Specific primers for identifying lactose-degrading bacteria and methods and uses thereof

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a specific primer for identifying lactose-degrading bacteria and a method and application thereof. The specific primer for identifying lactose-degrading bacteria has a forward nucleotide sequence as shown in SEQ ID NO. 3 and a reverse primer nucleotide sequence as shown in SEQ ID NO. 5. The specific primer is designed by targeting the conserved region of the beta-galactosidase gene of lactose-degrading bacteria of different bacterial phyla, and finally a pair of specific primers with broad spectrum and high specificity for identifying lactose-degrading bacteria are screened. The kit prepared by using the specific primers can amplify the sample to be tested by a PCR instrument, directly qualitatively identify lactose-degrading bacteria, effectively detect complex samples from different sources, and has a broad application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a specific primer for identifying lactose-degrading bacteria, its method, and its application. Background Technology

[0002] β-Galactosidase is a key enzyme catalyzing the hydrolysis of lactose into glucose and galactose, playing a crucial role in microbial carbon source utilization and lactose digestion in mammals. As a glycoside hydrolase widely found in various environmental microorganisms, the functional diversity of β-galactosidase has been extensively reported. Several studies have shown that *Streptococcus thermophilus* and its secreted β-galactosidase may have the potential to inhibit colorectal cancer, with mechanisms involving galactose-mediated Hippo pathway inhibition thereby inducing cancer cell apoptosis, and indirectly remodeling the gut microbiota through the enrichment of probiotics. Research has also shown that β-galactosidases derived from *Lactobacillus bulgaricus* and *Kluyveromyces kluyveromyces* exhibit dual functions as hydrolases and transgalactosidases, which can be used to catalyze the synthesis of prebiotic oligosaccharides, promoting the development of lactose-free dairy products and functional galactosyl oligosaccharide (GOS) foods. Furthermore, lactic acid bacteria strains enhance the value of acidic whey by producing β-galactosidase, showing potential application as lactose digestion aids. In recent years, a β-galactosidase gene derived from Bifidobacterium has been identified through metagenomic screening of human feces. This gene, after heterologous expression and purification, exhibits excellent transglycosylation activity and is considered an ideal candidate for the dairy industry and prebiotic manufacturing. Simultaneously, β-galactosidase is also highly expressed in Escherichia coli. Therefore, β-galactosidase, as a key functional gene molecular marker, is of great significance in the identification of environmental microbial communities.

[0003] Although various methods for detecting lactose-degrading bacteria have been developed, these technologies still have significant limitations. For example, metagenomics based on 16S rDNA gene amplicon analysis can rapidly resolve the species composition, relative abundance, and community diversity of bacteria in environmental samples, but it struggles to accurately identify bacterial communities with specific metabolic functions (such as lactose degradation). Furthermore, limited by sequencing depth and data noise reduction algorithms, low-abundance strains (relative abundance < 0.1%) are often missed due to insufficient coverage, creating blind spots in the detection of functional microorganisms. Traditional culture and plate counting techniques suffer from long cycles, large differences in enzyme activity, and low recovery rates. While researchers have attempted to use β-galactosidase activity detection technologies (such as chemiluminescence, fluorescence, and colorimetry) to identify lactose-degrading bacterial communities, chemiluminescence requires stringent experimental conditions and is costly, while fluorescence and colorimetry are insufficient for high-throughput screening, limiting their application in the comprehensive analysis of environmental microbial communities. Therefore, developing a simple, practical primer specific to the β-galactosidase gene that covers a wider range of bacterial groups has significant practical application value.

[0004] To achieve the above objectives, the present invention provides a specific primer for identifying lactose-degrading bacteria, a method thereof, and its application. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a specific primer, method, and application for identifying lactose-degrading bacteria. Existing technologies, such as metagenomics using 16S rDNA gene amplicon assays, struggle to identify specific metabolically active bacterial groups and easily miss low-abundance strains. Traditional culture and plate counting techniques suffer from long selection cycles, significant differences in enzyme activity, and insufficient recovery rates. Chemiluminescence assays for β-galactosidase activity detection are also hampered by demanding experimental conditions and high costs. The primary objective of this invention is to provide a specific primer for identifying lactose-degrading bacteria.

[0006] A second objective of this invention is to provide a method for identifying specific primers for lactose-degrading bacteria.

[0007] A third objective of this invention is to provide a specific primer for identifying lactose-degrading bacteria and its application in the preparation of a kit for identifying lactose-degrading bacteria.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A specific primer for identifying lactose-degrading bacteria, the forward nucleotide sequence of which is shown in SEQ ID NO.3 and the reverse nucleotide sequence of which is shown in SEQ ID NO.5.

[0009] The method for identifying specific primers for lactose-degrading bacteria described above includes the following steps: (1) Extract genomic DNA from the sample to be tested to obtain a DNA template; (2) Add ddH2O to the DNA template obtained in step (1), TaKaRa Ex Taq HS, dNTP Mixture, the specific primers mentioned above, and 10×Ex Tag Buffer to prepare the PCR amplification reaction system. (3) Place the PCR amplification reaction system prepared in step (2) into a PCR amplification instrument for reaction and detect it by agarose gel electrophoresis.

[0010] Further, in step (1), the sample to be tested is any one of rat feces, mouse feces, human feces, or soil.

[0011] Further, in step (2) of the PCR amplification reaction system, the volume of the DNA template is 1-3 μL; the volume of Ex Taq HS is 0.20-0.30 μL; the volume of dNTP Mixture is 3-5 μL; the volume of primers is 1-3 μL; the volume of 10×Ex Tag Buffer is 4-6 μL; the volume of ddH2O is 35-38 μL, and the total reaction system is 50 μL.

[0012] Further, the agarose gel electrophoresis in step (3) is 1-2 w / v% agarose gel electrophoresis.

[0013] The application of the specific primers described above for identifying lactose-degrading bacteria in the preparation of a kit for identifying lactose-degrading bacteria.

[0014] Compared with the prior art, the main advantages of the present invention are as follows: This invention provides specific primers, methods, and applications for identifying lactose-degrading bacteria. The invention designs conserved regions of β-galactosidase genes in lactose-degrading bacteria from different phyla, and through PCR amplification, agarose gel electrophoresis, and cloning sequencing, ultimately screens a pair of specific primers for targeting and identifying lactose-degrading bacterial communities. A kit prepared using the screened specific primers can directly and qualitatively identify lactose-degrading bacterial communities in complex samples (such as rat, mouse, human feces, and soil) using PCR technology. Phylogenetic analysis shows that the primers have broad spectrum and high specificity, capable of identifying highly diverse lactose-degrading bacterial communities covering 8 phyla, 12 classes, 16 orders, 19 families, and 28 genera. The method developed based on these primers is simple to operate, accurate in results, shortens the experimental cycle to approximately 6 hours, is low in cost, and has low requirements for bioinformatics technology, making it suitable for efficient identification and diversity analysis of lactose-degrading functional bacterial communities in different ecological environments. This invention provides microbiology professionals with molecular markers at the gene level for selecting lactose-degrading bacteria, and also provides researchers in clinical medicine, basic medicine and related fields with a convenient and reliable technical means to observe changes in species diversity of lactose-degrading bacteria in humans, animals and soil. Attached Figure Description

[0015] Figure 1 The first primer pair contains conserved regions of the pre-primer and post-primer sequences; where a is the conserved region of the pre-primer and b is the conserved region of the post-primer sequence. The black boxes contain conserved bases in either the pre-primer or post-primer region. Figure 2 The conserved regions of the front and back primer sequences of the second primer pair are shown in the black box. Here, a is the conserved region of the front primer, b is the conserved region of the back primer sequence, and the conserved bases in the front or back primer regions are shown in the black box. Figure 3The conserved regions of the front and back primer sequences of the third primer pair are shown in the black box. Here, a is the conserved region of the front primer, b is the conserved region of the back primer sequence, and the conserved bases in the front or back primer regions are shown in the black box. Figure 4 The conserved regions of the front and back primer sequences of the fourth primer pair are shown in the black box. Here, a is the conserved region of the front primer, b is the conserved region of the back primer sequence, and the conserved bases in the front or back primer regions are shown in the black box. Figure 5 The conserved regions of the front and back primer sequences of the fifth primer pair are shown in the black box. Here, a is the conserved region of the front primer, b is the conserved region of the back primer sequence, and the conserved bases in the front or back primer regions are shown in the black box. Figure 6 The conserved regions of the front and back primer sequences of the sixth primer pair are shown in the black box. Here, a is the conserved region of the front primer, b is the conserved region of the back primer sequence, and the conserved bases in the front or back primer regions are shown in the black box. Figure 7 The results of agarose gel electrophoresis of six primer pairs for the β-galactosidase gene are shown; where 1 is the first primer pair; 2 is the second primer pair; 3 is the third primer pair; 4 is the fourth primer pair; 5 is the fifth primer pair; and 6 is the sixth primer pair. Figure 8 The results of agarose gel electrophoresis of environmental samples with primers for the β-galactosidase gene are shown; the samples are rat fecal microbiota genomic DNA, mouse fecal microbiota genomic DNA, human fecal microbiota genomic DNA, and soil microbiota genomic DNA, respectively. Figure 9 Phylogenetic trees were constructed using the sequences of rat fecal microbiota, mouse fecal microbiota, and human fecal microbiota identified by the primer pairs and reference sequences; wherein, the reference sequence is the strain name plus the NCBI GenBank database reference sequence ID, and the β-galactosidase gene of Aspergillus niger (accession number CAK44114.1) is used as the outgroup; Figure 10 The phylogenetic tree was constructed using the soil microbial community sequences identified by the primer pair and a reference sequence; wherein the reference sequence is the strain name plus the NCBI GenBank database reference sequence ID, and the β-galactosidase gene of Aspergillus niger (accession number CAK44114.1) is used as the outgroup. Detailed Implementation

[0016] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0017] Example 1 Primer design for the β-galactosidase gene of lactose-degrading bacteria (1) Download the genome data of lactose-degrading bacteria from the NCBI Genome Database, and obtain the β-galactosidase protein information of glycoside hydrolase family 35 based on the list of carbohydrate-active enzyme proteins in the bacterial genome in the CAZy database, and obtain the corresponding β-galactosidase gene information. Use the Biopython tool to obtain the β-galactosidase gene sequence from the genome and construct a local database. Through species taxonomic analysis of the database, it was determined that the target β-galactosidase gene comes from 3 phyla (Acidobacteriota, Bacillota, Bacteroidota), 7 classes (Bacteroidia, Chitinophagia, Clostridia, Cytophagia, Flavobacteriia, Sphingobacteriia, Terriglobia), covering 8 orders (Chitinophagales, Bacteroidales, Cytophagales, Eubacteriales, Flavobacteriales, Lachnospirale). s, Sphingobacteriales, Terriglobales), including 13 families (Acidobacteriaceae, Bacteroidaceae, Chitinophagaceae, Flammeovirgaceae, Flavobacteriaceae, Hymeno bacteraceae, Lachnospiraceae, Odoribacteraceae, Oscillospiraceae, Prevotellaceae, Sphingobacteriaceae, Spirosomataceae, Weeksellaceae), covering 25 genera ( Arachidicoccus , Bacteroides , Chitinophaga , Chryseobacterium , Elizabethkingia , Fibrella , Flammeovirga , Flavisolibacter ,Flavobacterium , Hoylesella , Hymenobacter , Mucilaginibacter , Niabella , Odoribacter , Pedobacter , Phocaeicola , Pontibacter , Prevotella , Rufibacter , Ruminococcus , Solitalea , Terracidiphilus , Tyzzerella , Xylanibacter , Zunongwangia) A total of 81 bacterial species were included, ensuring broad coverage for subsequent primer design.

[0018] (2) Import all the above β-galactosidase gene sequences into BioEdit software. First, the nucleotide sequences were translated into amino acid sequences, and then multiple sequence alignment was performed using ClustalW. To enhance the conservation and universality of the designed primers, all homologous sequences were divided into multiple primer design groups based on the standard of intergenetic length difference (≤200 bp). Combined with the maximum parsimony strategy, representative sequences were retained to reduce redundancy, while ensuring a wide range of species coverage. After multiple screenings and alignments, 21 representative sequences were finally determined for core analysis. The species origin and protein sequence accession number of these sequences are as follows: Arachidicoccus terrestris 5GH13-10(UAY57377.1), Bacteroides fragilis BOB25 (AKA53674.1), Bacteroides helcogenes P36-108 (ADV42433.1), Chitinophaga sp .MD30(ASZ13595.1), Chryseobacterium sp .T16E-39(ASK32800.1), Chryseobacterium piperi ATCC BAA-1782 (ASW76465.1), Elizabethkingia anophelis 0422(AQW90926.1), Elizabethkingia ursingii G4123(AQX08491.1), Fibrella sp .ES10-3-2-2(ARK12862.1), Flavisolibacter ginsenosidimutans Gsoi l 636(QEC56180.1), Flavobacterium commune PK15 (APA00257.1), Hoylesella enoeca F0113(ALO47716.1), Hymenobacter sedentarius DG5B(ALW85225.1), Hymenobacter nivis NBRC 111535 (AWM32240.1), Mucilaginibacter xinganensisBJC16-A31 (ASU36328.1), Mucilaginibacter sp PAMC 26640 (AMR30202.1), Niabella soli DSM 19437 (AHF16258.1), Pedobacter ginsengisoli T01R-27 (ATP55662.1), Pontibacter korlensis X14-1T(AKD02294.1), Zunongwangia sp HGR-M22(WBL25219.1).

[0019] (3) Multiple comparisons were performed on the above representative sequences, and consensus sequences were generated through sequence analysis. Based on this, several highly conserved nucleotide regions suitable for primer design were identified (see [link to relevant documentation]). Figures 1 to 6 Within these highly conserved nucleotide regions, specific primers were designed, and six pairs of candidate primers were successfully obtained. Primer sequence details are shown in Table 1.

[0020] Table 1 Primer sequences designed for the β-galactosidase gene

[0021] Experimental Example 1 Screening and validation of primers for β-galactosidase gene This experiment aims to verify the effectiveness, sensitivity, and versatility of the primers designed in Example 1.

[0022] (1) Extraction of genomic DNA from the sample Fecal samples (200 mg each) were collected from rats, mice, and humans, as well as environmental soil samples, and temporarily stored at 4°C. Genomic DNA was extracted from fecal samples using the Tengen® Fecal Genomic DNA Extraction Kit (DP328-02) and from environmental soil samples using the Tengen® Soil Genomic DNA Extraction Kit (DP336-02) according to the manufacturer's instructions. After extraction, the concentration and purity of the DNA were determined spectrophotometrically using a Thermo Scientific™ NanoDrop™ One instrument. Qualified samples were stored at -20°C for later use.

[0023] (2) Primer sensitivity screening Using the rat fecal microbiota genomic DNA extracted in step (1) as a template, TaKaRa Ex Taq HS, dNTPMixture, the 6 pairs of primers obtained in Example 1, and 10×Ex Tag Buffer were added and mixed with ddH2O to prepare the PCR amplification reaction system, as shown in Table 2. The prepared PCR reaction system was placed on an Easy Cycler 96 PCR amplification instrument for reaction, and the PCR amplification reaction program is shown in Table 3. The PCR amplification products were stored at 4℃ and detected by 1w / v% agarose gel electrophoresis. The results are shown in Table 3. Figure 7 As shown.

[0024] Table 2 PCR amplification reaction system

[0025] Table 3 PCR amplification reaction procedure

[0026] The results are as follows Figure 7 The image shows the agarose gel electrophoresis results of six sets of β-galactosidase gene primer pairs. Figure 7 It was found that among all the primer pairs tested, only the fifth primer pair produced a single, bright, specific band at approximately 630 bp, while the other primer pairs failed to amplify the target band at the expected position. Therefore, the fifth primer pair was determined to be the preferred primer pair for subsequent experiments.

[0027] (3) Primer universality verification To verify the universality of the fifth primer pair for samples from different sources, fecal genomic DNA from rats, mice, and humans extracted in step (1), and environmental soil genomic DNA were used as templates. TaKaRa Ex Taq HS, dNTP Mixture, the six primer pairs obtained in Example 1, and 10×Ex Tag Buffer were added and mixed with ddH2O to prepare the PCR amplification reaction system, as shown in Table 2. The prepared PCR reaction system was placed on an EasyCycler 96 PCR amplification instrument for reaction, and the PCR amplification reaction program is shown in Table 3. The PCR amplification products were stored at 4℃ and detected by 1w / v% agarose gel electrophoresis. The results are shown in Table 3. Figure 8 As shown.

[0028] The results are as follows Figure 8 The image shows the agarose gel electrophoresis results of environmental samples using the β-galactosidase gene primer pair. Clear target bands were successfully amplified at 630 bp in samples from four different sources: rats, mice, humans, and soil, indicating the broad applicability of the fifth primer pair.

[0029] (4) Cloning, sequencing and sequence verification To confirm the accuracy of the amplified products, all samples were gel-extracted using the fifth primer pair to amplify the 630 bp band. The band was then purified using a universal DNA purification kit (DP204-2). The target fragment was transfected into *E. coli* DH5α receptor cells, positive clones were screened, and sequencing was performed at Shanghai Sangon Biotech Co., Ltd.

[0030] Experimental Example 2 To verify the correctness of the amplified sequence of the fifth primer pair of this invention and to evaluate its broad-spectrum identification of lactose-degrading bacteria, bioinformatics and phylogenetic analysis was performed on the sequencing product of Shanghai Sangon Biotech Co., Ltd. in step (4) of Experiment Example 1. The specific experimental steps are as follows: (1) Sequence alignment and reference sequence acquisition: All nucleotide sequences obtained from sequencing by Shanghai Sangon Biotech Co., Ltd. were analyzed online using the BLASTX tool on the NCBI database website. The reference nucleotide sequence with the highest comprehensive score for each sequence was retrieved and downloaded for subsequent alignment.

[0031] (2) Phylogenetic tree construction: The nucleotide sequences obtained from sequencing by Shanghai Sangon Biotech Co., Ltd., along with the downloaded reference sequences, were imported into MEGA 11.0.13 software. First, multiple sequence alignment and editing were performed using the CLUSTALW program. Then, based on the aligned sequences, a phylogenetic tree was constructed using the neighbor-joining method to determine the taxonomic position of the amplified sequences. The phylogenetic tree analysis results are as follows: Figure 9 , Figure 10 As shown.

[0032] The cloning and sequencing yielded 200 sequences. After alignment with the reference sequence obtained via NCBI's BlastX algorithm, 177 specific gene sequences were identified, accounting for 88.5%. The phylogenetic tree constructed from the rat, mouse, and human fecal microbiota sequences identified by the fifth primer pair and the reference sequence is shown below. Figure 9 As shown. The phylogenetic tree constructed from the soil microbial community sequences identified by the fifth primer pair and the reference sequence is shown below. Figure 10As shown in the figure, the fifth primer pair identified 8 phyla in the kingdom Bacteria (Acidobacteriota, Armatimonadota, Bacillota, Bacteroidota, Candidatus Binatota, Planctomycetota, Pseudomonadota, Verrucomicrobiota), 12 classes in the kingdom Blastocatellia, Candidatus Binatia, Chitinophagia, Chthonomonadia, Clostridia, Fimbriimonadia, Gammaproteobacteria, Bacteroidia, Sphingobacteriia, Terriglobia, unclassified Planctomycetota, Verrucomicrobiia), and 16 orders in the kingdom Bacteroidales, Blastocatellales, Candidatus, etc. Acidoferrales, Chitinophagales, Chthonomonadales, Eubacteriales, Fimbriimonadales, Lachnospirales, Lysobacterales, Limisphaerales, Sphingobacteriales, Terriglobales, unclassified Candidatus Binatia, unclassified Planctomycetota, unclassified Terriglobia, unclassified Verrucomicrobiia,19 bacterial families, Acidobacteriaceae, Acutalibacteraceae, Bacteroidaceae, Chitinophagaceae, Chthonomonadaceae, Lachnospiraceae, Lysobacteraceae, Oscillospiraceae, Prevotellaceae, Pyrinomonadaceae, Sphingobacteriaceae, Tannerellaceae, unclassified Candidatus Binatia, unclassified Fimbriimonadales, unclassified Limisphaerales, unclassified Planctomycetota, unclassified Sphingobacteriales, unclassified Terriglobia, unclassified Verrucomicrobiia, 28 genera, Alloprevotella , Bacteroides , Blautia , Candidatus Acidoferrum , Edaphobacter , Lysobacter , Mucilaginibacter , Parabacteroides , Paracidobacterium , Phocaeicola , Pinibacter , Puia , Ruminococcus , Terracidiphilus , Tyzzerella , unclassified Acutalibacteraceae , unclassified Bacteroidaceae , unclassified Candidatus Binatia , unclassified Chitinophagaceae , unclassified Chthonomonadaceae , unclassified Fimbriimonadales , unclassified Limisphaerales , unclassified Oscillospiraceae , unclassified Planctomycetota , unclassified Pyrinomonadaceae , unclassified Sphingobacteriales , unclassified Terriglobia , unclassified Verrucomicrobiia that can degrade lactose.

[0033] In summary, the specific primer pairs designed in this invention can effectively detect complex samples from various sources, including rat, mouse, and human feces and soil, via PCR amplification. Phylogenetic analysis confirmed that these primer pairs can identify lactose-degrading bacteria covering 8 phyla, 12 classes, 16 orders, 19 families, and 28 genera, exhibiting high species diversity. This fully demonstrates that the primers provided by this invention possess broad-spectrum and high-specificity characteristics, making them suitable for the efficient identification and diversity analysis of lactose-degrading functional bacteria in different ecological environments.

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A specific primer for identifying lactose-degrading bacteria, characterized in that, The specific primer is a specific primer targeting the β-galactosidase gene of glycosidase family 35; its forward nucleotide sequence is shown in SEQ ID NO.3, and its reverse primer nucleotide sequence is shown in SEQ ID NO.

5.

2. A method for identifying lactose-degrading bacteria for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested to obtain a DNA template; (2) The DNA template obtained in step (1), TaKaRa Ex Taq HS, dNTP Mixture, the specific primers described in claim 1, and 10×Ex Taq Buffer are mixed with ddH2O to prepare a PCR amplification reaction system. (3) Place the PCR amplification reaction system prepared in step (2) into a PCR amplification instrument for reaction and detect it by agarose gel electrophoresis.

3. The method according to claim 2, characterized in that, The sample to be tested in step (1) is any one of rat feces, mouse feces, human feces or soil.

4. The method according to claim 2, characterized in that, In step (2), the PCR amplification reaction system contains 1-3 μL of DNA template, 0.20-0.30 μL of Ex Taq HS, 3-5 μL of dNTP Mixture, 1-3 μL of primers, 4-6 μL of 10× Ex Taq Buffer, and 35-38 μL of ddH2O, for a total reaction volume of 50 μL.

5. The method according to claim 2, characterized in that, In step (3), the concentration of agarose is 1-2% (w / v).

6. The application of the specific primer for identifying lactose-degrading bacteria according to claim 1 in the preparation of a kit for identifying lactose-degrading bacteria.

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