CN118028505A - Dual PCR primer group for simultaneously detecting two probiotics and method and kit thereof - Google Patents

Abstract

The invention discloses a double PCR primer group for simultaneously detecting two probiotics, a method and a kit thereof. The double PCR system primer group comprises: the sequence of the primer group for the Lactobacillus casei dnaK gene and the primer group for the Lactobacillus acidophilus dnaK gene is SEQ ID No. 1-4 in sequence. The kit comprises a reaction solution A (2×PCR premix), a reaction solution B (primer mixture) and a PCR enzyme. The invention utilizes the specificity of target genes, and the primer combination only carries out specific amplification on target gene fragments of respective strains, is not interfered by other strains, realizes the purpose of detecting two probiotics in the same system, improves the detection efficiency, saves the detection cost, and is suitable for large-scale screening of probiotic components in probiotic products.

Description

Dual PCR primer group for simultaneously detecting two probiotics and method and kit thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a double PCR primer group for simultaneously detecting two probiotics, a method and a kit thereof.

Background

Probiotics are a kind of single microorganism or mixed microorganism with definite composition which can colonize in a host body, regulate the immune function of a host mucous membrane and a system or promote nutrition absorption and keep intestinal health by regulating the balance of flora in intestinal tracts, thereby producing health-beneficial effects. Common probiotics mainly include Lactobacillus, streptococcus, leuconostoc, pediococcus, etc. Lactobacillus is mostly a rod-like, straight or slightly curved Bacillus-free bacterium. Microaerophilic, but better growing in anaerobic environments. The most suitable temperature for growth is 30-40 ℃, the pH value for growth is 5.5-6.2, and the method is commonly used for producing animal and plant products. Lactobacillus casei (Lactobacilluscasei) can produce a large amount of extracellular polysaccharide, lactobacillus acidophilus (Lactobacillus acidophilus) can secrete antibiotics, and the two probiotics can improve the body defense mechanism, so that the lactobacillus casei can be widely applied to the fields of foods, medicines and clinical medicine.

With the development of modern molecular biology, the molecular biology method gradually replaces the traditional morphological and physiological detection method with the advantages of higher accuracy, higher stability and the like, and plays an important role in the detection and identification of probiotics. At present, the existing probiotics detection standard mainly adopts a conventional PCR or real-time fluorescence PCR method to identify single strains. In the detection of the actual probiotic product sample with deep processing and undefined components, the probiotic product sample can be determined through multiple detection and screening and exclusion, so that the sample detection period is increased, and the detection workload is increased. The multiplex PCR technology utilizes the specificity of primer combination to realize the directional detection of multiple targets in the same system. The method can be suitable for the actual detection of the compound probiotic preparation, and has important practical significance in both conventional food spot inspection and large-scale rapid screening.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention aims to design and provide a technical scheme of a double PCR primer group for simultaneously detecting two probiotics, a method and a kit thereof.

The invention adopts the following technical scheme:

The first aspect of the present invention provides a dual PCR primer combination for simultaneous detection of two probiotics, the primer combination comprising primers for Lactobacillus casei dnaK gene and Lactobacillus acidophilus dnaK gene;

the amplified fragment aiming at the Lactobacillus casei dnaK gene is 500bp, and the primers are shown as SEQ ID No.1 and SEQ ID No. 2;

SEQ ID No.1：Lca_F 5’-CACCTTGCAGAACGTGAATGTCAAC-3’；

SEQ ID No.2：Lca_R 5’-CCAGACCCAGATCAGTCTACCG-3’；

The amplified fragment aiming at the lactobacillus acidophilus dnaK gene is 988bp, and the primers are shown as SEQ ID No.3 and SEQ ID No. 4;

SEQ ID No.3：Lac_F 5’-GAAAAGACTTTGAAGGAAACTAAGGG-3’；

SEQ ID No.4：Lac_R 5’-ACGTCCATTACACTTGTCACAAGTG-3’。

in a second aspect, the invention provides a dual PCR kit for simultaneous detection of two probiotics, the kit comprising the primer combination described above.

Further, the dual PCR kit comprises a reaction solution A, a reaction solution B and PCR enzyme; the reaction solution A is 2 XPCR premix solution and comprises dNTP 0.4mM, tris-HCl pH8.3 mM, mgCl24mM, KCl 100mM, BSA1mg/mL, glycerol 1% (v/v), PEG8000 2% (v/v) and dimethyl sulfoxide 2% (v/v); the reaction solution B is a primer mixed solution and comprises primers Lca_ F, lca _ R, lac _F and Lac_R of two probiotics, wherein the concentration of each primer is 2.5 mu M; the high-fidelity Taq enzyme is 5U/. Mu.L.

Further, the PCR kit also comprises a positive control and a negative control; the positive control is a mixture of plasmids constructed according to target gene fragments of lactobacillus casei and lactobacillus acidophilus according to the ratio of 1:1; the negative control was DEPC water.

The third aspect of the present invention provides a dual PCR detection method for simultaneously detecting two probiotics, comprising the steps of:

1) Extracting genome DNA of a sample to be detected for standby;

2) Adding genomic DNA of a sample to be detected as a template into a double PCR reaction system for amplification, wherein the double PCR reaction system comprises the PCR kit;

3) PCR was programmed and amplified. The amplified products were subjected to 1% agarose gel electrophoresis, and the presence or absence of the two probiotics was determined by size analysis of the electrophoresis band.

Further, the dual PCR reaction system in the step 2) specifically comprises 12.5 mu L of reaction solution A, 4 mu L of reaction solution B and 0.5 mu L of high-fidelity Taq enzyme, 10-100 ng of DNA is added as a template, and water is added to 25 mu L;

further, the double PCR procedure in the step 3) is a pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 15s, annealing at 60℃for 30s, extension at 72℃for 60s for 35 cycles; finally, the extension is carried out for 5min at 72 ℃.

Further, the PCR reaction system in step 2) further comprises a PCR positive control and a negative control.

The fourth aspect of the invention provides the use of the two probiotic primer sets, methods and kits described above in any of the following:

(a) Detecting at least one probiotic of lactobacillus casei and lactobacillus acidophilus;

(b) Preparing a kit or product for the detection of (a).

Compared with the prior art, the invention has the beneficial effects that:

(1) The double PCR primer combination has higher specificity, and only target fragments of respective strains are subjected to specific amplification. Meanwhile, the double PCR detection method can detect two strains of lactobacillus casei and lactobacillus acidophilus at one time, thereby improving the detection efficiency and reducing the detection cost;

(2) The kit and the use method can rapidly identify two probiotics including lactobacillus casei and lactobacillus acidophilus, optimize a reaction system and a reaction program, and are particularly suitable for large-scale screening and rapid detection of probiotic products.

(3) The kit has the advantages of reasonable components and proportion, low specific cost, simple operation, convenient use and the like.

Drawings

FIG. 1 is the establishment of a dual PCR reaction system;

FIG. 2 is a dual PCR specificity assay;

FIG. 3 is a double PCR sensitivity assay.

Detailed Description

The technical means adopted by the invention and the effects thereof are further described by the following specific embodiments, but the invention is not limited to the scope of the examples; the materials used in the following examples are not limited to the above list, and other similar materials may be used instead, the apparatus is not specified, and the conditions are conventional or recommended by the manufacturer; unless otherwise indicated, the reagents and methods employed in the present invention are conventional in the art and conventional.

Example 1: design of specific primers

On the NCBI (National Center for Biotechnology Information ) website, the coding sequences of dnaK and other genes of common probiotic strains are retrieved and downloaded, and Vector NTI software is used for comparing and analyzing the gene sequences of the species and similar species. Finally, strain-specific primers were designed using Lactobacillus casei dnaK gene (HM 122261.1) and Lactobacillus acidophilus dnaK gene (AB 059359.1) as templates, and specificity was confirmed by searching using BLAST tool. After a series of pre-screening, specific primers are shown in the following table:

Example 2: establishment of double PCR method

1) The standard strain used in this experiment was purchased from the China industry microbiological culture collection center (CICC), cultured and subjected to extraction of bacterial genomic DNA (bacterial genomic DNA extraction kit or other extraction method recognized to have the same efficacy), and preserved at 20℃for later use.

2) The total volume of the double PCR reaction system is 25 mu L, and the configuration method is as follows: 12.5. Mu.L of reaction solution A, 4. Mu.L of reaction solution B, 0.5. Mu.L of high-fidelity Taq enzyme, and 1. Mu.L of DNA as a template were added and water was added to 25. Mu.L. The PCR procedure was 95℃for 5min of pre-denaturation; denaturation at 95 ℃ for 15s, annealing at 52 ℃ to 62 ℃ for 30s and extension at 72 ℃ for 60s, for 35 cycles; finally, the extension is carried out for 5min at 72 ℃.

3) The dual PCR products were analyzed by 1% agarose gel electrophoresis and the results are shown in FIG. 1. The annealing temperature is respectively 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃, 62 ℃, the amplification effect is good in the annealing temperature range of 56-62 ℃, the band specificity is good, and the band size meets the expectations. The corresponding bands were excised and recovered for sequencing, and the sequence results were consistent with the sequence of the fragment of interest, thus indicating that the amplified fragment was indeed the fragment of interest of the target gene.

Example 3: dual PCR reaction System specificity test

In order to verify the specificity of the double PCR detection system constructed by the invention, bacterial genome DNA of lactobacillus casei, lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus paracasei, bifidobacterium paraadolescentis and escherichia coli is extracted and used as a double PCR reaction template, so that the specificity of the constructed double PCR detection system is verified. The reaction system was configured and subjected to double PCR amplification according to the kit method established in example 2, wherein the annealing temperature was set to 60 ℃.

The results of the dual PCR reaction system specificity test are shown in FIG. 2. (M) DL1000 DNAMARKER; (B) a blank; (1-8) the mPCR template is Lactobacillus acidophilus, lactobacillus casei, lactobacillus acidophilus and Lactobacillus casei, lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus paracasei, bifidobacterium paraadolescentis and Escherichia coli, respectively. The result shows that the double PCR detection system has better specificity.

Example 4: sensitivity test of double PCR reaction system

To verify the sensitivity of the dual PCR detection system constructed according to the present invention, bacterial genomic DNA was subjected to 10-fold gradient dilution such that the DNA concentration gradients were 101, 100, 10-1, 10-2, 10-3, 10-4, 10-5 ng/. Mu.L and dual mPCR amplification was performed according to the kit and method established in example 2, with the annealing temperature set at 60 ℃.

The results of the sensitivity test of the dual PCR reaction system are shown in FIG. 3. (M) DL1000 DNAMARKER; (B) blank control. The results show that the detection sensitivity of both probiotics reaches 10 < -3 > ng/. Mu.L.

The basic principle and characteristics of the invention are shown and described in the above examples, and the results show that the kit has the characteristics of higher accuracy and better sensitivity, and is suitable for simultaneous detection of lactobacillus casei and lactobacillus acidophilus in probiotic products.

Claims (9)

1. A dual PCR primer combination for simultaneously detecting two probiotics, characterized in that: the primer combination comprises primers for lactobacillus casei dnaK gene and lactobacillus acidophilus dnaK gene;

primers for the Lactobacillus casei dnaK gene include Lca_F shown in SEQ ID No.1 and Lca_R shown in SEQ ID No. 2;

primers for the Lactobacillus acidophilus dnaK gene include Lac_F shown in SEQ ID No.3 and Lac_R shown in SEQ ID No. 4.

2. A dual PCR kit for simultaneously detecting two probiotics is characterized in that: the kit comprises the primer combination of claim 1.

3. The dual PCR kit of claim 2, wherein: comprises a reaction solution A, a reaction solution B and PCR enzyme; the reaction solution A is 2 XPCR premix, which comprises dNTP 0.4mM, tris-HCl pH8.3 mM, mgCl ₂ mM, KCl 100 mM, BSA 1mg/mL, glycerol 1% (v/v), PEG8000 2% (v/v) and dimethyl sulfoxide 2% (v/v); the reaction solution B is a primer mixed solution and comprises primers Lca_ F, lca _ R, lac _F and Lac_R of two probiotics, wherein the concentration of each primer is 2.5 mu M; the PCR enzyme was high-fidelity hot start Taq enzyme (5U/. Mu.L).

4. The dual PCR kit of claim 2, wherein: including positive and negative controls; the positive control is a mixture of plasmids constructed according to target gene fragments of lactobacillus casei and lactobacillus acidophilus according to the ratio of 1:1; the negative control was DEPC water.

5. A dual PCR method for simultaneously detecting two probiotics is characterized in that: the method comprises the following steps:

1) Extracting genome DNA of a sample to be detected for standby;

2) And adding the genomic DNA of the sample to be detected as a template into a double PCR reaction system for amplification. A dual PCR reaction system comprising the dual PCR kit of any one of claims 2-4;

3) PCR procedure was set and amplification was performed, and the amplified product was subjected to 1% agarose gel electrophoresis, and the presence or absence of the above two probiotics was determined by size analysis of the electrophoresis band.

6. The method of claim 5, wherein: the double PCR reaction system in the step 2) specifically comprises 12.5 mu L of reaction liquid A, 4 mu L of reaction liquid B, 0.5 mu L of high-fidelity Taq enzyme, and 10-100 ng of DNA (deoxyribonucleic acid) serving as a template, and water is added to 25 mu L.

7. The method of claim 5, wherein: the PCR procedure in the step 3) is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 15s, annealing at 60℃for 30s, extension at 72℃for 60s for 35 cycles; finally, the extension is carried out for 5min at 72 ℃.

8. The method according to any one of claims 5-7, wherein: PCR positive and negative controls were included.

9. The use of a primer combination according to any one of claims 1 and a kit according to any one of claims 2-4 in any one of the following:

(a) Detecting at least one probiotic of lactobacillus casei and lactobacillus acidophilus;

(b) Preparing a kit or product for the detection of (a).