A primer set for detecting 10 kinds of probiotics based on nucleic acid mass spectrometry and application thereof

Abstract

The application discloses a primer group for detecting 10 kinds of probiotics based on nucleic acid mass spectrometry and application thereof, and belongs to the field of molecular biology detection. The genome information of animal bifidobacterium lactis subsp, animal bifidobacterium animalis subsp, long bifidobacterium longum subsp, long bifidobacterium infantis subsp, bifidobacterium bifidum, bifidobacterium breve, bifidobacterium adolescentis, lactobacillus casei, lactobacillus paracasei and lactobacillus rhamnosus in the NCBI database is verified and compared, the single copy sequence with high inclusivity and exclusivity of each strain is determined, and amplification primers and extension primers are designed. Based on the nucleic acid mass spectrometry technology, a method for simultaneously performing high-throughput accurate identification and important subtype typing identification on the 10 kinds of probiotics in one reaction system is constructed, and the problems of low detection efficiency and high cost in the prior art are solved.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection, and in particular to a primer set for detecting 10 probiotics based on nucleic acid mass spectrometry and its application. Background Technology

[0002] Probiotics, as live microorganisms beneficial to the health of the host, are widely used in food, health products, and pharmaceuticals. Different species and subspecies of probiotics vary significantly in physiological functions and applicable scenarios. Accurate identification of probiotic species and subtypes is of great significance for product quality control, efficacy evaluation, and market supervision.

[0003] Currently, common methods for identifying probiotics include traditional culture-isolation and identification methods, biochemical identification methods, conventional PCR, and real-time quantitative PCR. Traditional culture-isolation and identification methods are cumbersome and time-consuming, and some probiotics are difficult to culture in pure form. Biochemical identification methods have poor specificity, are easily affected by culture conditions, and some probiotics cannot be distinguished by biochemical methods. While conventional PCR and real-time quantitative PCR have high specificity and sensitivity, multiple experiments are required for simultaneous identification of multiple species, resulting in low detection efficiency and high material and time costs. Currently, the commonly used multiplex PCR methods are duplex or triplex PCR, requiring fluorescently labeled probes, which already increases detection costs. Further increasing the number of multiplexes is not only limited by the number of fluorescent channels but also significantly increases the cost of fluorescent labeling.

[0004] Time-of-flight mass spectrometry (MALDI-TOF MS), as a novel detection technology, boasts advantages such as high throughput, high sensitivity, and rapid accuracy. It can simultaneously and accurately detect more than 10 PCR products and has shown promising application prospects in the field of microbial detection. Compound probiotic products often contain multiple probiotics, and detecting them separately would be extremely labor-intensive. MALDI-TOF MS perfectly meets the detection needs of compound probiotic products. However, there is currently no mature method using this technology to simultaneously detect multiple common probiotics and identify subtypes. Therefore, there is an urgent need to develop an efficient, accurate, and high-throughput probiotic identification technology to meet the industry's demand for rapid identification of multiple bacterial species.

[0005] This invention aims to provide a method for the simultaneous identification of 10 probiotic strains based on nucleic acid mass spectrometry, enabling the simultaneous detection and identification of important subtypes of Bifidobacterium animalis subsp. lactis, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus, thus solving the problems of low detection efficiency and high detection cost in existing technologies. Summary of the Invention

[0006] The purpose of this invention is to provide a primer set for detecting 10 probiotics based on nucleic acid mass spectrometry and its application, so as to solve the problems existing in the prior art. Based on nucleic acid mass spectrometry technology, this invention constructs a method for simultaneously performing high-throughput accurate identification and important subtype typing of 10 probiotics.

[0007] To achieve the above objectives, the present invention provides the following solution: The present invention provides a primer set for detecting probiotics, the primer set consisting of amplification primers with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.20 and extension primers with nucleotide sequences as shown in SEQ ID NO.21-SEQ ID NO.30.

[0008] The present invention also provides an application of the above-mentioned primer set in the preparation of products for detecting probiotics.

[0009] Furthermore, the probiotics include Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. animalis, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus.

[0010] The present invention also provides a product for detecting probiotics, using the above-mentioned primer set as the active ingredient.

[0011] Furthermore, the probiotics include Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. animalis, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus.

[0012] The present invention also provides an application of the above-described primer set or the above-described product in the detection of probiotics.

[0013] Furthermore, the probiotics include Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. animalis, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus.

[0014] The present invention also provides a method for detecting probiotics, comprising the following steps: Genomic DNA was extracted from the sample to be tested, and multiplex PCR amplification was performed using amplification primers with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.20. The amplification products were then collected. The amplification product was deoxyribonucleotides removed, and a single-base extension reaction was performed using extension primers with nucleotide sequences as shown in SEQ ID NO.21-SEQ ID NO.30. The extension product was collected, and mass spectrometry was performed to obtain and interpret the mass spectrometry analysis results. The interpretation is as follows: when a single peak appears at the molecular weight position of the expected extension product corresponding to the extension primer in the mass spectrometry analysis results and the signal-to-noise ratio is greater than 6, it is determined that the probiotic exists in the sample to be tested. If, in the mass spectrometry analysis results, no single peak appears at the molecular weight position of the expected extension product corresponding to the extension primer, or if a single peak appears and the signal-to-noise ratio is not greater than 6, it is determined that the probiotic does not exist in the sample to be tested.

[0015] Furthermore, the probiotics include Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. animalis, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus.

[0016] The present invention discloses the following technical effects: This invention verified and compared the genomic information of *Bifidobacterium animalis* subsp. *lacto*, *Bifidobacterium animalis* subsp. *animal*, *Bifidobacterium longum* subsp. *longum*, *Bifidobacterium longum* subsp. *infant*, *Bifidobacterium bifidum*, *Bifidobacterium breve*, *Bifidobacterium adolescentis*, *Lactobacillus casei*, *Lactobacillus paracasei*, and *Lactobacillus rhamnosus* from the NCBI database. Comparative genomics analysis identified highly inclusive and exclusive single-copy sequences for each species, and amplification and extension primers were designed. Based on nucleic acid mass spectrometry, a method for simultaneous high-throughput and accurate identification of these 10 probiotics and identification of important subtypes was constructed, solving the problems of low detection efficiency and high cost in existing technologies. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Image showing the results of Bifidobacterium lactis detection in animals; Figure 2 Image showing the results of animal subspecies detection of Bifidobacterium animalis; Figure 3 Image showing the results of Bifidobacterium longum infantile subspecies testing; Figure 4 This is a graph showing the results of the Bifidobacterium bifidum test. Figure 5 This is a graph showing the results of the Bifidobacterium breve test. Figure 6 This is a graph showing the detection results of Bifidobacterium longum subsp. longum; Figure 7 Image showing the results of Bifidobacterium adolescentis detection; Figure 8 The image shows the results of Lactobacillus casei detection. Figure 9 This is a graph showing the results of Lactobacillus paracasei testing. Figure 10 The image shows the detection results of Lactobacillus rhamnosus; Figure 11 This is a mass spectrometry result of partially exclusive samples; Figure 12 The image shows the sensitivity test results for Bifidobacterium lactis subsp. animalis. Figure 13 The image shows the sensitivity detection results of Bifidobacterium longum subsp. longum; Figure 14 This is a graph showing the detection results of mixed targets in a real sample. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] The primer sequence information is detailed in Table 1. The primers were diluted to a 100 μM stock solution. 5 μL of each primer was taken and mixed to prepare a 100 μL multiplex amplification primer mixture. The final concentration of each primer in the mixture was 5 μM.

[0025] Table 1. Amplification primer sequences and preparation information The sequence information of the extension primers is detailed in Table 2. The extension primers are diluted to a 500 μM stock solution. Take the volume of each primer stock solution listed in Table 2 and add water to make up to 100 μL to prepare the extension primer mixture.

[0026] Table 2. Extension primer sequences, molecular weights, and primer preparation information. Example 1 1. Operating Steps 1.1 Multiplex PCR amplification Genomic DNA was extracted from the sample to be tested. Using the sample genomic DNA as a template, multiplex PCR amplification was performed on the sample using 20 amplification primers listed in Table 1. A mixture of genomic DNA from 10 bacteria was used as a positive control, and ultrapure water was used as a negative control. The reaction system is shown in Table 3.

[0027] Table 3 PCR reaction system According to the instrument requirements, the prepared reaction system is placed in the PCR amplification instrument, and amplification is carried out according to the reaction procedure shown in Table 4.

[0028] Table 4 PCR reaction procedure 1.2 Dephosphorylation reaction To eliminate excess dNTPs after multiplex PCR amplification and ensure the accuracy of single-base extension detection by mass spectrometry, shrimp alkaline phosphatase (SAP) was used for treatment. The SAP digestion enzyme reaction system is shown in Table 5.

[0029] Table 5 SAP Reaction System According to the instrument requirements, the prepared reaction system was placed in the PCR amplification instrument, and the digestion reaction was carried out according to the reaction procedure shown in Table 6.

[0030] Table 6 SAP Response Procedure 1.3 Single-base extension reaction After thawing and mixing the extension reaction mixture and extension enzyme mixture at room temperature, centrifuge at 2000 rpm for 10 seconds. Add 2 μL of the prepared single-base extension reaction solution to each well of the SAP product. The extension primers can be premixed. The preparation of each test reaction system is shown in Table 7.

[0031] Table 7 Single-base extension reaction system Place the prepared reaction system in a PCR amplification instrument and perform a single-base extension reaction according to the reaction procedure shown in Table 8.

[0032] Table 8. Single-base extension reaction procedure 1.4 Resin desalting purification and MALDI-TOF mass spectrometry detection Pre-fill each well in the 384 reaction plate with 18 μL of DEPC-treated water. Distribute 9 μL of the single-base extension product to each well containing DEPC-treated water, mix thoroughly, seal with a sealing film, and centrifuge to ensure the mixture is evenly distributed at the bottom of the wells. Ensure the Assay file accurately contains the molecular weight information of all single-base extension primers and their extension products, and import this information into the mass spectrometer software. Open the nucleic acid flight mass spectrometer deck, remove the sealing film from the 384 reaction plate, and place it in the designated position. Set the resin dosage to 10 μL per well. The instrument will automatically perform product desalting and purification steps, as well as automatic sample loading and mass spectrometry detection.

[0033] The result is determined based on whether the known single-base extension primers extend and produce specific extension peaks and bases. The mass spectrometry analysis results for each target show two peak positions: one for the single-base extension primer and one for the single-base extension product. If a single peak appears at the molecular weight position corresponding to any extension product of a target and the signal-to-noise ratio is greater than 6, the result is considered positive for that bacterium (subspecies); otherwise, it is considered negative.

[0034] 2. Inclusivity Validation 2.1 Information on strains used for inclusion validation The strain information is shown in Table 9.

[0035] Table 9. Information on Inclusive Strains Samples 2.2 Test Results When the target bacterial species is present, the corresponding extension primer peak weakens or disappears, and the peak corresponding to the extension product appears. The molecular weights of the extension primers and extension products for each bacterial species are consistent with those in Table 2. A schematic diagram of the mass spectrometry detection results of *Bifidobacterium animalis* subsp. *lactam* is shown below. Figure 1 As shown in the diagram. A schematic diagram of the mass spectrometry detection results of *Bifidobacterium animalis* subspecies is shown below. Figure 2 As shown in the diagram. A schematic diagram of the mass spectrometry detection results of *Bifidobacterium longum* subsp. *infantii* is shown below. Figure 3 As shown in the diagram. A schematic diagram of the mass spectrometry detection results of Bifidobacterium bifidum is shown below. Figure 4 As shown in the diagram. A schematic diagram of the mass spectrometry detection results of Bifidobacterium breve is shown below. Figure 5 As shown in the diagram. A schematic diagram of the mass spectrometry detection results of *Bifidobacterium longum* subsp. *longum* is shown below. Figure 6 As shown in the diagram. A schematic diagram of the mass spectrometry detection results of Bifidobacterium adolescentis is shown below. Figure 7 As shown in the diagram. A schematic diagram of the mass spectrometry detection results of *Lactobacillus casei* is shown below. Figure 8 As shown in the diagram. A schematic diagram of the mass spectrometry detection results of *Lactobacillus paracasei* is shown below. Figure 9 As shown in the diagram. A schematic diagram of the mass spectrometry detection results of *Lactobacillus rhamnosus* is shown below. Figure 10 As shown.

[0036] 3. Exclusivity verification 3.1 Information on strains used for exclusion verification The information on the standard strains for exclusion verification is shown in Table 10.

[0037] Table 10 Information on standard strains used for exclusion verification 3.2 Test Results The nucleic acid extraction and detection steps are the same as in "1. Operation Steps". The above samples were tested for nucleic acid, using a mixture of positive genomic DNA from 10 probiotics corresponding to the detection target as a positive control, and ultrapure water as a negative control. The results are as follows: Figure 11 As shown, it can be seen that for other bacterial species not within the detection range, only the extension primer peak is observed in the detection results, and no extension product peak is observed at the target site. This fully demonstrates that the detection method, primer combination and kit provided by the present invention have good exclusivity.

[0038] 4. Detection sensitivity In this embodiment, after digital PCR quantification of the genomic DNA of each target, it was diluted to obtain target sensitivity references of 1000 copies / μL, 100 copies / μL, 50 copies / μL, 10 copies / μL, and 5 copies / μL. Detection was performed according to the methods and steps in "1. Operation Procedures", with each gradient performed in triplicate. The final detection sensitivity results for each target are shown in Table 11.

[0039] The results of sensitivity testing are illustrated using *Bifidobacterium animalis* subsp. *lactamella* and *Bifidobacterium longum* subsp. *longum* as examples. Figure 12 As shown, when detecting Bifidobacterium lactis subsp., when the sample concentration was reduced to 10 copies / μL, the extension primer peak did not undergo any extension reaction to transform into the extension product peak. Therefore, the previous concentration of 50 copies / μL was taken as its limit of detection.

[0040] like Figure 13 As shown, when detecting *Bifidobacterium longum* subsp. *longum*, when the sample concentration is reduced to 10 copies / μL, the extension primer peak partially converts into an extension product peak after the extension reaction. When the sample concentration is reduced to 5 copies / μL, the extension primer peak does not convert into an extension product peak at all. This indicates that the target can detect samples at 10 copies / μL, i.e., 10 copies / μL is its limit of detection.

[0041] Table 11 Sensitivity Detection Results 5. Actual sample testing Using real probiotic products as samples, tests were conducted according to the steps outlined in "1. Operating Procedures". The test results are as follows: Figure 14 As shown in Table 12, the test results are consistent with the results displayed on the label.

[0042] Table 12 Actual Sample Test Results The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer set for detecting probiotics, characterized in that, The primer set consists of amplification primers with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.20 and extension primers with nucleotide sequences as shown in SEQ ID NO.21-SEQ ID NO.

30.

2. The application of the primer set according to claim 1 in the preparation of products for detecting probiotics.

3. The application as described in claim 2, characterized in that, The probiotics include Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. animalis, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus.

4. A product for detecting probiotics, characterized in that, Includes the primer set as described in claim 1.

5. The product as described in claim 4, characterized in that, The probiotics include Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. animalis, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus.

6. The use of the primer set of claim 1 or the product of claim 4 or 5 in the detection of probiotics for non-diagnostic purposes.

7. The application as described in claim 6, characterized in that, The probiotics include Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. animalis, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus.

8. A method for detecting probiotics for non-diagnostic purposes, characterized in that, Includes the following steps: Genomic DNA was extracted from the sample to be tested, and multiplex PCR amplification was performed using amplification primers with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.

20. The amplification products were then collected. The amplification product was deoxyribonucleotides removed, and a single-base extension reaction was performed using extension primers with nucleotide sequences as shown in SEQ ID NO.21-SEQ ID NO.

30. The extension product was collected, and mass spectrometry was performed to obtain and interpret the mass spectrometry analysis results. The interpretation is as follows: when a single peak appears at the molecular weight position of the expected extension product corresponding to the extension primer in the mass spectrometry analysis results and the signal-to-noise ratio is greater than 6, it is determined that the probiotic exists in the sample to be tested. If, in the mass spectrometry analysis results, no single peak appears at the molecular weight position of the expected extension product corresponding to the extension primer, or if a single peak appears and the signal-to-noise ratio is not greater than 6, it is determined that the probiotic does not exist in the sample to be tested.

9. The method as described in claim 8, characterized in that, The probiotics include Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. animalis, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus.

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