A multiplex fluorescent quantitative PCR primer probe set, method and kit for simultaneously detecting three bifidobacteria

Abstract

The application discloses a multiplex fluorescent quantitative PCR primer probe set, a method and a kit for simultaneously detecting three kinds of bifidobacteria. The multiplex fluorescent PCR system comprises three groups of primer probe combinations, i.e., a primer probe combination for a PurF gene of Bifidobacterium infantis, a primer probe combination for a PurF gene of Bifidobacterium longum and a primer probe combination for a RecA gene of Bifidobacterium breve, and the sequences are SEQ ID No. 1-9 in turn. The application combines real-time fluorescent quantitative PCR and multiplex fluorescent quantitative PCR technology, the primer probe combinations are only specific to the amplification of the target gene fragments of the respective bifidobacteria, the detection efficiency is improved, the detection cost is saved, and the application is suitable for large-scale rapid detection of bifidobacterial components in probiotic products.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a multiplex fluorescent quantitative PCR primer and probe set, method, and kit for the simultaneous detection of three types of Bifidobacteria. Background Technology

[0002] Bifidobacteria are present in both humans and animals and are important intestinal microorganisms. They produce short-chain fatty acids, vitamins, bacteriocins, and antibiotic-like substances, and play important physiological roles in maintaining microecological balance, biological antagonism, immune regulation, nutrition, and synergistic degradation of polysaccharides. Among them, *Bifidobacterium infantis*, *Bifidobacterium longum*, and *Bifidobacterium breve* are widely used in food supplements to exert "probiotic" functions in the body, such as regulating gastrointestinal function, reducing and controlling endotoxins, improving immune function, and producing enzymes that are beneficial to host metabolism.

[0003] In recent years, the probiotic industry has experienced explosive growth, with a large number of probiotic products flooding the market. These products claim to contain labeled strains and a large number of live bacteria. However, due to limitations in research and production technology, the quality of probiotic products varies greatly. To date, there is no effective standard or method for accurately and quickly detecting Bifidobacterium products. This directly affects the research and development and sales of Bifidobacterium preparations. Therefore, in order to correctly evaluate and monitor the quality of Bifidobacterium products, it is imperative to establish a reliable, rapid, and easy-to-use Bifidobacterium detection and identification technology.

[0004] Early taxonomy of Bifidobacteria was largely based on physiological phenotypes, but this approach often lacked reliability, particularly regarding the phylogenetic relationships among closely related species, which remained controversial. Accurate and reliable classification and identification are not only the primary goals of taxonomic research but also the foundation for developing Bifidobacteria test kits and facilitating scientific communication. Real-time fluorescence PCR (qPCR) technology is a rapidly developing nucleic acid detection technique widely used in recent years, offering advantages such as high sensitivity, short detection cycle, and strong specificity. Multiplex qPCR, developed based on conventional single-system qPCR, utilizes combinations of various fluorescent groups and the instrument's ability to detect fluorescence in different channels to achieve real-time quantitative detection of multiple targets within the same system. Currently, there is an urgent need to establish a qPCR system capable of simultaneously detecting multiple Bifidobacteria and develop it into a test kit. This has significant practical value and relevance for both routine food sampling and large-scale rapid screening. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to design and provide a technical solution for the simultaneous detection of three types of Bifidobacteria using multiplex fluorescent quantitative PCR primer and probe sets, methods, and kits.

[0006] The present invention specifically adopts the following technical solution:

[0007] The first aspect of the present invention provides a primer and probe set for simultaneous detection of three types of Bifidobacteria, wherein the primer and probe set comprises primer and probe sets targeting the PurF gene and RecA gene of Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium breve, respectively, and fluorescent reporter groups and fluorescent quencher groups at both ends of the probes.

[0008] Among them, the upstream primer for the PurF gene of Bifidobacterium infantis is Binfa_purF_F with the sequence shown in SEQ ID No.1, the downstream primer is Binfa_purF_R with the sequence shown in SEQ ID No.2, and the probe is Binfa_purF_P with the sequence shown in SEQ ID No.3;

[0009] The upstream primer for the PurF gene of Bifidobacterium longum is Blong_purF_F with the sequence shown in SEQ ID No. 4, the downstream primer is Blong_purF_R with the sequence shown in SEQ ID No. 5, and the probe is Blong_purF_P with the sequence shown in SEQ ID No. 6.

[0010] The upstream primer for the RecA gene of Bifidobacterium breve is Bbrev_recA_F with the sequence shown in SEQ ID No. 7, the downstream primer is Bbrev_recA_R with the sequence shown in SEQ ID No. 8, and the probe is Bbrev_recA_P with the sequence shown in SEQ ID No. 9.

[0011] A second aspect of the present invention provides a multiplex real-time PCR kit for the simultaneous detection of three types of Bifidobacteria, the kit comprising the primer and probe set described above.

[0012] Furthermore, the kit also includes reaction solution A, reaction solution B, and reaction solution C; reaction solution A is a 2×PCR Mix containing: Taq enzyme 0.2 U / μL, dNTP 0.4 mM, Tris-HCl (pH 8.3) 20 mM, MgCl2 4 mM, KCl 100 mM, and BSA 2 mg / mL; reaction solution B is a Primer Mix containing three primers for Bifidobacterium: Binfa_purF_F, Binfa_purF_R, Blong_purF_F, Blong_purF_R, Bbrev_recA_F, and Bbrev_recA_R, each with a concentration of 1.7 nM; reaction solution C is a ProbeMix containing probes Binfa_purF_P, Blong_purF_P, and Bbrev_recA_P, each with a concentration of 3.3 nM.

[0013] Furthermore, the kit also includes a negative control and a positive control; the negative control is DEPC water, and the positive control is a mixture of plasmids constructed from the PurF gene fragment of Bifidobacterium infantis, the PurF gene fragment of Bifidobacterium longum, and the RecA gene fragment of Bifidobacterium breve in a 1:1:1 ratio.

[0014] A third aspect of the present invention provides a multiplex real-time PCR detection method for simultaneously detecting three types of Bifidobacteria, comprising the following steps:

[0015] 1) Extract genomic DNA from the sample to be tested for later use;

[0016] 2) Use the genomic DNA of the sample to be tested as a template to add to the multiplex real-time PCR reaction system for PCR amplification. The PCR reaction system includes the PCR kit mentioned above.

[0017] 3) Set up the PCR program and collect the fluorescence signal during the amplification process. Analyze the fluorescence signal to determine whether the above three types of Bifidobacteria are present.

[0018] Furthermore, the PCR reaction system in step 2) specifically includes 12.5 μL of reaction solution A, 6 μL of reaction solution B, 3 μL of reaction solution C, 50 ng of DNA template, and water added to 25 μL;

[0019] Furthermore, in step 3), the PCR program is to perform a quantitative real-time PCR reaction in 40 cycles of 95℃ for 10s, 95℃ for 5s for denaturation, and 60℃ for annealing / extension. At the end of each cycle, the fluorescence signal during the PCR amplification process is collected, and the positive result is determined by the Ct value of a certain channel gene in the sample and the amplification curve.

[0020] Furthermore, the judgment criterion in step 3) is:

[0021] a) If a gene channel of the sample to be tested shows an amplification curve and the Ct value is ≤35, then the sample to be tested is determined to contain that type of Bifidobacterium.

[0022] b) If a gene channel in the sample to be tested does not have an amplification curve and the Ct value is ≥38, then the sample to be tested is determined not to contain that type of Bifidobacterium.

[0023] c) If a gene channel in the sample to be tested has an amplification curve and 35 < Ct < 38, it is judged as an uncertain sample and needs to be retested.

[0024] The fourth aspect of this invention provides for the application of the above-described primer-probe set and the above-described kit in any of the following ways:

[0025] (a) Detection of at least one Bifidobacterium among Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium breve;

[0026] (b) Prepare a kit or product for the detection of (a).

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] (1) The multiplex fluorescent quantitative PCR primer-probe combination of the present invention has high specificity, specifically amplifying only the target fragments of their respective bacterial species. Furthermore, the multiplex fluorescent quantitative PCR detection method of the present invention can simultaneously detect three Bifidobacterium species: Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium breve, significantly improving detection efficiency and reducing detection costs.

[0029] (2) The method involved in this invention can quickly identify three Bifidobacterium species: Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium breve. It optimizes the specific PCR reaction system and procedure, and the PCR process only takes 45 minutes. It is particularly suitable for large-scale screening and rapid detection of Bifidobacterium products in actual kits.

[0030] (3) The reagent kit of the present invention has reasonable components and ratios, is simple to operate and easy to use, and minimizes cross-contamination to the greatest extent. Attached Figure Description

[0031] Figure 1 (A) Positive amplification curve of Bifidobacterium infantis, blank control and negative control (Bifidobacterium longum and Bifidobacterium breve); (B) Positive amplification curve of Bifidobacterium longum, blank control and negative control (Bifidobacterium infantis and Bifidobacterium breve); (C) Positive amplification curve of Bifidobacterium breve, blank control and negative control (Bifidobacterium infantis and Bifidobacterium longum).

[0032] Figure 2Triple real-time PCR amplification curves for Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium breve, blank control, and negative control (Lactobacillus plantarum and Streptococcus thermophilus);

[0033] Figure 3 Sensitivity test of real-time PCR for (A) Bifidobacterium infantis, (B) Bifidobacterium longum, and (C) Bifidobacterium breve;

[0034] Figure 4 The standard curves for real-time PCR of (A) Bifidobacterium infantis, (B) Bifidobacterium longum, and (C) Bifidobacterium breve are shown. Detailed Implementation

[0035] To further illustrate the technical means and effects of the present invention, the technical solution of the present invention will be further explained through specific embodiments below. However, the present invention is not limited to the scope of the embodiments. The materials used in the following embodiments are not limited to those listed above and can be replaced by other similar materials. If the specific conditions of the instruments are not specified, they shall be performed according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the reagents and methods used in the present invention are conventional reagents and methods in this technical field.

[0036] Example 1: Design of Specific Primers and Probes

[0037] The PurF gene sequences of *Bifidobacterium infantis* (HQ438388.1) and *Bifidobacterium longum* (HQ438401.1) were downloaded from the Genebank database. The target gene sequences of the two *Bifidobacterium* species were compared and analyzed using the AlignX method in Vector NTI software. Regions with significant differences were selected as target sites to design specific primers and probes for *Bifidobacterium infantis* and *Bifidobacterium longum*. The specificity of the designed primers and probes across all bacterial species was confirmed using the PrimerBLAST tool on the NCBI website. Using the same method, the RecA gene sequences of *Bifidobacterium infantis* (U50269.1), *Bifidobacterium longum* (JF776111.1), and *Bifidobacterium breve* (U50268.1) were downloaded, and specific primers and probes for *Bifidobacterium breve* were designed. While meeting the primer and probe design principles, the amplified fragment was ensured to be within 200 bp. After a series of preliminary screenings, the primer and probe screening results are shown below:

[0038] (1) Specific primers and probes for Bifidobacterium infantis

[0039] SEQ ID No. 1 upstream primer Binfa_purF_F 5'-GCGTCTGGCGCAGGAGTC-3'

[0040] SEQ ID No. 2 downstream primer Binfa_purF_R 5'-CACGCACGGCGGACAGC-3'

[0041] SEQ ID No. 3 Fluorescent probe Binfa_purF_P 5'-FAM-TACGCGGAGGCCGCCGGGC-BHQ1-3'

[0042] (2) Specific primers and probes for Bifidobacterium longum

[0043] SEQ ID No. 4 upstream primer Blong_purF_F 5'-ACGTCTCGCCGCCGAATC-3'

[0044] SEQ ID No. 5 downstream primer Blong_purF_R 5'-AACCACGCTGCGCACAGCA GAG-3'

[0045] SEQ ID No. 6 Fluorescent probe Blong_purF_P 5'-HEX-ATCCGGCTATGCGGAAG CAGCAGGC-BHQ1-3'

[0046] (3) Specific primers and probes for Bifidobacterium breve

[0047] SEQ ID No. 7 upstream primer Bbrev_recA_F 5'-CTGATTCGCTTATCGTTTCTCA G-3'

[0048] SEQ ID No. 8 downstream primer Bbrev_recA_R 5'-CAGAGCCGCCACCGAATC-3'

[0049] SEQ ID No. 9 Fluorescent probe Bbrev_recA_P 5'-ROX-CGGCGAACAGGCTCTTG AAATTGCA-BHQ1-3'

[0050] The blank control was DEPC water, and the three types of Bifidobacteria served as negative controls for each other.

[0051] Example 2: Construction of a multiplex real-time PCR reaction system

[0052] 1) Extract DNA from the sample to be tested (using a bacterial genomic DNA extraction kit or other recognized extraction methods with equivalent efficacy) and store at -20°C for later use.

[0053] 2) After optimizing the conditions of the multiplex quantitative PCR reaction system, the final total reaction volume (25 μL) was as follows: Reaction solution A 12.5 μL, Reaction solution B 6 μL, Reaction solution C 3 μL, Sample DNA template 50 ng, and the remainder made up with water. The reaction conditions were: 95℃ pre-denaturation for 10 s; 95℃ denaturation for 5 s, 60℃ annealing / extension for 30 s, for a total of 40 cycles.

[0054] 3) Result interpretation: a) If a gene channel of the sample to be tested shows an amplification curve and the Ct value is ≤35, then the sample to be tested is determined to contain the Bifidobacterium species; b) If a gene channel of the sample to be tested does not show an amplification curve and the Ct value is ≥38, then the sample to be tested does not contain the Bifidobacterium species; c) If a gene channel of the sample to be tested shows an amplification curve and 35 < Ct < 38, then the sample is determined to be an uncertain sample and needs to be retested.

[0055] Example 3: Establishment of a multiplex quantitative PCR detection kit for rapid identification of Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium breve.

[0056] This multiplex quantitative PCR kit for rapid identification of Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium breve includes reaction solution A, reaction solution B, reaction solution C, a positive control, a negative control, instructions, and the kit itself. Reaction solution A is a 2×PCR Mix containing: Taq enzyme 0.2 U / μL, dNTP 0.4 mM, Tris-HCl (pH 8.3) 20 mM, MgCl2 4 mM, KCl 100 mM, and BSA 2 mg / mL. Reaction solution B contains primers for the three Bifidobacterium species: Binfa_purF_F, Binfa_purF_R, Blong_purF_F, Blong_purF_R, Bbrev_recA_F, and Bbrev_recA_R, each at a concentration of 1.7 nM. Reaction solution C is a Probe Mix containing probes Binfa_purF_P, Blong_purF_P, and Bbrev_recA_P, each at a concentration of 3.3 nM. The positive control was a mixture of plasmids constructed from the PurF gene fragments of *Bifidobacterium infantis*, *Bifidobacterium longum*, and *Bifidobacterium breve* in a 1:1:1 ratio, with a plasmid concentration of 1.0 ng / μL and corresponding Ct values ​​between 18 and 21. The negative control was DEPC water.

[0057] Example 4: Specificity Validation of Multiplex Quantitative Real-Time PCR Detection System

[0058] To verify the specificity of the multiplex quantitative PCR detection system constructed in this invention, bacterial genomic DNA was extracted from Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus plantarum, and Streptococcus thermophilus as PCR reaction templates to verify the specificity of the constructed multiplex quantitative PCR detection system.

[0059] DNA extraction, PCR reaction system, and reaction conditions were the same as in Example 2. The specificity verification results of multiplex quantitative PCR are as follows: Figure 1 As shown, (A) *Bifidobacterium infantis*, (B) *Bifidobacterium longum*, and (C) *Bifidobacterium breve* showed amplification curves only in their respective fluorescence channels; the blank control and negative control showed no amplification curves in any of the three channels. This result indicates that the multiplex quantitative PCR detection system of this invention has high specificity.

[0060] Example 5: Sensitivity Validation of Multiplex Quantitative PCR Detection System

[0061] Genomic DNA from (A) *Bifidobacterium infantis*, (B) *Bifidobacterium longum*, and (C) *Bifidobacterium breve* was collected, and its concentration was determined using a micro-volume ultraviolet spectrophotometer. The DNA was then diluted 10-fold, and a concentration of 1.0 × 10⁻⁶ was selected. 0 ~1.0×10 -6 DNA at a concentration of ng / μL was used as a PCR template and detected using the kit and method of this invention.

[0062] The results are as follows Figure 3 As shown, the minimum detectable concentration of this kit for (A) Bifidobacterium infantis, (B) Bifidobacterium longum, and (C) Bifidobacterium breve is 1.0 × 10⁻⁶. -5 ng / μL; a standard curve constructed based on the average Ct value is shown below. Figure 4 As shown, the slopes of the standard curves for the three types of Bifidobacteria were 3.11, 2.36, and 2.93, respectively, with correlation coefficients R0. 2 All are greater than 0.99.

[0063] The above embodiments show and describe the basic principles and features of the present invention. The results show that the kit of the present invention has the characteristics of good accuracy and high sensitivity, and is suitable for rapid detection of Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium breve in probiotic products.

Claims

1. A primer and probe set for simultaneous detection of three types of Bifidobacteria in multiplex real-time PCR, characterized in that: The primer-probe set includes: The upstream primer for the PurF gene of Bifidobacterium infantis is Binfa_purF_F 5'- GCGTCTGGCGCAGGAGTC-3', as shown in SEQ ID No. 1; the downstream primer is Binfa_purF_R 5'- CACGCACGGCGGACAGC-3', as shown in SEQ ID No. 2; and the fluorescent probe is Binfa_purF_P 5'-FAM- TACGCGGAGGCCGCCGGGC-BHQ1-3', as shown in SEQ ID No.

3. The upstream primer for the PurF gene of Bifidobacterium longum is Blong_purF_F 5'- ACGTCTCGCCGCCGAATC-3', as shown in SEQ ID No. 4; the downstream primer is Blong_purF_R 5'- AACCACGCTGCGCACAGCAGAG-3', as shown in SEQ ID No. 5; and the fluorescent probe is Blong_purF_P 5'-HEX- ATCCGGCTATGCGGAAGCAGCAGGC-BHQ1-3', as shown in SEQ ID No.

6. The upstream primer for the RecA gene of Bifidobacterium breve is Bbrev_recA_F 5'- CTGATTCGCTTATCGTTTCTCAG-3', as shown in SEQ ID No. 7; the downstream primer is Bbrev_recA_R 5'- CAGAGCCGCCACCGAATC-3', as shown in SEQ ID No. 8; and the fluorescent probe is Bbrev_recA_P 5'-ROX- CGGCGAACAGGCTCTTGAAATTGCA-BHQ1-3', as shown in SEQ ID No.

9.

2. A multiplex real-time PCR kit for simultaneous detection of three types of Bifidobacteria, characterized in that: The kit includes the primer and probe set as described in claim 1.

3. The multiplex real-time PCR kit as described in claim 2, characterized in that: The reaction mixture includes reaction solution A, reaction solution B, and reaction solution C. Reaction solution A is a 2×PCR Mix, containing 0.2 U / μL Taq enzyme, 0.4 mM dNTP, 20 mM Tris-HCl at pH 8.3, 4 mM MgCl2, 100 mM KCl, and 2 mg / mL BSA. Reaction solution B is a Primer Mix, containing three primers for Bifidobacterium: Binfa_purF_F, Binfa_purF_R, Blong_purF__F, Blong_purF__R, Bbrev_recA__F, and Bbrev_recA_R, each with a concentration of 1.7 nM. Reaction solution C is a Probe Mix, containing probes Binfa_purF_P, Blong_purF_P, and Bbrev_recA_P, each with a concentration of 3.3 nM.

4. The PCR kit according to claim 3, characterized in that... It also includes a negative control and a positive control; the negative control is DEPC water, and the positive control is a mixture of plasmids constructed based on the target genes of Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium breve in a 1:1:1 ratio.

5. A multiplex real-time PCR detection method for simultaneously detecting three types of Bifidobacteria, said method being used for non-diagnostic purposes, characterized in that... Includes the following steps: 1) Extract genomic DNA from the sample to be tested for later use; 2) The genomic DNA of the sample to be tested is added as a template to the multiplex real-time PCR reaction system for PCR amplification. The PCR reaction system includes the PCR kit as described in any one of claims 2-4. 3) Set up the PCR program and collect the fluorescence signal during the amplification process. Analyze the fluorescence signal to determine whether the above three types of Bifidobacteria are present.

6. The method according to claim 5, characterized in that: The PCR reaction system in step 2) specifically includes 12.5 μL of reaction solution A, 6 μL of reaction solution B, 3 μL of reaction solution C, 50 ng of DNA as a template, and water added to a final volume of 25 μL.

7. The method according to claim 5, characterized in that: In step 3), the PCR program is as follows: 40 cycles of 95℃ for 10s, 95℃ for 5s for denaturation, and 60℃ for annealing / extension. At the end of each cycle, the fluorescence signal during the PCR amplification process is collected, and the positive result is determined by the Ct value of a certain channel gene in the sample and the amplification curve.

8. The method according to claim 7, characterized in that: The positive control Ct value is between 15 and 20, and the negative control Ct is ≥ 38, according to the judgment criteria in step 3): a) If an amplification curve is observed after amplification of a certain gene channel in the sample to be tested, and the Ct value is ≤35, then the sample to be tested is determined to contain that type of Bifidobacterium. b) If there is no amplification curve after amplification of a certain gene channel in the sample to be tested, and the Ct value is ≥38, then it is determined that the sample to be tested does not contain this type of Bifidobacterium. c) If a gene channel in the sample to be tested has an amplification curve and 35 < Ct < 38, it is judged as an uncertain sample and the amount of DNA template needs to be appropriately increased for retesting.

9. The use of the primer and probe set according to claim 1 in the preparation of a kit for the detection of Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium breve.

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