A probe combination, gene chip, kit and method for high-resolution detection of probiotics

Abstract

This application discloses a high-resolution probe combination, gene chip, kit, and method for detecting probiotics, belonging to the field of microbial detection technology. The probe combination of this application is designed for the entire genome and can simultaneously detect 15 probiotic species, offering higher resolution and sensitivity compared to commonly used PCR methods in the prior art. Simultaneously, the probe combination, gene chip, kit, and method of this application can assess the relative abundance of probiotics, thereby achieving quantitative detection.

Description

Technical Field

[0001] This application relates to the field of microbial detection technology, and in particular to a high-resolution probe combination, gene chip, kit, and method for detecting probiotics. Background Technology

[0002] Probiotics are defined as "live microorganisms that, when administered in adequate amounts, provide a health benefit to the host." Besides requiring sufficient dosage, the effects of probiotics on the host can be species-, subspecies-, or even combination-specific. Therefore, taxonomic consistency of the probiotic composition is crucial for product quality and efficacy. However, many studies have reported discrepancies between the contents of commercial product labels and the actual strains contained. This inconsistency may be due to limited taxonomic resolution and detection sensitivity of existing methods, insufficient viable cell counts or reduced activity, and contamination by other strains. Furthermore, accurately distinguishing closely related probiotic subspecies remains relatively difficult. For example, *Bifidobacterium animalis* subsp. *lactobacter* (… Bifidobacterium animal subsp. milk ) and animal subspecies ( Bifidobacterium animalum subsp animal The high similarity between probiotic strains and their 16S rRNA gene sequences, even at the whole genome level, limits the taxonomic resolution of existing microbial identification methods. These limitations are particularly important for the analysis and quality control of multi-strain probiotic products and constitute a key unresolved issue in the probiotic industry.

[0003] Traditional culture-based methods for probiotic product detection are typically slow and labor-intensive, making them unsuitable for high-throughput screening. Molecular methods such as qPCR and MALDI-TOF MS offer better specificity, but require advanced instrumentation, limiting their widespread application. In recent years, 16S rRNA gene sequencing has been widely used to identify various microbial communities, including probiotics, due to its simplicity and cost-effectiveness. However, the inherent conservation of 16S rRNA gene sequences among probiotic species hinders accurate identification at the species level, and in some cases, even fails to distinguish them correctly at the genus level. Next-generation sequencing (NGS) offers greater sequencing depth, but it is computationally demanding, analytically complex, susceptible to sequencing errors, and difficult to implement in routine quality control. Therefore, there is an urgent need for an accurate and efficient method to overcome the limitations of existing methods.

[0004] DNA microarrays (gene chips), consisting of tens of thousands of oligonucleotide probes arranged on a glass slide, have been successfully applied to microbial identification and functional analysis. Compared with NGS, DNA microarrays offer high specificity, sensitivity, and reproducibility, while significantly reducing time and computational requirements and possessing quantitative potential. However, current technologies lack high-resolution gene chips for detecting probiotics. Summary of the Invention

[0005] To solve at least one of the above-mentioned technical problems, the technical solution adopted in this application is as follows.

[0006] The first aspect of this application provides a probe assembly for high-resolution detection of probiotics, comprising: As shown in SEQ ID No. 1~350, used for detection Bifidobacterium animalum subsp . animal The probe; As shown in SEQ ID No. 351~1068, this is used for detection. Bifidobacterium animalum subsp. milk The probe; As shown in SEQ ID No. 1069~1418, this is used for detection. Bifidobacterium bifidum The probe; As shown in SEQ ID No. 1419~1768, this is used for detection. Bifidobacterium breve The probe; As shown in SEQ ID No. 1769~2118, this is used for detection. Bifidobacterium longum subsp. long The probe; As shown in SEQ ID No. 2119~2468, this is used for detection. Bifidobacterium longum subsp. child The probe; As shown in SEQ ID No. 2469~2818, this is used for detection. Lactobacillus casei The probe; As shown in SEQ ID No. 2819~3168, this is used for detection. Lactobacillus paracasei The probe; As shown in SEQ ID No. 3169~3518, this is used for detection. Lactobacillus rhamnosus The probe; As shown in SEQ ID No. 3519~3868, this is used for detection. Lactiplantibacillus plantarum The probe; As shown in SEQ ID No. 3869~4218, this is used for detection. Lactobacillus acidophilus The probe; As shown in SEQ ID No. 4219~4568, this is used for detection. Lactococcus lactis subsp. milk The probe; As shown in SEQ ID No. 4569~4918, this is used for detection. Lactococcus lactis subsp. milk Probes for biovar diacetylactis; As shown in SEQ ID No. 4919~5268, this is used for detection. Limosilactobacillus reuteri subsp. reuteri The probe; As shown in SEQ ID No. 5269~5618, this is used for detection. Streptococcus salivarius subsp. thermophilic The probe.

[0007] The second aspect of this application provides a high-resolution gene chip for detecting probiotics, wherein the gene chip is arranged with the probe combination described in the first aspect of this application.

[0008] In some embodiments of this application, the gene chip further includes globally distributed control probes. In some specific embodiments of this application, the nucleotide sequences of the globally distributed control probes are shown in SEQ ID No. 5619. The globally distributed control probes are uniformly distributed on the chip.

[0009] In some embodiments of this application, the gene chip further includes a positive control probe. In some specific embodiments of this application, the nucleotide sequence of the positive control probe is shown in SEQ ID No. 5620~5853.

[0010] In some embodiments of this application, negative control probes are also arranged on the gene chip. In some specific embodiments of this application, the nucleotide sequences of the negative control probes are shown in SEQ ID No. 5854~5884.

[0011] A third aspect of this application provides a high-resolution detection kit for probiotics, the kit comprising the probe combination described in the first aspect of this application or the gene chip described in any of the second aspects of this application.

[0012] In some embodiments of this application, the kit further includes reagents for extracting genomic DNA from the sample to be tested, nucleic acid amplification reagents, fluorescent labeling reagents, and / or purification reagents.

[0013] The fourth aspect of this application provides a method for high-resolution detection of probiotics, comprising the following steps: S1, Obtain the DNA sample of the sample to be tested; S2, the obtained DNA sample is fluorescently labeled and purified through a single nucleic acid amplification; S3, perform hybridization detection using the gene chip described in any of the second aspects of this application; S4. Determine the detection result based on the detected probe signal.

[0014] In some embodiments of this application, the sample to be tested is a food or medicine containing probiotics.

[0015] In some embodiments of this application, the detection result is determined based on the proportion of the number of probes that detected signals to the total number of probes.

[0016] The probe combination detection rate is defined as the proportion of probes that detect signals out of the total number of probes. If the detection rate of a probe combination is greater than a set threshold, it is classified as positive to ensure that only true positive results are considered and to reduce the false positive rate.

[0017] In some specific embodiments of this application, the ratio threshold is set to 0.30-0.75, for example, 0.30, 0.50, 0.60 or 0.70.

[0018] Compared with the prior art, this application has the following advantages: 1. Current technologies for assessing the quality of probiotic products primarily rely on traditional isolation and culture methods. However, due to the relatively small differences between probiotic strains, it is difficult to accurately distinguish and identify them based on simple morphological and physiological / biochemical characteristics. Furthermore, isolation and culture often involve significant randomness, making it impossible to accurately assess product quality. This application innovates a method for detecting probiotics in products, filling a gap in related detection technologies.

[0019] 2. The probe suite of this application is designed for the entire genome and can simultaneously detect 15 probiotic species, offering higher resolution and sensitivity compared to commonly used PCR methods in the prior art. Furthermore, the probe suite, gene chip, kit, and method of this application can assess the relative abundance of probiotics, thereby achieving quantitative detection.

[0020] 3. The gene chip of this application is a novel DNA microarray tool for efficient identification of probiotic species and subspecies. The gene chip contains eight identical subarrays, each containing 5618 specific probes targeting 15 commonly used probiotic species. This application validated its specificity, sensitivity, quantitative performance, and reproducibility using gDNA from pure probiotic cultures and mixed microbial communities (simulated communities), and further applied it to evaluate commercial probiotic products. Compared to traditional culture-based methods and 16S rRNA gene sequencing, the gene chip of this application enables rapid and accurate component analysis at the species and subspecies levels, providing a promising tool for the quality assessment and regulatory monitoring of probiotic products.

[0021] 4. The method described in this application has the advantage of simple operation. Compared with sequencing-based detection methods, it eliminates the steps of PCR and sequencing of the target region, and data processing is also simpler and more convenient. This high-throughput, high-resolution, and convenient detection method has important application value for the quality assessment of probiotic products, can guide the monitoring and supervision of the probiotic industry, and protect the rights and interests of consumers.

[0022] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0023] The above and other objects, features, and advantages of exemplary embodiments of this application will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings. Several embodiments of this application are illustrated in the drawings by way of example and not limitation, in which: Figure 1 This document shows an overview of the distribution of various types of probes in the gene chip subarray used for detecting probiotics in Embodiment 2 of this application; Figure 2 The performance of the gene chip in Example 3 of this application for detecting probiotic species under different probe detection rate (PDR) thresholds (0-1) is shown. A and C are ROC curves, and B and D are PR curves. The data in A and B are from pure probiotic cultures, and the data in C and D are from simulated communities. Figure 3 The detection results of probiotic strains in the simulated community in Example 3 of this application are shown; Figure 4 The results of gene chip capability and sensitivity evaluation in Example 4 of this application are shown, where A and B represent respectively. B. animal subsp. milk HN019 and L. rhamnosusThe linear relationship between the total probe fluorescence intensity of GG and the amount of DNA input, where C and D represent the following: B. animal subsp. milk HN019 and L. rhamnosus The number of detection probes for GG is linearly related to the amount of DNA input. Detailed Implementation

[0024] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, particularly the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0025] To make the technical problems, technical solutions and beneficial effects solved by this application clearer, the following detailed description is provided in conjunction with embodiments.

[0026] The following examples are used to illustrate preferred embodiments of this application. Those skilled in the art will understand that the techniques disclosed in the examples represent technologies discovered by the inventors that can be used to implement this application, and therefore can be considered preferred embodiments of this application. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of this application.

[0027] Unless otherwise defined, 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 application pertains, and all materials cited herein and referenced by them are incorporated herein by reference.

[0028] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0030] Example 1: Design of Probiotic-Specific Probes This embodiment designs specific probes for 15 types of probiotics. The 15 types of probiotics are as follows: Bifidobacterium animalis subspecies ( Bifidobacterium animalum subsp animal Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalum subsp. milk Bifidobacterium bifidum ( Bifidobacterium bifidum ), Bifidobacterium breve ( Bifidobacterium breve Bifidobacterium longum subsp. ( Bifidobacterium longum subsp. long Bifidobacterium longum infantis subspecies ( Bifidobacterium long subsp. child Lactobacillus casei ( Lactobacillus casei Lactobacillus paracasei ( Lactobacillus paracasei Lactobacillus rhamnosus ( Lactobacillus rhamnosus Lactobacillus plantarum ( Lactiplantibacillus plantarum ), Lactobacillus acidophilus ( Lactobacillus acidophilus Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. milk Lactococcus lactis subsp. (diacetyl) Lactococcus lactis subsp. milk biovar diacetylactis), Lactobacillus reuteri subsp. reuteri ( Limosilactobacillus reuteri subsp. reuteri ) and Streptococcus salivarius thermophilus ( Streptococcus salivarius subsp. thermophilic ).

[0031] The probe designed in this embodiment is an oligonucleotide fragment of a certain length that can bind to a specific target molecule and be detected by a specific detection method. The purpose of specific probe design is to find a probe that, for a given probiotic, binds only to the DNA sequence of that probiotic species and not to the DNA sequence of any other bacterial species. The probe design method includes the following steps: 1. Target sequence collection Reference or commonly used complete genomes (target genomes) of 15 probiotic strains were collected from public databases, along with 413 complete or best-assembled genomes of the most closely related strains (background genomes). The target genome information is shown in Table 1.

[0032] Table 1: Target genome information used for designing specific probes ; 2. Preliminary selection of probiotic strain-specific probes Break the target probiotic genome sequence and the background genome sequence into fragments of 50-mer length k-mer and establish a hash library of the target sequence and background sequence k-mer fragments, recording k-mer the occurrence frequency and the information of the strain to which they belong. Compare the k-mer library of the target sequence with the k-mer library of the background sequence. Select those k-mer that only exist in the k-mer library of the target sequence and do not appear in the k-mer library of the background sequence as the candidate specific probe library for this strain.

[0033] 3. Secondary structure screening Probes should avoid containing palindromic sequences that can form stable secondary structures, otherwise it may affect the hybridization efficiency with the target sequence.

[0034] 4. Elimination of potentially non-specific binding probes in candidate specific probes The designed probes should only specifically bind to the target sequence and not to any other non-target DNA sequences. Some studies have pointed out that if there are more than 20 consecutive base matches between the probe and the non-target sequence, potential non-specific binding may occur. Use the Blast program of NCBI to perform sequence alignment of the candidate specific probes with the k-mer library of the background. According to the alignment results, eliminate the probes in the candidate specific probes that have more than 20 consecutive base matches with the k-mer library of the background.

[0035] 5. Screening of probe physicochemical properties The strain-specific probes obtained after the above step are unique to the genome of this strain and have no more than 20 consecutive base matches with the genomes of other species. Subsequently, physicochemical property screening is performed on the remaining specific probes, and the main conditions include: (1) The nucleic acid free energy (Free energy, unit: kcal / mol) of the probe sequence and the target sequence. If the nucleic acid free energy is less than -30, then remove this probe sequence; (2) If there are 5 consecutive identical bases in the probe, then the complexity of this probe is too low, and remove this probe sequence; (3) Finally, screen the strain-specific probes according to the melting temperature Tm value and GC content GC content: The GC content of the designed probes should be optimized, and it is necessary to ensure that the overall GC content of all designed probes tends to be consistent to ensure the stability of hybridization. In addition, it is also necessary to consider whether the GC content of the target strain is too high or too low, and select a suitable GC content range accordingly. In this embodiment, set: 0.3 < GC content < 0.7.

[0036] Tm value: The Tm value of the probes should be set according to the experimental conditions, and it should be ensured that the Tm values ​​of all probes are generally consistent to guarantee the stability of hybridization. Appropriate GC content and Tm value help improve the success rate of hybridization experiments. In this embodiment, the temperature is set to 65℃. <Tm<82℃。

[0037] Ultimately, 5618 probiotic-specific probes distributed across the entire genome were screened, except for... B. animal subsp milk In addition to 718 probes, the remaining probiotics each contain 350 probes, which can accurately distinguish the target species from its closely related subspecies, as shown in Table 2.

[0038] Table 2: Probiotic-Specific Probes ; Example 2: Preparation of Gene Chips The probes designed in Example 1 were synthesized and integrated into a DNA chip containing eight identical subarrays. The probes in one subarray were arranged as follows: Figure 1 As shown. Wherein: UCP represents a Universal Control probe, with the sequence shown in SEQ ID No. 5619; PCP represents a Positive Control probe, with sequences shown in SEQ ID Nos. 5620-5853. Specifically, SEQ ID Nos. 5620-5648 are designed based on the *E. coli* genome AB548579; SEQ ID Nos. 5649-5678 are designed based on the *E. coli* genome CVOH01000136; SEQ ID Nos. 5679-5708 are designed based on the *E. coli* genome CBWT010001155; SEQ ID Nos. 5709-5738 are designed based on the *E. coli* genome LBBM01000104; SEQ ID Nos. 5739-5767 are designed based on the *E. coli* genome KC254646; SEQ ID Nos. 5768-5796 are designed based on the *E. coli* genome CP007799; SEQ ID No. SEQ IDs 5797-5824 were designed based on the E. coli genome KF500595; SEQ IDs 5825-5853 were designed based on the E. coli genome KC504010; the probe shown in SEQ ID 5644 also targets the KF500595 genome. NCP stands for Positive Control probe, with sequences shown in SEQ IDs 5854-5884; pos and neg represent probes included during chip synthesis.

[0039] The arrangement principle of each probe is as follows: The probes in the subarray are arranged according to... Figure 1 The area is further divided into 32 regions, with UCP distributed across these 32 regions in a "nine-square grid," belonging to 8 different regions. E. coli The strain's PCPs are distributed longitudinally in the subarray, with every other region horizontally; each region has one NCP; pos and neg are arranged by the Agilent chip itself.

[0040] Example 3: Rapid detection of probiotics using gene chips 1. gDNA extraction When using the Qiagen bacterial genomic DNA (gDNA) extraction kit, please note the following before use: ① Solution CD2 should be stored at 2-8℃, and the rest at room temperature (15-25℃); ② If CD3 precipitates, heat at 60℃ until completely dissolved; ③ All centrifugation steps should be performed at 15-25℃. Wear sterile gloves throughout the extraction process and ensure the operating area is clean.

[0041] (1) Instantly disconnect the Power Bead Pro Tube to confirm that the grinding beads have settled to the bottom, add up to 250 mg of bacterial powder and 800 μL of CD1 solution, and vortex to mix; (2) All 2mL Power Bead Pro Tubes were vortexed at the maximum speed of the vortex mixer for 10 minutes. If there were many samples, the vortexing time was extended by 5 to 10 minutes. (3) Centrifuge the Power Bead Pro Tube at 15000×g for 1 min; (4) Transfer approximately 500-600 μL of the supernatant to a clean 2 mL Microcentrifuge Tube; (5) Add 200 μL of CD2 solution and vortex for 5 seconds; (6) Centrifuge at 15000×g for 1 min, discard the precipitate and transfer about 600 μL of supernatant to a new 2 mL Microcentrifuge Tube; (7) Add 600 μL of CD3 solution and vortex for 5 seconds; (8) Add 650 μL of the lysis buffer from the previous step to the MB Spin Column and centrifuge at 15000×g for 1 min; (9) Discard the waste liquid in the collection tube and repeat the above steps; (10) Carefully transfer the MB Spin Column to a new 2mL Collection tube to avoid contaminating the MB Spin Column with waste liquid; (11) Add 500 μL of solution EA to the MB Spin Column and centrifuge at 15000×g for 1 min; (12) Discard the waste liquid and return the MB Spin Column to the same 2mL Collection tube; (13) Add 500 μL of solution C5 to the MB Spin Column and centrifuge at 15000×g for 1 min; (14) Discard the waste liquid and place the MB Spin Column into a new 2mL collection tube; (15) Centrifuge at 16000×g for 2 min, and carefully transfer the MB Spin Column into a new 1.5mL Elution tube; (16) Carefully add 100 μL of EDTA-free solution CD6 to the center white filter membrane; (17) Centrifuge at 15000×g for 1 min, discard the MB Spin Column, and store the gDNA at -30~-15℃ or -90~-65℃.

[0042] 2. DNA magnetic bead purification The main steps in DNA processing are: (1) Equilibrate OnePure MagBeads at room temperature for 30 min beforehand and shake thoroughly to ensure no obvious magnetic bead precipitation; (2) Add 50-100 μL of DNA sample to be purified by magnetic beads to the PCR tube / eight-tube, then add an equal volume of OnePure MagBeads, vortex to mix, briefly collect the liquid on the tube wall, and let stand at room temperature for 5 min. (3) Place the PCR tube / eight-tube strip on the magnetic rack and wait for the solution in the tube to become clear before discarding the supernatant; (4) Add 200 μL of 80% freshly prepared ethanol to the PCR tube / eight-tube, let it stand for 30 seconds and then discard the supernatant. Repeat the operation until the supernatant is relatively clean. (5) Place the PCR tube / eight-tube on a magnetic rack and let it stand at room temperature for 1-2 minutes until the magnetic beads dry and crack, or open the tube and place it on a 45°C metal bath until there is no water on the surface of the magnetic beads and no ethanol residue at the bottom of the tube. (6) Remove the PCR tube / eight-tube from the magnetic rack, add 43 μL of sterile water to resuspend the magnetic beads, vortex or pipette to mix, collect the liquid on the tube wall and let it stand at room temperature for 3 min. (7) Place the PCR tube / eight-tube strip on the magnetic rack and wait for the solution in the tube to become clear. Transfer 42 μL of supernatant to a new EP tube for the next step of labeling.

[0043] Using the steps described above, the inventors extracted gDNA from 11 pure cultures of probiotic strains. Furthermore, the inventors also extracted gDNA from 5 closely related subspecies strains and used these gDNAs to construct 15 simulated probiotic communities, as shown in Table 3.

[0044] Table 3: Information on pure cultures and simulated communities of probiotics ; 3. DNA fluorescent labeling This example uses the Agilent SureTag Complete DNA Labeling Kit and includes the following steps: (1) Take 250 ng of purified gDNA from magnetic beads and make up the volume to 14.75 μL with sterile water. Then add 2.75 μL of random primer, mix well, and carry out the following denaturation reaction: 98℃ for 10 min, and heat-cover at 105℃. (2) After the time is reached, immediately place the sample on ice to cool; (3) After the above sample is momentarily separated, add the following reagents directly: 1 μL of sterile water, 5.5 μL of 5×Reactionbuffer, 2.75 μL of 10×dNTP mix, 0.25 μL of Cyanine 3-dUTP, and 0.5 μL of Exo(-)Klenow, for a total of 27.5 μL; (4) After mixing by pipetting or vortexing, quickly centrifuge to collect the liquid on the tube wall and remove air bubbles; (5) Place the reaction system on the PCR instrument, set the hot lid temperature to 105℃, and run the following program: 37℃ 4h, 95℃ 3min, 4℃ hold.

[0045] 4. Purification of fluorescently labeled DNA products This embodiment uses the Agilent Oligo aCGH / ChIP-on-chip Hybridization Kit and includes the following steps: (1) Centrifuge the labeled product, confirm the column order, add 125 μL of 1×TE and the sample three times each time, rinse the labeled tube and transfer it into the purification column, centrifuge at 14000×g for 10 min; (2) Discard the filtrate, put the collection tube back into the collection column, add 480 μL of 1×TE (pH 8.0) into the collection column, cover the column, and centrifuge at 14000×g for 10 min; (3) Take out the collection column and invert it into a new 2mL centrifuge tube. Mark the tube accordingly. Centrifuge at 1000×g for 1min. The purified sample (samples from the same hybridization region are purified and recycled into the same collection tube) (the volume is about 40-64μL). Transfer the purified product into a PCR tube. (4) Using Nanodrop oneC Determine the total nucleic acid concentration and the corresponding dye labeling concentration; (5) Use a concentrator to dry the sample to a volume of 10 μL.

[0046] 5. Hybridization of fluorescent DNA from the target sample with the gene chip. This embodiment uses the Agilent Oligo aCGH / ChIP-on-chip Hybridization Kit and includes the following steps: (1) After preparing the gene chip hybridization system according to Table 4, add 45 μL of hybridization system to the above concentrated 10 μL sample and mix with a pipette. After a short time, place the reaction system on the PCR instrument, set the hot cover temperature to 105℃, and run the following program: 98℃ 3min, 37℃ 30min, 37℃ hold. Table 4: Gene chip hybridization system ; (2) Hybridization a. First, place a clean gasket into the Agilent chamber with the gasket label facing up, aligning it with the rectangular part at the bottom of the chamber, ensuring that the gasket is flush with the chamber base; b. Then, take 47 μL of the 37°C sample from the previous step and put it into the middle of the rubber ring on the pad to avoid air bubbles. Then, place the chip upside down on the pad. c. Next, replace the chamber lid and tighten the knob; d. Place each assembled device into the rotating rack of the incubator, take a balanced chamber, rotate the hybridization chamber vertically to wet the slide, and evaluate the flowability of the bubbles; e. Set the hybridization rotator to a rotation speed of 20 rpm and hybridize at 67°C for 22 hours.

[0047] (3) Chip cleaning: After hybridization, the gene chip was removed at room temperature and placed in washing solution 1 (a reagent in the Agilent kit). The solution was set to 250 rpm and the chip was shaken and cleaned for 5 min at room temperature. Then, washing solution 2 (also a reagent in the Agilent kit) was used at 200 rpm and the chip was shaken and cleaned for 1 min at 39°C. Finally, the liquid on the surface of the chip was removed and the chip was scanned within 4 h.

[0048] 6. Fluorescence Result Scanning and Signal Analysis Data import: (1) Import the microarray data file into the microarray data preprocessing software.

[0049] (2) Verify the integrity of the data file and ensure that the file format is compatible with the preprocessing software.

[0050] Quality control and data cleaning: (3) Conduct quality control checks to assess the overall quality of the data and identify potential problems (such as outliers and artifacts).

[0051] Parameter: Outlier detection threshold (set to ±3 standard deviations in this embodiment) (4) Remove or mark low-quality or unreliable probes or spots from the data.

[0052] Parameter: Signal strength threshold for probe removal (in this embodiment, probes with a signal strength <100 are removed). (5) Apply background correction methods to remove non-specific hybridization signals.

[0053] Parameters: Background correction algorithm (based on a negative control probe-based method), correction parameters (based on the correction results of an Agilent chip in this embodiment). (6) Perform data normalization to adjust for systematic variations between arrays.

[0054] Parameters: Normalization method (Lowess locally weighted regression based on Agilent chips) (7) Export the preprocessed microarray data in an appropriate format for subsequent analysis.

[0055] Parameters: Export file format (e.g., CSV, Excel) 7. Analysis and Judgment of Probiotic Detection Results For pure cultures of probiotics and simulated community gDNA, the ROC values ​​of the PDR value and the AUC values ​​of the PR curve were 0.9848 and 0.9834, and 0.9948 and 0.9683, respectively, indicating that gene chip detection of target probiotic species and subspecies has a high confidence level. Figure 2 ).

[0056] Furthermore, the inventors evaluated the specificity of the gene chip using gDNA from 11 pure cultures of probiotics and 15 simulated communities. When the probe detection threshold (defined by probe fluorescence intensity) was set between 500 and 3000, the PDR showed a significant difference between target and non-target species, indicating that the probe had high specificity. To obtain better detection results, the inventors selected a fluorescence intensity threshold of 700 signal units for probe detection.

[0057] because B. animal subsp. milk The total number of probes differed from the other 14 bacterial species. Specific identification (probiotic species or subspecies) was based on a comprehensive analysis of probe count and PDR value.

[0058] The results of gDNA detection in pure cultures of probiotics are shown in Table 5. The results of detection in 15 simulated communities are also shown in Table 5. Figure 3 As shown.

[0059] For 15 probiotic species, the inventors developed a stepwise detection process based on probe number and PDR, which is as follows: (i~iii) The PDR thresholds were set to 0.30, 0.50 and 0.60, respectively, corresponding to 105, 175 and 210 detected species-specific probes. This enabled the chip to specifically detect 15 probiotic species, as well as the most closely related species. B. longum subsp. long and B. longum subsp. child as well as B. bifidumand B. short ; (iv) Set the PDR threshold to 0.75, corresponding to 263 detected targets. B. animal subsp. animal Specific probes to exclude B. animal subsp. animal To detect false positives and achieve B. animal subsp. milk Specific detection, at this time for B. animal subsp. milk The specific probe PDR was 0.37, corresponding to 266 detected probes.

[0060] Table 5: Results of gDNA detection using pure cultures of probiotics ; As shown in Table 5, only the probes in the corresponding probe groups have the highest fluorescence signal, indicating that the probe groups all have high specificity.

[0061] Depend on Figure 3 As can be seen, following the established detection process, namely the probe positive thresholds for different bacterial species, these target bacterial species were successfully detected, while background interference from five closely related subspecies was also excluded.

[0062] Example 4: Quantitative capability and sensitivity testing of gene chips To assess the quantitative capabilities of the gene chip, the inventors used two widely used probiotic strains. B. animal subsp. Milk HN019 and L. rhamnosus GG's gDNA was tested.

[0063] The results showed a strong linear correlation between the total probe fluorescence intensity and the amount of gDNA used (0.05-10 ng) (HN019: average R). 2 = 0.95; GG: Average R 2 = 0.94; Figure 4 (A and B in the original text). These findings demonstrate the strong potential of gene chips in the quantitative assessment of probiotic species.

[0064] The inventors discovered that even at a DNA input level of 0.05 ng, targeting B. animal subsp. Milk HN019 and L. rhamnosus 318 and 242 species-specific probes from GG's probe set were successfully detected, respectively. Figure 4(C and D), which meet the established species-specific detection workflow, indicating its high sensitivity.

[0065] The above results indicate that the sensitivity of the gene chip may allow it to perform detection even with DNA input levels as low as 0.05 ng in single-strain assays. Furthermore, when the DNA input level is ≥1 ng, B. animal subsp. Milk HN019 and L. rhamnosus GG's species-specific probes have a PDR close to 1, defining their optimal working range.

[0066] The results of this embodiment confirm that the gene chip prepared in Example 2 has the required sensitivity and high quantification ability for detecting probiotic strains.

[0067] Example 5: Detection and analysis of probiotic strains from probiotic products Five probiotic product samples (P1-P5) were selected, with labels indicating 10, 9, 15, 11, and 18 probiotic species, respectively. The probiotics in the products were detected using the gene chip prepared in Example 2 and the method in Example 3.

[0068] In this embodiment, the inventors analyzed five commercial probiotic products using isolation culture, full-length 16S rRNA gene amplicon sequencing, and gene chip analysis, respectively. The test results are shown in Tables 6 to 10.

[0069] Table 6: Results of detection of bacterial species in probiotic product P1 by gene chip, 16S amplicon sequencing and conventional isolation culture ; Table 7: Results of detection of bacterial species in probiotic product P2 by gene chip, 16S amplicon sequencing and conventional isolation culture ; Table 8: Results of detection of bacterial strains in probiotic product P3 by gene chip, 16S amplicon sequencing and conventional isolation culture ; Table 9: Results of gene chip, 16S amplicon sequencing and conventional isolation culture for detecting bacterial species in probiotic product P4 ; Table 10: Results of detection of bacterial strains in probiotic product P5 by gene chip, 16S amplicon sequencing and conventional isolation culture ; Tables 6-10 show significant differences in consistency between the products and their label claims. P1 and P2 almost perfectly matched the label claims across all methods. Most labeled species were successfully isolated (90% and 88.9%, respectively), confirmed by sequencing after BLAST alignment (100% and 88.9%, respectively), and fully validated by gene chip analysis (10 / 10 and 9 / 9, respectively). In contrast, P3 and P5 showed significant differences, with only 13.3% (2 / 15) and 11.1% (2 / 18) of the labeled species being isolated, respectively. Most labeled species were undetectable: sequencing and gene chip analysis consistently indicated the absence of most claimed species (sequencing detection rates were 13.3% and 27.8%, while gene chip detection rates were 6.7% and 27.8%, respectively). P4 showed moderate consistency, with some species present at very low abundance. Specifically, 100% (11 / 11) of the tagged species were detected by sequencing, 81.8% by gene chip, and only 18.2% by culture.

[0070] Following the established detection workflow, the gene chip not only detected the probiotic species in the product but also provided higher taxonomic resolution. Compared to 16S rRNA gene sequencing, it accurately resolved subspecies, such as those in P2. L. milk subsp. milk biovar diacetylactis.

[0071] The inventors also compared the relative abundance of gDNA-derived fluorescence signals based on the detected species, using both normalized probe fluorescence intensity and 16S rRNA gene sequencing read counts. Although differences exist between the measured signals and the labeled composition, potentially influenced by factors such as DNA extraction efficiency and product matrix effects, the gene chips consistently showed a closer consistency with the declared species composition, superior to sequencing-based analyses, such as those in sample P1. L. casei .

[0072] These results demonstrate that gene chips, by avoiding biases introduced by PCR amplification, can assess the composition and relative abundance of probiotics in commercial products more rapidly and at higher resolution compared to 16S rRNA gene sequencing and traditional culture methods.

[0073] Furthermore, it should be understood that after reading the foregoing teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A probe assembly for high-resolution detection of probiotics, characterized in that, include: As shown in SEQ ID No. 1~350, used for detection Bifidobacterium animalis subsp animalis The probe; As shown in SEQ ID No. 351~1068, this is used for detection. Bifidobacterium animalis subsp. lactis The probe; As shown in SEQ ID No. 1069~1418, this is used for detection. Bifidobacterium bifidum The probe; As shown in SEQ ID No. 1419~1768, this is used for detection. Bifidobacterium breve The probe; As shown in SEQ ID No. 1769~2118, this is used for detection. Bifidobacterium longum subsp. longum The probe; As shown in SEQ ID No. 2119~2468, this is used for detection. Bifidobacterium longum subsp. infantis The probe; As shown in SEQ ID No. 2469~2818, this is used for detection. Lacticaseibacillus casei The probe; As shown in SEQ ID No. 2819~3168, this is used for detection. Lacticaseibacillus paracasei The probe; As shown in SEQ ID No. 3169~3518, this is used for detection. Lacticaseibacillus rhamnosus The probe; As shown in SEQ ID No. 3519~3868, this is used for detection. Lactiplantibacillus plantarum The probe; As shown in SEQ ID No. 3869~4218, this is used for detection. Lactobacillus acidophilus The probe; As shown in SEQ ID No. 4219~4568, this is used for detection. Lactococcus lactis subsp. lactis The probe; As shown in SEQ ID No. 4569~4918, this is used for detection. Lactococcus lactis subsp. lactis Probes for biovar diacetylactis; As shown in SEQ ID No. 4919~5268, this is used for detection. Limosilactobacillus reuteri subsp. reuteri The probe; As shown in SEQ ID No. 5269~5618, this is used for detection. Streptococcus salivarius subsp. thermophilus The probe.

2. A high-resolution gene chip for detecting probiotics, characterized in that, The gene chip is arranged with the probe combination as described in claim 1.

3. The gene chip according to claim 2, characterized in that, The gene chip also contains global quality control probes, the nucleotide sequences of which are shown in SEQ ID No. 5619.

4. The gene chip according to claim 2, characterized in that, The gene chip also includes a positive control probe, the nucleotide sequence of which is shown in SEQ ID No. 5620~5853.

5. The gene chip according to claim 2, characterized in that, The gene chip also contains negative control probes, the nucleotide sequences of which are shown in SEQ ID No. 5854~5884.

6. A high-resolution detection kit for probiotics, characterized in that, The kit comprises the probe combination of claim 1 or the gene chip of any one of claims 2 to 5.

7. The reagent kit according to claim 6, characterized in that, The kit also includes reagents for extracting genomic DNA from the sample to be tested, nucleic acid amplification reagents, fluorescent labeling reagents, and / or purification reagents.

8. A method for high-resolution detection of probiotics, characterized in that, Includes the following steps: S1, Obtain the DNA sample of the sample to be tested; S2, the obtained DNA sample is fluorescently labeled and purified through a single nucleic acid amplification; S3, Hybridization detection is performed using the gene chip described in any one of claims 2 to 5; S4. Determine the detection result based on the detected probe signal.

9. The method according to claim 8, characterized in that, The sample to be tested is a food or medicine containing probiotics.

10. The method according to claim 8 or 9, characterized in that, The detection result is determined by the proportion of the number of probes that detected signals to the total number of probes.

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