] 1142-2-F: 5'-CAATCCATCAGTCAGAATGTGGAAGC-3',

[0023] 1142-2-R: 5'-CGAATGCACATGAGGATATCATTTCAGC-3';

[0024] The primer pair 1142-aF and 1142-aR is designed based on the nucleotide sequence shown in SEQ ID NO.1; the primer pair 1142-2-F and 1142-2-R is designed based on the nucleotide sequence shown in SEQ ID NO.2.

[0025] Preferably, the kit further comprises a genome extraction reagent and a PCR amplification reagent;

[0026] Further preferably, the genome extraction reagent comprises alkaline lysis solution I, lysozyme, RNase A enzyme, lysis solution II, lysis solution III, phenol, chloroform, isoamyl alcohol and anhydrous ethanol; the alkaline lysis solution I is composed of 50 mmol L -1 Glucose, 25mmolL-1pH 8.0Tris-Cl, 10mmol L-1pH 8.0EDTA; Lysis buffer II consists of 0.2mol L -1 NaOH solution, 1% SDS; Lysis solution III consists of 2 mol L -1 Potassium acetate and 2 mol L -1 Composition of glacial acetic acid.

[0027] The fourth aspect of the present invention provides a method for rapid identification of Lactobacillus casei, the method comprising the following steps: extracting genomic DNA of a sample to be tested; using the extracted genomic DNA as a template, and performing PCR amplification using a primer pair designed according to the nucleotide sequence shown in SEQ ID NO.1 and / or a primer pair designed according to the nucleotide sequence shown in SEQ ID NO.2; performing agarose gel electrophoresis on the amplified product to obtain;

[0028] Preferably, the primer pair comprises any one or two of the following primer pairs;

[0029] 1142-aF: 5'-CTAAGGTAGCTGATCGGTGGCACGATC-3',

[0030] 1142-aR: 5'-CACTCTTGCCAATGGTCATCGCTG-3';

[0031] 1142-2-F: 5'-CAATCCATCAGTCAGAATGTGGAAGC-3',

[0032] 1142-2-R: 5'-CGAATGCACATGAGGATATCATTTCAGC-3';

[0033] The primer pair 1142-aF and 1142-aR is designed based on the nucleotide sequence shown in SEQ ID NO.1; the primer pair 1142-2-F and 1142-2-R is designed based on the nucleotide sequence shown in SEQ ID NO.2.

[0034] Preferably, the method for extracting genomic DNA from the sample to be tested is as follows: collect bacteria from the sample to be tested, add 100 μL of alkaline lysis solution I, lysozyme and RNase A enzyme to the collected bacteria in sequence, and shake well; add alkaline lysis solution II, mix well, and place in an ice bath; add alkaline lysis solution III after ice bath, mix well, and place in an ice bath, centrifuge to obtain the supernatant, add phenol, chloroform and isoamyl alcohol, mix well, centrifuge to obtain the supernatant, add anhydrous ethanol, and mix well; let stand, centrifuge, and dry; add ethanol solution, mix well, and centrifuge to dry the precipitate; resuspend with 1×TE solution containing RNase A enzyme to obtain;

[0035] More preferably, the alkaline lysis solution I is composed of 50 mmol L -1 Glucose, 25 mmol L-1 pH 8.0 Tris-Cl, 10 mmol L-1 pH 8.0 EDTA; Lysis buffer II consisted of 0.2 mol L -1 NaOH solution, 1% SDS; Lysis solution III consists of 2 mol L -1 Potassium acetate and 2 mol L -1 Composition of glacial acetic acid.

[0036] Preferably, the PCR reaction system is medium, and the genome concentration is 210 μg·L -1 , primer concentration was 21 μmol·L -1 Mg 2+Concentration: 1.50mmol·L -1 ;

[0037] The PCR reaction conditions were as follows: pre-denaturation: 92-99°C, 8-15 min; denaturation: 93-100°C, 1-4 min; annealing: 40-45°C, 30s-1 min; extension: 68-78°C, 2-4 min; 30-35 cycles; final extension: 68-78°C, 10 min; storage: 12-17°C; the obtained PCR products were verified by 0.8% agarose gel electrophoresis.

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] The present invention screens two new gene fragments that can specifically identify Lactobacillus casei based on ERIC-PCR technology, as shown in SEQ ID NO. 1 and SEQ ID NO. 2. By designing primers to amplify all or part of the two gene fragments, Lactobacillus casei can be quickly and effectively identified.

[0040] The primer probe and the kit product of the present invention can quickly detect whether a product contains Lactobacillus casei, and the detection result has high accuracy and strong stability. The application of the primer probe and the kit product of the present invention in production, life, medicine and health care and other aspects has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 :Agarose gel electrophoresis diagram of primer and probe design test. The primer and probe design test was carried out using the genomic DNA of Lactobacillus casei HP-B1142 as a template, and 0.8% agarose gel electrophoresis was used for verification.

[0042] M: 2K plusⅡDNA marker; 1 in Figure A: 1142-2-F / 1142-2-R as primers, Lactobacillus casei HP-B1142 genomic DNA as template for verification. 1 in Figure B: 1142-aF / 1142-aR as primers, Lactobacillus casei HP-B1142 genomic DNA as template for verification.

[0043] Figure 2 :Agarose gel electrophoresis diagram of primer probe specificity test. Primer probe specificity test was performed using genomic DNA of Lactobacillus casei HP-B1142 and genomic DNA of five strains as templates.

[0044] M: 2K plusⅡDNA marker; 1, 3, 5, 7, 9, 11 in the figure: all use 1142-aF / 1142-aR as primers, and the templates used are respectively Lactobacillus casei HP-B1142, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, and Lactobacillus helveticus genomic DNA. 2, 4, 6, 8, 10, 12 in the figure: all use 1142-2-F / 1142-2-R as primers, and the templates used are respectively Lactobacillus casei HP-B1142, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, and Lactobacillus helveticus genomic DNA.

[0045] Figure 3 : Agarose gel electrophoresis of the primer probe to detect the concentration limit of Lactobacillus casei. M: 2K PlusⅡDNA marker, lanes 1-2 are Lactobacillus casei content 100% (absolute content 900ng·mL -1 ) verification, lanes 3-4 are Lactobacillus casei content 70% (absolute content 630ng·mL -1 ), Lanes 5-6 represent Lactobacillus casei content of 30% (absolute content 270 ng·mL -1 ), Lanes 7-8 represent Lactobacillus casei content 0% (absolute content 0 ng·mL -1 The primer probes used in lanes 1, 3, 5, and 7 are 1142-aF / 1142-aR, and the primer probes used in lanes 2, 4, 6, and 8 are 1142-2-F / 1142-2-R.

[0046] Figure 4 :Agarose gel electrophoresis diagram for testing the universality of primer probes. M: 2K PlusⅡDNA marker, lanes 1-2, 3-4, 5-6, 7-8, 9-10, and 11-12 use genomic DNA of Lactobacillus casei HP-B1142, Lactobacillus casei DSM20011, Lactobacillus case JCM 8129, Lactobacillus case IDCC 3451, Lactobacillus case JX 20225, and Lactobacillus case ATCC 11443 as templates, respectively. The primer probes used in lanes 1, 3, 5, 7, 9, and 11 are 1142-aF / 1142-aR, and the primer probes used in lanes 2, 4, 6, 8, 10, and 12 are 1142-2-F / 1142-2-R.

[0047] Figure 5 :Agarose gel electrophoresis diagram of the verification of using two primer probes to detect Lactobacillus casei in three commercially available probiotic products. 1-2, 3-4, 5-6 use the genomic DNA of commercially available mixed probiotic product 1, commercially available mixed probiotic product 2 and commercially available probiotic product 3 as templates respectively; the primer probes used in lanes 1, 3, and 5 are 1142-aF / 1142-aR, and the primer probes used in lanes 2, 4, and 6 are 1142-2-F / 1142-2-R.

[0048] Figure 6 : The genomic DNA of Lactobacillus casei HP-B1142 was extracted using CHELEX method.

[0049] M: 2K plusⅡDNA marker; 1 is the PCR result of genomic DNA extracted using the CHELEX method. DETAILED DESCRIPTION

[0050] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0051] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and "including" are used in this specification, they indicate the presence of features, steps, operations and combinations thereof.

[0052] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0053] Example 1

[0054] In the early stage of the study on Lactobacillus casei, two gene fragments 1142-a and 1142-2 were screened: 1142-a is 2672bp in size, and the base sequence is shown in SEQ ID NO.1; 1142-2 is 528bp in size, and the base sequence is shown in SEQ ID NO.2. The sequences of the two gene fragments 1142-a and 1142-2 have been uploaded to the GenBank database, and the received sequence numbers are OP700792 and OP700793, respectively. The two gene fragments can specifically identify Lactobacillus casei.

[0055] Design specific primer probes for the specific gene fragments shown in SEQ ID NO.1 and / or SEQ ID NO.2:

[0056] Specific primers were designed according to the primer design principles using Primer Premier 5 and Oligo 7 software.

[0057] Example 2

[0058] Primer probe for rapid identification of Lactobacillus casei:

[0059] 1142-aF: (5'-CTAAGGTAGCTGATCGGTGGCACGATC-3'),

[0060] 1142-aR: (5'-CACTCTTGCCAATGGTCATCGCTG-3');

[0061] The 1142-aF and 1142-aR primer pairs were designed based on the nucleotide sequence shown in SEQ ID NO. 1. The amplified fragment size was 1815 bp, as shown in the sequence table SEQ ID NO. 3.

[0062] Primer probe for rapid identification of Lactobacillus casei:

[0063] 1142-2-F: (5'-CAATCCATCAGTCAGAATGTGGAAGC-3'),

[0064] 1142-2-R: (5'-CGAATGCACATGAGGATATCATTTCAGC-3').

[0065] The primer pair 1142-2-F and 1142-2-R is designed according to the nucleotide sequence shown in SEQ ID NO. 2. The amplified fragment size is 428 bp, see the sequence table SEQ ID NO. 4 for details.

[0066] Lactobacillus casei HP-B1142 DNA was used as template, and primer pair 1142-aF / 1142-aR and primer pair 1142-2-F / 1142-2-R were used for PCR reaction. The template concentration was 210 μg·L -1 , primer concentration was 21 μmol·L -1 Mg 2+ Concentration: 1.50mmol·L -1 .

[0067] The PCR reaction system involved was pre-denaturation (92-99°C, 8-15min); denaturation (93-100°C, 1-4min); annealing (40-45°C, 30s-1min); extension (68-78°C, 2-4min, 30-35 cycles); final extension (68-78°C, 10min); storage (12-17°C). The obtained PCR product was verified by 0.8% agarose gel electrophoresis. The results were as follows: Figure 1 shown.

[0068] The primers were synthesized by Genewise, and the designed primers were used for further experiments. According to SnapGene analysis, the designed primers can amplify the fragment lengths of Lactobacillus casei to 1815 bp and 428 bp respectively.

[0069] Primer probe specificity test

[0070] Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, and Lactobacillus helveticus were used as controls for primer-probe specificity testing. PCR was performed on the above strains and Lactobacillus casei HP-B1142 using primer pairs 1142-aF / 1142-aR and 1142-2-F / 1142-2-R, respectively. The template concentration was 210 μg·L -1 , primer concentration was 21 μmol·L -1 Mg 2+ Concentration: 1.50mmol·L -1 The PCR reaction system involved was pre-denaturation (92-99°C, 8-15min); denaturation (93-100°C, 30s-4min); annealing (40-55°C, 30s-1min); extension (68-78°C, 30s-5min, 30-35 cycles); final extension (68-78°C, 10min); storage (12-17°C). The obtained PCR product was verified by 0.8% agarose gel electrophoresis. The results are as follows: Figure 2 shown.

[0071] Primer probe can detect the content limit test of genomic DNA

[0072] Lactobacillus casei HP-B1142 was mixed with Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus helveticus and Lactobacillus casei HP-B1142 to prepare the mixture. The content of Lactobacillus casei HP-B1142 was 100% (absolute content 900 ng·mL -1)、70%(absolute content 975ng·mL -1 )、30%(absolute content 270ng·mL -1 )、0%(absolute content 0ng·mL -1 ) was prepared, and the mixed DNA solution was used as a template. The primer pairs 1142-aF / 1142-aR and 1142-2-F / 1142-2-R were used to perform PCR on the above-mentioned strains and Lactobacillus casei HP-B1142, respectively. The PCR reaction system involved was pre-denaturation (92-99°C, 8-15min); denaturation (93-100°C, 1-4min); annealing (40-50°C, 30s-1min); extension (68-78°C, 3-5min); cycle (30-35 times); final extension (68-78°C, 10min); storage (12-17°C). The obtained PCR product was verified by 0.8% agarose gel electrophoresis, and the results were as follows: Figure 3 shown.

[0073] Primer probe universality test

[0074] Five strains of Lactobacillus casei, Lactobacillus casei DSM 20011, Lactobacillus case JCM 8129, Lactobacillus case IDCC 3451, Lactobacillus case JX 20225, and Lactobacillus case ATCC 11443, were used as controls for the universality test of the primer probe. PCR was performed on the above strains and Lactobacillus casei HP-B1142 using primer pairs 1142-aF / 1142-aR and 1142-2-F / 1142-2-R, respectively. The PCR reaction system involved was pre-denaturation (92-99°C, 8-15min); denaturation (93-100°C, 1-4min); annealing (40-50°C, 30s-1min); extension (68-78°C, 3-5min); cycle (30-35 times); final extension (68-78°C, 10min); storage (12-17°C). The obtained PCR product was verified by 0.8% agarose gel electrophoresis. The results were as follows: Figure 4 shown.

[0075] Using the two pairs of specific primers to test products containing Lactobacillus casei in commercially available public formulas

[0076] Three commercially available probiotic products containing Lactobacillus casei in their public formulas were selected, and the genomic DNA of the products was extracted as templates. The primer pairs 1142-aF / 1142-aR and 1142-2-F / 1142-2-R were used to perform PCR on the products. The PCR reaction system involved was pre-denaturation (92-99°C, 8-15min); denaturation (93-100°C, 1-4min); annealing (40-45°C, 30s-1min); extension (68-78°C, 2-4min); cycle (30-35); final extension (68-78°C, 10min); storage (12-17°C). The obtained PCR products were verified by 0.8% agarose gel electrophoresis, and the results are as follows: Figure 5 shown.

[0077] Example 3

[0078] A kit for rapid identification of Lactobacillus casei, the kit comprising the following primer probes:

[0079] Using the nucleotide sequence shown in SEQ ID NO.1 as a template, using Primer Premier 5 and Oligo 7 software, according to the primer design principle, specific primers were designed; the primers can amplify the entire nucleotide sequence shown in SEQ ID NO.1.

[0080] The kit also includes a genome extraction reagent and a PCR amplification reagent; the genome extraction reagent includes alkaline lysis solution I, lysozyme, RNase A enzyme, lysis solution II, lysis solution III, phenol, chloroform, isoamyl alcohol and anhydrous ethanol; the alkaline lysis solution I is composed of 50 mmol L -1 Glucose, 25 mmol L-1 pH 8.0 Tris-Cl, 10 mmol L-1 pH 8.0 EDTA; Lysis buffer II consisted of 0.2 mol L -1 NaOH solution, 1% SDS; Lysis solution III consists of 2 mol L -1 Potassium acetate and 2 mol L -1 Composition of glacial acetic acid.

[0081] The reagents also include 2×Taq Master Mix (Dye Plus), Mg 2+ solution and water.

[0082] Example 4

[0083] A kit for rapid identification of Lactobacillus casei, the kit comprising the following primer probes:

[0084] Using the nucleotide sequence shown in SEQ ID NO.2 as a template, using Primer Premier 5 and Oligo 7 software, according to the primer design principle, specific primers were designed; the primers can amplify the entire nucleotide sequence shown in SEQ ID NO.2.

[0085] The rest are the same as in Example 3.

[0086] Example 5

[0087] A kit for rapid identification of Lactobacillus casei, the kit comprising the following primer probes:

[0088] 1142-aF: 5'-CTAAGGTAGCTGATCGGTGGCACGATC-3',

[0089] 1142-aR: 5'-CACTCTTGCCAATGGTCATCGCTG-3'.

[0090] The rest are the same as in Example 3.

[0091] Example 6

[0092] A kit for rapid identification of Lactobacillus casei, the kit comprising the following primer probes:

[0093] 1142-2-F: 5'-CAATCCATCAGTCAGAATGTGGAAGC-3',

[0094] 1142-2-R: 5'-CGAATGCACATGAGGATATCATTTCAGC-3'.

[0095] The rest are the same as in Example 3.

[0096] Example 7

[0097] A kit for rapid identification of Lactobacillus casei, the kit comprising the following primer probes:

[0098] 1142-aF: 5'-CTAAGGTAGCTGATCGGTGGCACGATC-3',

[0099] 1142-aR: 5'-CACTCTTGCCAATGGTCATCGCTG-3';

[0100] 1142-2-F: 5'-CAATCCATCAGTCAGAATGTGGAAGC-3',

[0101] 1142-2-R: 5'-CGAATGCACATGAGGATATCATTTCAGC-3'.

[0102] The rest are the same as in Example 3.

[0103] Example 8

[0104] A method for rapidly identifying Lactobacillus casei comprises the following steps: extracting genomic DNA of a sample to be tested; using the extracted genomic DNA as a template, and performing PCR amplification using a primer probe designed according to a nucleotide sequence shown in SEQ ID NO.1 and / or a primer probe designed according to a nucleotide sequence shown in SEQ ID NO.2; and performing agarose gel electrophoresis on the amplified product to obtain the product.

[0105] The method for extracting genomic DNA of the sample to be tested is as follows: collect bacteria in the sample to be tested, add 100 μL of alkaline lysis solution I, lysozyme and RNase A enzyme to the collected bacteria in sequence, shake well; add alkaline lysis solution II, mix well, and place in an ice bath; add alkaline lysis solution III after ice bath, mix well and place in an ice bath, centrifuge to obtain supernatant, add phenol, chloroform and isoamyl alcohol, mix well, centrifuge to obtain supernatant, add anhydrous ethanol, mix well; let stand, centrifuge, and dry; add ethanol solution, mix well and centrifuge, dry the precipitate; resuspend with 1×TE solution containing RNase A enzyme to obtain;

[0106] The alkaline lysis solution I is composed of 50 mmol L -1 Glucose, 25mmol L-1pH 8.0Tris-Cl, 10mmol L-1pH8.0EDTA; Lysis buffer II consists of 0.2mol L -1 NaOH solution, 1% SDS; Lysis solution III consists of 2 molL -1 Potassium acetate and 2 mol L -1 Composition of glacial acetic acid.

[0107] The PCR reaction system was medium, and the genome concentration was 210 μg·L -1 , primer concentration was 21 μmol·L -1 Mg 2+ Concentration: 1.50mmol·L -1 ;

[0108] The PCR reaction conditions were as follows: pre-denaturation: 92-99°C, 8-15 min; denaturation: 93-100°C, 1-4 min; annealing: 40-45°C, 30s-1 min; extension: 68-78°C, 2-4 min; 30-35 cycles; final extension: 68-78°C, 10 min; storage: 12-17°C; the obtained PCR products were verified by 0.8% agarose gel electrophoresis.

[0109] Comparative Example 1

[0110] The difference from Example 8 is that the CHELEX method is used to extract genomic DNA.

[0111] Figure 6 This is a PCR experiment to extract the genomic DNA of Lactobacillus casei HP-B1142 using the CHELEX method. Figure 6 It was found that the genomic DNA of Lactobacillus casei was not successfully extracted using the CHELEX method, which may be due to the low purity of the DNA template obtained by the CHELEX method.

[0112] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of a reagent for detecting the nucleotide sequence shown in SEQ ID NO.1 in identifying Lactobacillus casei.

2. A method for rapid identification of Lactobacillus casei, characterized in that: The method comprises the following steps: extracting genomic DNA of a sample to be tested; using the extracted genomic DNA as a template and using a primer pair designed according to the nucleotide sequence shown in SEQ ID NO.1 to perform PCR amplification; and performing agarose gel electrophoresis on the amplified product to obtain the product; The primer pairs are: 1142-aF: 5'-CTAAGGTAGCTGATCGGTGGCACGATC-3', 1142-aR: 5'-CACTCTTGCCAATGGTCATCGCTG-3'.

3. The method according to claim 2, characterized in that: The method for extracting genomic DNA of the sample to be tested is as follows: collect the bacteria in the sample to be tested, add 100 μL of alkaline lysis solution I, lysozyme and RNase A enzyme to the collected bacteria in sequence, shake well; add alkaline lysis solution II, mix well, and place in an ice bath; add alkaline lysis solution III after ice bath, mix well and place in an ice bath, centrifuge to obtain the supernatant, add phenol, chloroform and isoamyl alcohol, mix well, centrifuge to obtain the supernatant, add anhydrous ethanol, mix well; let stand, centrifuge, and dry; Add ethanol solution, mix well, centrifuge, dry the precipitate, and resuspend it with 1×TE solution containing RNase A enzyme to obtain; The alkaline lysis solution I is composed of 50 mmol L -1 Glucose, 25 mmol L-1 pH 8.0 Tris-Cl, 10 mmol L-1 pH8.0 EDTA; Lysis buffer II consisted of 0.2 mol L -1 NaOH solution, 1% SDS; Lysis solution III consists of 2 mol L -1 Potassium acetate and 2 mol L -1 Composition of glacial acetic acid.

4. The method according to claim 2, characterized in that: In the PCR reaction system, the genome concentration was 210 μg·L -1 The primer concentration was 21 μmol·L -1 Mg 2+ The concentration is 1.50 mmol·L -1 ; PCR reaction conditions were as follows: initial denaturation: 92-99°C, 8-15 min; Denaturation: 93-100°C, 1-4 min; Annealing: 40-45°C, 30s-1min; Extension: 68-78℃, 2-4min; 30-35 cycles; Final extension: 68-78℃, 10min; Storage: 12-17°C; verify the PCR product by 0.8% agarose gel electrophoresis.

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