CN116121422A - Primer probe, kit and method for identifying lactobacillus paracasei - Google Patents
Primer probe, kit and method for identifying lactobacillus paracasei Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention relates to the technical field of biology, in particular to a primer probe, a kit and a method for rapidly identifying lactobacillus paracasei. The primer probe is designed according to a nucleotide sequence shown in SEQ ID NO. 1. The primer probe and the kit product can be used for rapidly detecting whether lactobacillus paracasei (Lactobacillus paracasei) is contained in the product, and the detection result has high accuracy and strong stability. The primer probe has strong specificity, high sensitivity and high stability.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer probe, a kit and a method for identifying lactobacillus paracasei.
Background
The development and utilization of microbial resources are the hot spot of current research because microorganisms have the advantages of short growth cycle, easy strain transformation and the like. Lactic acid bacteria are microorganisms which utilize fermentable sugar and produce a large amount of lactic acid, are various, are widely distributed in nature, are safe and harmless to people and livestock, have health care and medical effects, have the effects of improving the flavor of food, improving the nutritive value of the food, resisting bacteria, preventing corrosion and the like, and are favored by researchers at home and abroad. In recent years, the application specific gravity of lactobacillus in the fields of food, light industry, medicine, feed and the like is greatly increased, and novel lactic acid fermentation food and beverage, lactobacillus preservative, lactobacillus preparation, lactobacillus probiotics, lactobacillus feed additive and the like are layered endlessly, so that the life quality of people is improved, the physical health of people is promoted, and meanwhile, great economic benefits are created, and according to statistics, the economic value created by the food industry taking lactobacillus as the fermentation bacteria only accounts for 20% of the global fermentation food. It follows that lactic acid bacteria have an irreplaceable priority as the most important industrial microorganism.
Lactobacillus paracasei belongs to the genus Lactobacillus, and is widely used in fermented foods such as cheese and kimchi, and in the oral cavity and intestinal tract of human body, and few reports indicate that the strain can be isolated from some alcoholic beverages such as low alcohol sake and brandy. In recent years, the qualitative, screening, metabolism, physiology and other aspects of lactobacillus paracasei are widely studied, and a great amount of data show that the lactobacillus paracasei is suitable for various applications, and has wide market prospect in the aspects of production, life, medical health care and the like. However, a rapid, accurate and effective identification method for lactobacillus paracasei is still lacking at present.
Disclosure of Invention
The invention mainly aims at providing a primer probe, a kit and a method for identifying lactobacillus paracasei. The invention screens and obtains a gene fragment which can specifically identify the lactobacillus paracasei, and the gene fragment is shown as SEQ ID NO. 1. The primer probe and the kit are adopted to identify the lactobacillus paracasei, so that the specificity is high and the stability is high.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the first aspect of the invention provides the use of the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO.2 for identifying Lactobacillus paracasei (Lactobacillus paracasei). The nucleotide sequence shown in SEQ ID NO.2 is a part of the sequence shown in SEQ ID NO. 1.
In a second aspect, the invention provides a primer probe for identifying Lactobacillus paracasei (Lactobacillus paracasei), said primer probe being designed according to the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO. 2.
Further, the primer probe comprises the following primer pairs:
1145-S-F：5’-GCAGGATGCTGATTGTTAGCAG-3’；
1145-S-R：5’-CTCCGACCAAAGCGACTATGAC-3。
in a third aspect the invention provides a kit for identifying lactobacillus paracasei (lactobacillus paracasei), said kit comprising the following primer probes:
the primer probe can amplify all or part of the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
In a fourth aspect the invention provides a method of identifying lactobacillus paracasei (lactobacillus paracasei), the method comprising the steps of: extracting genome DNA of a sample to be detected; using the extracted genome DNA as a template, and adopting a primer probe designed according to the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 to carry out PCR amplification; performing agarose gel electrophoresis on the amplified product to obtain the amplification product; the primer probe can amplify all or part of the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
Compared with the prior art, the invention has the following advantages:
the invention screens and obtains a gene fragment shown in SEQ ID NO.1 which can specifically identify lactobacillus paracasei, the sequence of the gene fragment is uploaded to a GenBank database, and the receiving sequence number is OP681785. The primer probe is designed by taking the sequence shown in SEQ ID NO.1 as a template, so that lactobacillus paracasei (Lactobacillus paracasei) can be rapidly and accurately identified. The primer probe has strong specificity, high sensitivity and high stability.
The primer probe, the kit product and the method can be suitable for identifying lactobacillus paracasei (Lactobacillus paracasei) in relatively complex environments. The primer probe and the kit product have wide market prospect in the aspects of production, living, medical health care and the like.
Drawings
Fig. 1:1145-S, 1145-S Gene fragment 0.8% agarose gel electrophoresis pattern:
m:2K plus II DNA marker; in fig. a 1:1145-S gene fragment; fragment 1145-B-a in Panel B;
fig. 2: the genomic DNA of Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 was extracted by the CHELEX method and the alkaline lysis method, and the 16S rRNA gene amplification was performed.
M:2Kplus II DNAmarker;1 is the result of 16S rRNA gene amplification by extracting HP-B1145 genomic DNA by alkaline lysis; 2 is the result of 16S rRNA gene amplification by extraction of HP-B1145 genomic DNA by the CHELEX method.
Fig. 3: and (3) designing and detecting agarose gel electrophoresis patterns by using a primer probe. The genome DNA of Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 is used as a template for primer probe design and detection, and 0.8% agarose gel electrophoresis is used for verification.
M:2Kplus II DNAmarker; in fig. a 1: the verification is carried out by taking 1145-S-F/1145-S-R as a primer and taking Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 genomic DNA as a template. In fig. b 1: verification was performed using 1145-B-a-F/1145-B-a-R as primers and Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 genomic DNA as a template.
Fig. 4: the primer probe specificity test agarose gel electrophoresis pattern. Primer probe specificity was examined using the genomic DNA of Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 and the genomic DNA of 7 strains as templates.
M:2Kplus II DNAmarker; 1-9 in the drawings: all 1145-S-F/1145-S-R are used as primers, and the templates are genomic DNA of Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145, pediococcus acidilactici, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus pentosus, lactobacillus animalis, lactobacillus casei and Escherichia coli, respectively.
Fig. 5: the primer probe can detect the content limit agarose gel electrophoresis pattern of the genome DNA. The content of genome DNA of Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 was 100% (absolute content 900 ng.mL) -1 ) 70% (absolute 975ng mL) -1 ) 30% (absolute content 270 ng.mL) -1 ) 0% (absolute content 0 ng.mL) -1 ) The mixed DNA solution of (2) is used as a template, the limit test of the content of the detectable genome DNA of the primer probe is carried out, and the verification is carried out by adopting 0.8% agarose gel electrophoresis.
M:2Kplus II DNAmarker; 1-5 in the figure: all 1145-S-F/1145-S-R were used as primers, and the content of Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 genomic DNA was 100% respectively (absolute content 900 ng. ML) -1 ) 70% (absolute 975ng mL) -1 ) 30% (absolute content 270 ng.mL) -1 ) 0% (absolute content 0 ng.mL) -1 ) Is a mixed DNA solution of (A) and (B).
Fig. 6: and detecting agarose gel electrophoresis patterns by primer probe universality. Primer probe universality test is carried out by taking genome DNA of Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 and genome DNA of a closely related strain as templates.
M:2Kplus II DNAmarker; 1-8 in the figure: all of which are 1145-S-F/1145-S-R primers, and the templates selected are the genomic DNA of Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145, lactobacillus paracasei TMPC 46M17,Lactobacillus paracasei CD-M-1,Lactobacillus paracasei BCRC-16100,Lactobacillus paracasei AK508,Lactobacillusparacasei KPP377, respectively.
Fig. 7: agarose gel electrophoresis patterns of products containing Lactobacillus paracasei in the commercial public formulas were tested using 1145-S-F/1145-S-R primer probes. M:2K plus II DNAmarker; in the figures, 1145-S-F/1145-S-R is used as a primer probe in each of the first lanes, the first lane uses a template of Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145, and the second and third lanes are products containing Lactobacillus paracasei in commercially available public formulas.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular forms also are intended to include the plural forms unless the context clearly indicates otherwise, and furthermore, it is to be understood that when the terms "comprises", "comprising" are used in this specification, they specify the presence of stated features, steps, operations, and combinations thereof.
In order to solve the problems described in the background art, the first aspect of the present invention provides the use of the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 for identifying Lactobacillus paracasei (Lactobacillus paracasei).
In a second aspect of the present invention, there is provided a primer probe for identifying Lactobacillus paracasei (Lactobacillus paracasei), said primer probe being designed according to the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO. 2.
As a preferred embodiment of the present invention, the primer probe is capable of amplifying all or part of the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO. 2.
As a preferred embodiment of the present invention, the primer probe includes the following primer pairs:
1145-S-F：5’-GCAGGATGCTGATTGTTAGCAG-3’；
1145-S-R：5’-CTCCGACCAAAGCGACTATGAC-3。
in a third aspect the invention provides a kit for identifying lactobacillus paracasei (lactobacillus paracasei), said kit comprising the following primer probes:
the primer probe can amplify all or part of the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
As a preferred embodiment of the present invention, the kit comprises the following primer probes:
1145-S-F：5’-GCAGGATGCTGATTGTTAGCAG-3’；
1145-S-R：5’-CTCCGACCAAAGCGACTATGAC-3。
as a preferred embodiment of the present invention, the kit further comprises a genome extraction reagent and a PCR amplification reagent.
The genome extraction reagent comprises an alkaline lysate I, lysozyme, RNaseA enzyme, lysate II, lysate III, phenol, chloroform, isoamyl alcohol and absolute ethyl alcohol; the alkaline lysis solution I consists of 50mmol L -1 Glucose, 25mmol L -1 pH 8.0Tris-Cl，10mmol L -1 pH 8.0 EDTA; lysate II consists of 0.2mol L -1 NaOH solution, SDS with the mass concentration of 1%; the lysate III is composed of 2mol L -1 Potassium acetate and 2mol L -1 Glacial acetic acid.
In a fourth aspect the invention provides a method of identifying lactobacillus paracasei (lactobacillus paracasei), the method comprising the steps of: extracting genome DNA of a sample to be detected; using the extracted genome DNA as a template, and adopting a primer probe designed according to the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 to carry out PCR amplification; performing agarose gel electrophoresis on the amplified product to obtain the amplification product; the primer probe can amplify all or part of the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
As a preferred embodiment of the invention, the method for extracting genomic DNA of a sample to be tested comprises the following steps: collecting thalli in a sample to be detected, sequentially adding 100 mu L of alkaline lysate I, lysozyme and RNase A enzyme into the collected thalli, and uniformly vibrating; adding an alkali pyrolysis liquid II, uniformly mixing, and carrying out ice bath; adding an alkali lysate III after ice bath, carrying out ice bath after uniform mixing, centrifuging to obtain a supernatant, adding phenol, chloroform and isoamyl alcohol, uniformly mixing, centrifuging to obtain a supernatant, adding absolute ethyl alcohol, and uniformly mixing; standing, centrifuging and drying; adding ethanol solution, mixing, centrifuging, and drying to obtain precipitate; re-suspending with 1×TE solution containing RNase A enzyme;
as a preferred embodiment of the present invention, the alkaline lysis solution I is composed of 50mmol L -1 Glucose, 25mmol L-1pH 8.0Tris-Cl,10mmol L-1pH 8.0 EDTA; lysate II consists of 0.2mol L -1 NaOH solution, SDS with the mass concentration of 1%; the lysate III is composed of 2mol L -1 Potassium acetate and 2mol L -1 Glacial acetic acid.
As a preferred embodiment of the present invention, the PCR reaction system is such that the genomic concentration is 210. Mu.g.L -1 Primer concentration was 21. Mu. Mol.L -1 、Mg 2+ The concentration is 1.50 mmol.L -1 。
The PCR reaction conditions were: pre-denaturation: 92-99 ℃ for 8-15min; denaturation: 93-100deg.C for 1-4min; annealing: 42-50 ℃ for 30-60s; extension: 68-78deg.C for 3-5min; cycling 30-35 times; final extension: 68-78deg.C for 10min; and (3) preserving: 12-17 ℃; the obtained PCR product was confirmed by 0.8% agarose gel electrophoresis.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Example 1
Screening in the earlier study to obtain two specific bands, namely nucleotide sequences shown as SEQ ID NO.1 (designated 1145-S) and SEQ ID NO.3 (designated 1145-B-a), wherein agarose gel electrophoresis diagram is shown in FIG. 1; 1145-S size is 848bp and 1145-B-a size is 1716bp. Respectively using 1145-S and 1145-B-a as templates, and designing specific primers by using Primer Premier 5 and Oligo 7 software according to a Primer design principle. The specific primer probes designed for the two segments are respectively as follows:
(1) Designing the obtained primer probe by taking 1145-S as a template:
1145-S-F：5’-GCAGGATGCTGATTGTTAGCAG-3’；
1145-S-R：5’-CTCCGACCAAAGCGACTATGAC-3’。
(2) Designing a primer probe by taking 1145-B-a as a template:
1145-B-a-F：5’-CTCATCATCACCAATCCATTGAGCAC-3’；
1145-B-a-R：5’-GCAGGATGCTGATTGTTAGCAG-3’。
primers were synthesized by Jin Weizhi company and the experiment was continued with the designed primers. As shown by Snap Gene analysis, the designed primers can amplify the fragment length of Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 to 489bp (the nucleotide sequence of which is shown as SEQ ID NO. 2) and 789bp (the nucleotide sequence of which is shown as SEQ ID NO. 4) respectively.
A PCR reaction was performed using Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 genomic DNA as a template, 1145-S-F/1145-S-R primer probe and 1145-B-a-F/1145-B-a-R primer probe, respectively, and 2 XTaq Master Mix (Dye Plus) as Taq enzyme.
Extraction of genomic DNA by alkaline lysis:
lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 was cultured in MRS solid medium for 24h, and a portion of the cells was taken into a 1.5ml EP tube. Sequentially adding 100. Mu.L of alkaline lysate I (50 mmol/L glucose, 25mmol/L Tris-Cl pH 8.0, 10mmol/L EDTA pH 8.0), lysozyme (50 mg. Multidot.mL) -1 ) And RNase A enzyme (10 mg/L), 900rpm min -1 Shaking for 1min. 200 mu L of alkaline lysis solution II (0.2 mol/L NaOH solution, 1% SDS) is added, the alkaline lysis solution II is prepared immediately before use, and the mixture is inverted upside down for 5 times to be uniformly mixed, and the ice bath is performed for 30min. 160. Mu.L of precooled alkaline lysate III (5 mol/L) potassium acetate 60.0mL, glacial acetic acid 11.5mL, sterilized ultra-pure water 28.5mL were added and turned upside down5 times, after 5min ice bath 12000rpm min -1 Centrifuging for 5min. 460 mu L of supernatant is taken, added with equal volume of phenol/chloroform/isoamyl alcohol and evenly mixed, 12000 rpm.min -1 Centrifuging for 5min, collecting 460 μL supernatant, adding 2 times volume of frozen absolute ethanol, mixing, precipitating DNA at room temperature for 10min, and standing at 12000rpm min -1 The pellet was dried by centrifugation for 5min at 37 ℃. 1mL of 70% ethanol was added, and the mixture was stirred upside down, centrifuged, and the precipitate was dried. Re-suspended with 50. Mu.L of 1 XTE solution containing RNase A enzyme (10 mg/L) and stored at-20℃for use.
Genomic DNA was extracted by the CHELEX method: small amount of Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 thallus is selected and mixed with sterile CHELEX-100 gel, and the mixture is cracked for 15 minutes at 95 ℃, and the supernatant is taken to obtain the genome DNA of HP-B1145.
The genomic DNA extracted by the alkaline lysis method and the CHELEX method were subjected to 16S rRNA gene amplification, respectively, and the results are shown in FIG. 2, and it was found that the genomic DNA extracted by the alkaline lysis method was more advantageous when used as a PCR template.
The PCR reaction system is pre-denatured at 92-99 ℃ for 8-15min; denaturation at 93-100deg.C for 1-4min; annealing at 42-50deg.C for 30s-1min; extending at 68-78deg.C for 3-5min; cycling 30-35 times; final extension at 68-78deg.C for 10min; preserving at 12-17 ℃.
The template concentration was 210. Mu.g.L -1 Primer concentration was 21. Mu. Mol.L -1 、Mg 2+ The concentration is 1.50 mmol.L -1 . The PCR reaction described below was carried out such that the template concentration was 210. Mu.g.L unless otherwise specified -1 Primer concentrations were 21. Mu. Mol.L -1 、Mg 2 + The concentrations are 1.50 mmol.L -1 。
The obtained PCR product was subjected to 0.8% agarose gel electrophoresis and the result is shown in FIG. 3. The target band can be effectively amplified by adopting 1145-S-F/1145-S-R primer probes.
1145-S-F/1145-S-R primer probe specificity test
Pediococcus acidilactici, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus pentosus, bifidobacterium animalis, lactobacillus casei and escherichia coli are used as a reference for the specificity test of a primer, PCR is respectively carried out on the strain and lactobacillus paracasei (Lactobacillus paracasei) HP-B1145, and the PCR is carried out by adopting 1145-S-F/1145-S-R primer probes, wherein the related PCR reaction system is pre-denatured (92-99 ℃ for 8-15 min); denaturation (93-100deg.C, 1-4 min); annealing (42-50 ℃ for 30s-1 min); extending (68-78deg.C, 3-5 min); cycling (30-35); final extension (68-78deg.C, 10 min); preserving (12-17 ℃). The obtained PCR product was subjected to 0.8% agarose gel electrophoresis, and the result is shown in FIG. 4. Therefore, the 1145-S-F/1145-S-R primer probe has better specificity.
Detection of the limit of the amount of genomic DNA detectable by the 1145-S-F/1145-S-R primer probe
Mixing Pediococcus acidilactici, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus pentosus, lactobacillus animalis, lactobacillus casei, escherichia coli and Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 to obtain Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 with 100% (absolute 900ng mL) -1 ) 70% (absolute 975ng mL) -1 ) 30% (absolute content 270 ng.mL) -1 ) 0% (absolute content 0 ng.mL) -1 ) The mixed DNA solution of the mixed DNA solution is used as a template, and a 1145-S-F/1145-S-R primer probe is adopted to carry out PCR reaction by taking 2 XTaq Master Mix (Dye Plus) as Taq enzyme:
the PCR reaction system is pre-denatured at 92-99 ℃ for 8-15min; denaturation at 93-100deg.C for 1-4min; annealing at 42-50 ℃ for 30-60s; extending at 68-78deg.C for 3-5min; cycling 30-35 times; final extension at 68-78deg.C for 10min; preserving at 12-17 ℃. The obtained PCR product was subjected to 0.8% agarose gel electrophoresis, and the result is shown in FIG. 5. Therefore, the primer probe has better sensitivity.
1145-S-F/1145-S-R primer probe universality test
Performing PCR on the strain and Lactobacillus paracasei (Lactobacillus paracasei) HP-B1145 by using five strains Lactobacillusparacasei TMPC 46M17,Lactobacillusparacasei CD-M-1,Lactobacillus paracasei BCRC-16100,Lactobacillus paracasei AK508,Lactobacillusparacasei KPP3777 as primer and probe universality test control, wherein the related PCR reaction system is pre-denatured at 92-99 ℃ for 8-15min by using 1145-S-F/1145-S-R as primers; denaturation at 93-100deg.C for 1-4min; annealing at 42-50 ℃ for 30-60s; extending at 68-78deg.C for 3-5min; cycling 30-35 times; final extension at 68-78deg.C for 10min; preserving at 12-17 ℃. The obtained PCR product was subjected to 0.8% agarose gel electrophoresis, and the result is shown in FIG. 6. Therefore, the primer probe 1145-S-F/1145-S-R is used for identifying lactobacillus paracasei (Lactobacillus paracasei) and has good universality.
Identification of products containing Lactobacillus paracasei in commercial formulas using 1145-S-F/1145-S-R primer probe
3 products containing lactobacillus paracasei in a commercial public formula are selected, genomic DNA of the products is extracted as a template, and 1145-S-F/1145-S-R primer probes are adopted for PCR reaction. The PCR reaction system is pre-denatured at 92-99 ℃ for 8-15min; denaturation at 93-100deg.C for 1-4min; annealing at 42-50 ℃ for 30-60s; extending at 68-78deg.C for 3-5min; cycling 30-35 times; final extension at 68-78deg.C for 10min; preserving at 12-17 ℃. The obtained PCR product was subjected to 0.8% agarose gel electrophoresis, and the result is shown in FIG. 7.
Example 2
A kit for identifying lactobacillus paracasei (lactobacillus paracasei), said kit comprising the following primer probes:
1145-S-F：5’-GCAGGATGCTGATTGTTAGCAG-3’；
1145-S-R：5’-CTCCGACCAAAGCGACTATGAC-3’。
the kit also comprises a genome extraction reagent and a PCR amplification reagent; the genome extraction reagent comprises an alkaline lysate I, lysozyme, RNaseA enzyme, lysate II, lysate III, phenylphenol, chloroform, isoamyl alcohol and absolute ethyl alcohol; the alkaline lysis solution I consists of 50mmol L -1 Glucose, 25mmol L-1pH 8.0Tris-Cl,10mmol L-1pH 8.0 EDTA; lysate II consists of 0.2mol L -1 NaOH solution, SDS with the mass concentration of 1%; the lysate III is composed of 2mol L -1 Potassium acetate and 2mol L -1 Glacial acetic acid.
The reagent is also packagedIncludes 2 XTaq Master Mix (Dye Plus), mg 2+ Solution and water.
Example 3
A method of identifying lactobacillus paracasei (lactobacillussp paramasasei), the method comprising the steps of:
extracting genome DNA of a sample to be detected by adopting an alkaline lysis method; using the extracted genome DNA as a template, and adopting a primer probe designed according to a nucleotide sequence shown in SEQ ID NO.1 to carry out PCR amplification; and (3) carrying out agarose gel electrophoresis on the amplified product to obtain the final product.
The primer probe:
1145-S-F：5’-GCAGGATGCTGATTGTTAGCAG-3’；
1145-S-R：5’-CTCCGACCAAAGCGACTATGAC-3’。
the method for extracting the genome DNA of the sample to be detected comprises the following steps: collecting thalli in a sample to be detected, sequentially adding 100 mu L of alkaline lysate I, lysozyme and RNase A enzyme into the collected thalli, and uniformly vibrating; adding an alkali pyrolysis liquid II, uniformly mixing, and carrying out ice bath; adding an alkali lysate III after ice bath, carrying out ice bath after uniform mixing, centrifuging to obtain a supernatant, adding phenol, chloroform and isoamyl alcohol, uniformly mixing, centrifuging to obtain a supernatant, adding absolute ethyl alcohol, and uniformly mixing; standing, centrifuging and drying; adding ethanol solution, mixing, centrifuging, and drying to obtain precipitate; re-suspending with 1×TE solution containing RNase A enzyme;
the alkaline lysis solution I consists of 50mmol L -1 Glucose, 25mmol L-1pH 8.0Tris-Cl,10mmol L-1pH 8.0 EDTA; lysate II consists of 0.2mol L -1 NaOH solution, SDS with the mass concentration of 1%; the lysate III is composed of 2mol L -1 Potassium acetate and 2mol L -1 Glacial acetic acid.
The PCR reaction system is that the genome concentration is 210 mug.L -1 Primer concentration was 21. Mu. Mol.L -1 、Mg 2+ The concentration is 1.50 mmol.L -1 ；
The PCR reaction conditions were: pre-denaturation: 92-99 ℃ for 8-15min; denaturation: 93-100deg.C for 1-4min; annealing: 40-45 ℃ for 30s-1min; extension: 68-78deg.C for 2-4min; cycling 30-35 times; final extension: 68-78deg.C for 10min; and (3) preserving: 12-17 ℃; the obtained PCR product was confirmed by 0.8% agarose gel electrophoresis.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
- The application of the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.2 in identifying lactobacillus paracasei.
- 2. The primer probe for identifying the lactobacillus paracasei is characterized by being designed according to a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
- 3. The primer-probe of claim 2, wherein the primer-probe is capable of amplifying all or part of the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No. 2.
- 4. A primer probe according to claim 2 or 3, characterized in that the primer probe comprises the following primer pairs:1145-S-F：5’-GCAGGATGCTGATTGTTAGCAG-3’；1145-S-R：5’-CTCCGACCAAAGCGACTATGAC-3。
- 5. the kit for identifying lactobacillus paracasei is characterized by comprising the following primer probes:the primer probe can amplify all or part of the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
- 6. The kit of claim 5, comprising the following primer probes:1145-S-F：5’-GCAGGATGCTGATTGTTAGCAG-3’；1145-S-R：5’-CTCCGACCAAAGCGACTATGAC-3。
- 7. the kit of claim 5, further comprising a genome extraction reagent and a PCR amplification reagent;the genome extraction reagent comprises an alkaline lysate I, lysozyme, RNaseA enzyme, lysate II, lysate III, phenol, chloroform, isoamyl alcohol and absolute ethyl alcohol; the alkaline lysis solution I consists of 50mmol L -1 Glucose, 25mmol L -1 pH 8.0Tris-Cl，10mmol L -1 pH 8.0 EDTA; lysate II consists of 0.2mol L -1 NaOH solution, SDS with the mass concentration of 1%; the lysate III is composed of 2mol L -1 Potassium acetate and 2mol L -1 Glacial acetic acid.
- 8. A method for identifying lactobacillus paracasei, characterized in that it comprises the steps of: extracting genome DNA of a sample to be detected; using the extracted genome DNA as a template, and adopting a primer probe designed according to the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 to carry out PCR amplification; performing agarose gel electrophoresis on the amplified product to obtain the amplification product; the primer probe can amplify all or part of the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO. 2.
- 9. The method according to claim 8, wherein the method for extracting genomic DNA of the sample to be tested comprises the steps of: collecting thalli in a sample to be detected, sequentially adding 100 mu L of alkaline lysate I, lysozyme and RNase A enzyme into the collected thalli, and uniformly vibrating; adding an alkali pyrolysis liquid II, uniformly mixing, and carrying out ice bath; adding an alkali lysate III after ice bath, carrying out ice bath after uniform mixing, centrifuging to obtain a supernatant, adding phenol, chloroform and isoamyl alcohol, uniformly mixing, centrifuging to obtain a supernatant, adding absolute ethyl alcohol, and uniformly mixing; standing, centrifuging and drying; adding ethanol solution, mixing, centrifuging, and drying to obtain precipitate; re-suspending with 1×TE solution containing RNase A enzyme;preferably, the alkaline lysis solution I consists of 50mmol L -1 Glucose, 25mmol L-1pH 8.0Tris-Cl,10mmol L-1pH 8.0 EDTA; lysate II is prepared from0.2mol L -1 NaOH solution, SDS with the mass concentration of 1%; the lysate III is composed of 2mol L -1 Potassium acetate and 2mol L -1 Glacial acetic acid.
- 10. The method of claim 8, wherein the PCR reaction system is such that the genome concentration is 210. Mu.g.L -1 Primer concentration was 21. Mu. Mol.L -1 、Mg 2+ The concentration is 1.50 mmol.L -1 ；The PCR reaction conditions were: pre-denaturation: 92-99 ℃ for 8-15min; denaturation: 93-100deg.C for 1-4min; annealing: 42-50 ℃ for 30-60s; extension: 68-78deg.C for 3-5min; cycling 30-35 times; final extension: 68-78deg.C for 10min; and (3) preserving: 12-17 ℃; the obtained PCR product was confirmed by 0.8% agarose gel electrophoresis.
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| CN111286550A (en) * | 2019-07-18 | 2020-06-16 | 大连民族大学 | Specific primer for amplifying lactobacillus paracasei and application thereof |
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| CN117487943A (en) * | 2023-12-29 | 2024-02-02 | 汤臣倍健股份有限公司 | Application of gene fragment in identification of cheese bacillus paracasei |
| CN117487943B (en) * | 2023-12-29 | 2024-03-19 | 汤臣倍健股份有限公司 | Application of gene fragment in identification of cheese bacillus paracasei |
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