High-throughput sequencing-based detection primer and method for cyphellophora and application

Abstract

The application discloses a high-throughput sequencing-based detection primer and method for Stemonitis and application, and belongs to the technical field of microorganism detection. The technical problem to be solved is that there is a lack of a method for detecting Stemonitis resources in the environment, which is high in specificity, sensitivity, coverage of species and efficiency. The technical solution provides a primer group for detecting Stemonitis, and the primer group is based on high-throughput sequencing. The primer group can be used for efficiently and rapidly detecting Stemonitis resources in the environment and evaluating the relative abundance of Stemonitis resources. The primer group is high in specificity and sensitivity, and has great application potential in the detection of Stemonitis.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, and relates to *Slimecium stenoptera*, specifically to primers, methods and applications for detecting *Slimecium stenoptera* based on high-throughput sequencing. Background Technology

[0002] Dictyostelid cellular slime molds (or simply dictyostelids) belong to the Protist kingdom and are widely distributed in various types of soils, including forests, grasslands, tundra, polar regions, and farmlands, as well as in fallen leaves and branches. Their distribution ranges from tropical to frigid zones, exhibiting a global distribution.

[0003] The life cycle of *Dystomium repens* includes stages such as spore germination, aggregation of myxomorphs, formation of pseudoprotoplasmic masses, and development of spore-bearing fruits. It is divided into asexual vegetative growth stages and sexual reproduction stages. Both the vegetative body and the fruiting body are extremely small, approximately 70 μm to 20 mm, making them difficult to observe directly with the naked eye. In laboratory cultivation, observation and identification require instruments such as optical microscopes. When environmental conditions (moisture, temperature, pH) are unsuitable, the amoebas of *Dystomium repens* will form spherical, thick-walled structures called "microcysts" to resist the adverse conditions.

[0004] Dictyophora are important model organisms in eukaryotes. During their vegetative growth stage, most Dictyophora species feed on environmental bacteria, while a small number evolve a primitive "cultivation" behavior by capturing, carrying, and "seeding" bacteria. In *Dictyophora discoidea* (… Dictyostelium discoideum In this study, about one-third of the wild-type *Dendrobium distichum* exhibit this behavioral pattern. After capturing bacteria, the vegetative body engulfs and digests only a portion, preserving the rest. As *Dendrobium distichum* develops, the preserved bacteria are transported to the top of the *Dendrobium distichum*, where they live in symbiosis with the spores within the *Dendrobium distichum* spore mass. When mature *Dendrobium dispersal* spores, the preserved bacteria are also dispersed into the environment. When bacteria are scarce in the environment, the dispersed bacteria can provide nutrients for newly germinating *Dendrobium distichum* spores.

[0005] Dystosomes are important soil protozoa, and their abundance is a key indicator of ecosystem stability. Their predation and utilization of bacteria directly affect the bacterial abundance in their growth substrates. They play a crucial role in regulating the dynamic balance of microbial communities in soil and other environments, acting as key players in soil material cycling and energy exchange. They have significant application potential in assessing soil health, evaluating harmful environmental microorganisms, and implementing biological control.

[0006] Although *Dermatophytes* is distributed worldwide, only 178 species have been reported to date, leaving a large number of species awaiting discovery by researchers. Current research on *Dermatophytes* diversity largely relies on traditional methods such as field soil sample collection, laboratory culture, and microscopic comparison. This extensive laboratory work significantly delays the isolation, identification, diversity analysis, and microbial community interaction studies of *Dermatophytes*, and makes it difficult to obtain precise data on *Dermatophytes* diversity in natural environments. Therefore, developing rapid and effective methods to detect the presence of *Dermatophytes* resources in soil and the environment, and further assessing the relative abundance of these resources, is essential for integrating *Dermatophytes* research into the era of big data.

[0007] Currently, most species classification of *Dermatophytes* relies on molecular systematics, constructing phylogenetic trees based on 18S rRNA sequence data and classifying families and genera according to the similarity of different 18S fragments. However, commonly used universal primers for eukaryotes have poor specificity for *Dermatophytes*, resulting in low amplification efficiency and difficulty in detecting *Dermatophytes* in the environment. Currently, both domestically and internationally, the method of isolating and culturing *Dermatophytes* is still used to study its diversity. These methods require sufficient *Dermatophytes* DNA, which must be amplified by PCR and first-generation sequencing to obtain the *Dermatophytes* gene sequence. The experimental cycle is long, and the composition of the *Dermatophytes* community after culture is easily altered, introducing human interference to ecological and biogeographical studies of *Dermatophytes* and affecting the research results.

[0008] High-throughput sequencing technology, as a relatively mature detection technology, has been widely used in the detection of pathogens in humans, animals and plants, and has played an important role in the diagnosis of diseases in food crops and economic crops. However, no high-throughput detection method for slime molds in the environment has been reported so far.

[0009] Relevant patent documents retrieved: This document, published in China (CN117025811A) on November 10, 2023, discloses a pair of primers for the rapid detection of wild-type slime molds in soil. The steps include: Step 1: Illumina sequencing experimental procedure; Step 2: Genomic DNA quality control; total DNA quality control was performed using a Thermo NanoDrop 2000 UV micro-spectrophotometer and 1% agarose gel electrophoresis; Step 3: Primer design and synthesis: 18S rDNA amplification of selected regions, using universal primers D542F and D1340; Step 4: Sequencing of PCR products. This primer pair has poor specificity and low sensitivity, making it unsuitable for efficient assessment of soil microbial community diversity.

[0010] Relevant non-patent literature retrieved: The journal or book title is *Journal of Jilin Agricultural University*, and the article title is "Molecular Systematic Study of *Slimecium reticulatum* in China," volume number 41, published in 2019. This article discloses a taxonomic study of *Slimecium reticulatum* in my country based on ITS and 18S rRNA gene fragments. Eleven species of *Slimecium reticulatum* were used as research subjects. Multiple gene fragments were amplified and analyzed, and a phylogenetic tree was constructed. For DNA PCR, two gene fragments, ITS and 18S rRNA, were selected, with primer sequences of the commonly used primers ITS1, ITS4, NS1, and NS8, respectively. A total of 42 gene sequences were obtained, including 26 ITS gene sequences and 16 18S rRNA gene sequences. The phylogenetic tree shows that molecular systematics methods have high accuracy in species identification, but the phylogenetic relationships are not entirely consistent with morphological taxonomy.

[0011] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: Chinese patent CN117025811A uses a single primer pair and only performs 18S analysis on soil, resulting in poor specificity and low sensitivity. The study "Molecular Systematics of Slimecarpa in China" used 11 species of Slimecarpa as research subjects, employing universal primers ITS1, ITS4, NS1, and NS8 for analysis. However, the resulting phylogenetic relationships did not fully align with morphological taxonomy, indicating poor specificity. Furthermore, the V4 and V9 region amplicon primer pairs, commonly used for protozoa, exhibit low amplification efficiency for Slimecarpa in the environment. Due to the conservatism of primer positions, the amplified fragments are only suitable for annotation at the order level, making them unsuitable for genus-level differentiation. Therefore, the diversity and specificity of Slimecarpa often render existing primers insufficient for their amplification needs, necessitating the development of more efficient primers and amplification methods to improve the amplification efficiency of Slimecarpa. Summary of the Invention

[0012] The purpose of this invention is to provide: A primer set for detecting slime mold cells with reticulate stems and related technologies are provided to address the technical problems, such as the lack of a method or combination thereof in the prior art that is highly specific, sensitive, covers a wide range of species, and is efficient and rapid for detecting slime mold resources with reticulate stems in the environment.

[0013] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0014] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0015] The definition of standard chemical terms can be found in the reference "Guidelines for Validation and Verification of Microbial Detection Methods" (State Administration for Market Regulation, 2020-12-01).

[0016] Unless otherwise stated, conventional methods within the scope of the art, such as DNA extraction and DNA sequencing, shall be used.

[0017] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0018] The term "primer" refers to a short nucleic acid sequence (usually DNA or RNA), typically 15-30 nucleotides in length, that plays a crucial role as the starting point for nucleic acid synthesis in molecular biology techniques. Its core function is to provide the 3-OH terminus for DNA polymerase to initiate synthesis, guiding the enzyme to extend the new nucleic acid chain along the template strand.

[0019] The primers provided by this invention contain degenerate bases, and the identification of degenerate bases strictly follows the IUPAC standard. A, C, G, T, and U represent single non-degenerate bases, suitable for marking conserved sites in sequences; R represents two purine bases (A / G), Y represents two pyrimidine bases (C / T), M represents two amino bases (A / C), K represents two keto bases (G / T), S represents two strongly paired bases (G / C), and W represents two weakly paired bases (A / T). These symbols are often used for describing codon degeneracy, labeling primer degenerate sites, and analyzing common sequences; B represents C / G / T (not A), D represents A / G / T (not C), H represents A / C / T (not G), and V represents A / C / G (not T). These symbols are commonly used for labeling specific non-single base types in sequencing data; N represents any of the four bases (A / C / G / T).

[0020] The term "upstream primer" refers to a short oligonucleotide sequence that binds complementary to the non-coding strand (antisense strand) of the target DNA template during a PCR reaction, located at the 5' end of the target region. Its function is to guide DNA polymerase to synthesize a new DNA strand from the 5'-3' direction, initiating at the upstream boundary of the target region.

[0021] The term "PCR" stands for Polymerase Chain Reaction, a technique for amplifying specific DNA fragments in vitro. It achieves exponential amplification of DNA fragments through a cycle of high-temperature denaturation, low-temperature annealing, and temperature-appropriate extension.

[0022] The term "amplification" refers to the process of significantly increasing the copy number of a specific nucleic acid fragment through biotechnology. In molecular biology, the most common amplification method is PCR, but other techniques (such as rolling circle amplification) are also included.

[0023] The term "reagent kit" refers to a standardized product that combines various experimental materials and reagents for a specific detection, analysis, or experimental procedure. This includes: molecular biology reagent kits (such as PCR kits), immunoassay kits (such as ELISA kits), and clinical diagnostic kits (such as novel coronavirus nucleic acid detection kits).

[0024] The term "biofilm sample" refers to an environmental sample collected from the natural environment, artificial facilities, or experimental systems, with microbial biofilms as the primary research object. Microbial biofilms are complex, structured community aggregates formed at interfaces by various microorganisms, including bacteria, fungi, algae, and protozoa, and their secreted extracellular polymers (such as polysaccharides, proteins, and nucleic acids). They are widely found on the surfaces of aquatic sediments, rocks, plant roots, soil particles, pipe walls, and the surfaces of plants and animals. Unlike free-floating microorganisms, microorganisms within biofilms exhibit a highly aggregated state, stable community structure, and synergistic metabolic functions, playing a crucial role in environmental material cycling, pollutant degradation, and ecosystem stability. The collection, nucleic acid extraction, and high-throughput sequencing analysis of biofilm samples can more accurately reflect the community composition, diversity, and functional characteristics of microorganisms and protozoa (such as *Dystomata*) in the interfacial microenvironment, making them an important sample type for environmental microbial ecology research.

[0025] The term "high-throughput sequencing" refers to a technology capable of simultaneously sequencing massive amounts of nucleic acid molecules in parallel, obtaining hundreds of thousands to millions of sequence information at once. It is characterized by high throughput, high speed, and low cost. This technology has been widely applied in research on environmental microbial community composition, species diversity, and functional gene mining. It can directly sequence and analyze total DNA in environmental samples without relying on isolation and culture, thus accurately reflecting the community structure of organisms in the natural environment. In protist and soil microbial diversity research, high-throughput sequencing can effectively overcome the shortcomings of traditional isolation and culture methods, such as long cycles, susceptibility to distortion, and low coverage. For specific methods, please refer to Goodwin S, et al. Coming of age: ten years of next-generation sequencing technologies[J]. Nature Reviews Genetics, 2016.

[0026] The term "alpha diversity analysis" refers to the species richness and evenness within a single ecosystem or sample, used to describe the complexity of the biological community in a single sample. Alpha diversity analysis comprehensively assesses the number, abundance distribution, and phylogenetic relationships of species within a sample through a series of indices, and is the most fundamental analytical content in environmental microbiology and protobiodiversity research. For soil protozoa such as *Dystropharia*, alpha diversity can directly reflect the level of diversity within the community under different habitats and treatments, providing quantitative indicators for evaluating soil health and ecosystem stability.

[0027] The term "alpha diversity dilution curve" is a curve plotted with the amount of sequencing data on the x-axis and the number of observed species / ASVs on the y-axis. It is used to determine whether the sequencing depth is sufficient to cover the vast majority of biological groups in a sample. When the curve flattens out and approaches saturation, it indicates that the amount of sequencing data is sufficient to reflect the true diversity of the sample relatively completely; if the curve still shows a significant upward trend, it indicates that the sequencing depth is insufficient, and a large number of groups remain undetected. The dilution curve is a key basis for evaluating the reliability of high-throughput sequencing data and whether it meets the requirements for subsequent diversity analysis. ASVs, or amplicon sequence variations, are precise molecular sequences obtained by denoising and clustering high-throughput amplicon sequencing sequences based on single-nucleotide precision. They can distinguish sequence variations differing by only one base. Compared to traditional OTUs (Operational Taxonomic Units) clustered with 97% similarity, ASVs offer higher resolution, more stable results, and cross-study comparisons, making them more suitable for distinguishing closely related species, infraspecific units, and cryptic species. In the study of protists such as Dystrophiae, which are small in size and have many closely related species, ASV can more realistically and accurately reflect the species composition and diversity in the environment.

[0028] The term "alpha diversity box plot" is a statistical graph used to visually display the distribution characteristics of alpha diversity indices across multiple sample groups. It can present information such as the median, upper and lower quartiles, extreme values, and outliers. Box plots allow for rapid comparison of the overall distribution, dispersion, and significance of alpha diversity indices among different groups (e.g., habitat, geographic gradient, treatment groups), providing a visual basis for determining whether significant differences exist in community diversity between groups. In studies of *Dendrobium* diversity, box plots are commonly used to illustrate differences in indicators such as the Shannon index and Faith phylogenetic diversity among different soil samples.

[0029] The term "Shannon index" is a classic alpha diversity index that considers both species richness and evenness. A higher index value indicates a greater number of species, a more even distribution of abundance, and a more complex and stable community structure. In *Dendrobium* community studies, the Shannon index can be used to compare the overall diversity level of *Dendrobium* communities under different soil types, geographical regions, or environmental stresses, objectively reflecting differences in community complexity and stability.

[0030] The term "Faith phylogenetic diversity" is an alpha diversity index calculated based on phylogenetic trees. It reflects the evolutionary differences and genetic diversity of species within a community by summing the branch lengths of all corresponding branches for each species within a sample. Unlike traditional indices that only consider species abundance, phylogenetic diversity indices emphasize the evolutionary relationships between taxa, providing a more comprehensive measure of community phylogenetic diversity. In protist research, such as *Dystrophia*, the Faith phylogenetic diversity index can reflect the evolutionary diversity of *Dystrophia* groups in different environments, providing a more comprehensive basis for assessing their phylogenetic resources and ecological functions.

[0031] The term "Shannon entropy" is synonymous with the Shannon index, essentially an informatics indicator used to measure the disorder, diversity, or uncertainty of a system. In biodiversity analysis, a higher Shannon entropy indicates a more complex and evenly distributed species composition within the community. Applying Shannon entropy to the analysis of protozoan communities such as *Dystropharia* can quantify the diversity level of communities in environmental samples, reveal the changing patterns of community structure under different habitats and disturbance conditions, and provide an entropy-based indicator for ecosystem health assessment.

[0032] The term "SNP, Single Nucleotide Polymorphism" refers to variations such as base substitutions, insertions, or deletions at a single nucleotide site in the genome. It is the most abundant and stable genetic marker in biological populations. SNP polymorphisms can be used for species identification, population genetic structure, evolutionary relationships, and adaptive analysis. In the molecular ecology of *Dermatophytes*, SNP polymorphism analysis can reveal the levels of genetic variation in different geographical populations and habitats, providing high-resolution molecular evidence for species classification, population differentiation, and the study of environmental adaptation mechanisms.

[0033] Amoebozoa is a group of eukaryotic organisms including slime molds with reticulate cell-like structures. NCBIGenBank contains a large number of gene sequences from protists, microorganisms, and plants and animals. Blast alignment of high-throughput sequencing-obtained 18S rRNA sequences of *Amoebozoa* with published *Amoebozoa* sequences in GenBank enables taxonomic annotation, species classification, and homologous sequence retrieval. This method is a core reference tool for the molecular identification, diversity analysis, and discovery of new taxa in *Amoebozoa*.

[0034] To achieve the above objectives, the present invention provides the following technical solution: On one hand, the present invention provides a primer set for the detection of slime molds with reticulate stems, the primer set comprising SEQ ID NO.1-9.

[0035] Preferably, the primer set further comprises SEQ ID NO.10 and SEQ ID NO.11.

[0036] On the other hand, the present invention provides a method for detecting slime molds with reticulate stems, using the above-mentioned primer set for detection.

[0037] Preferably, the method includes the following steps: Genomic DNA was extracted from the sample to be tested, and the genomic DNA was amplified by PCR using the primer set to obtain PCR products. Sequencing was performed using the PCR products as templates.

[0038] Preferably, the sample to be tested is selected from one or more of biofilm samples, water samples, and soil samples.

[0039] Preferably, the PCR amplification of genomic DNA using the primer set includes the following steps: first, the genomic DNA is amplified for the first time using SEQ ID NO.10 and SEQ ID NO.11 to obtain the first amplification product, and then the first amplification product is amplified using SEQ ID NO.1-9 to obtain the PCR product.

[0040] Preferably, the reaction program for PCR amplification includes: 95℃ for 3 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 45 s, 20 cycles; 72℃ for 10 min.

[0041] On the other hand, the present invention provides a kit for microbial detection, the kit comprising the primer set described above.

[0042] Preferably, the kit further includes DNA polymerase and DNA extraction reagent.

[0043] On the other hand, the present invention provides the application of the above-described primer set, the above-described method, or the above-described kit in microbial detection.

[0044] The present invention has at least the following beneficial effects: 1. High specificity: This invention designs 9 specific primers targeting the 18S sequence of 12 genera of *Dendrobium* as the target gene. The amplification regions of these primers include conserved and specific regions, enabling efficient isolation of *Dendrobium* from complex samples such as environmental DNA. 2. High sensitivity: The detection sensitivity of this invention for the 18S rRNA gene fragment of *Dendrobium nobile* in the environment can reach 11,746 read pairs at the DNA level, which is highly sensitive. 3. High efficiency and speed of detection: The detection method of this invention can perform high-throughput detection of a large number of soil and other environmental samples in a short time. In contrast, traditional laboratory isolation methods require several days or even weeks of culture to obtain a sample of soil containing *Dendrobium*. The method described in this invention significantly shortens the operation time and is convenient and fast. 4. High applicability and low cost: This invention uses second-generation amplicon high-throughput sequencing technology, which has advantages such as maturity, accurate data, high market acceptance, and low price; In summary, high-throughput detection methods have advantages such as high specificity, high sensitivity, simple operation, and low detection cost. They can be used to efficiently assess soil microbial community diversity, providing a scientific basis for regulating soil health and controlling harmful microorganisms. They also have important guiding significance, broad application prospects, and economic benefits. Attached Figure Description

[0045] Figure 1The images show the agarose gel electrophoresis results of genomic DNA and PCR amplification products from different samples in Example 1; the top image is the genomic DNA electrophoresis image, and the bottom image is the PCR amplification product electrophoresis image; M: DNA marker (5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp), BC: blank control, 1-3: soil samples, 4-6: water samples, 7-9: biofilm samples.

[0046] Figure 2 This is a schematic diagram of the nested PCR amplification protocol in Example 3.

[0047] Figure 3 The annotation results of amplicon high-throughput sequencing of *Dendrobium reticulosum* ASVs for 16 different samples in Example 3 are shown, corresponding to the genus-level classification of *Dendrobium reticulosum* ASVs. Each taxonomic group is represented by a single color; among them, the "HY" sample corresponds to blank data, indicating that *Dendrobium reticulosum* ASVs were not detected in the sample.

[0048] Figure 4 The annotation results of amplicon high-throughput sequencing of *Dendrobium truncatum* ASVs for 16 different samples in Example 3 are shown. The species classification of *Dendrobium truncatum* ASVs is shown, with each taxonomic group represented by a different color. The "HY" sample corresponds to blank data, indicating that *Dendrobium truncatum* ASVs were not detected in the sample.

[0049] Figure 5 The dilution curves for the α diversity of *Dendrobium tumefaciens* 18S amplicon sequencing in Example 3 are shown, with each quadrat represented by a separate color.

[0050] Figure 6 This is a box plot of α-diversity for *A. reticulatus* ASV obtained by amplicon high-throughput sequencing in Example 3; where A represents the Shannon entropy analysis results of α-diversity for different samples, and B represents the Faith phylogenetic diversity analysis results for different samples; where n represents the number of sample replicates for each group in the statistics; where Jianfengling non-protected area includes samples from groups HBA and HBI; Jianfengling protected area includes samples from groups HY, HS, HN, HC, and HH; Shennongjia protected area includes samples from groups BF, BN, BC, BH, and BZ; Changbaishan non-protected area includes samples from groups K and Z; and Changbaishan protected area includes samples from groups Y1 and H. Detailed Implementation

[0051] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0053] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0054] Example 1: Primer set and detection method By reviewing the published ribosomal sequences of *Slimecium cytomorphum* from GenBank, BLAST alignment analysis was performed to compare the polymorphism of different ribosomal gene fragments at the species level, and SSU rDNA was selected as the target sequence for designing high-throughput sequencing primers. Organized by genus, the SSU sequences of all species within the genus were assembled using SeqMan software to form a ensemble sequence covering multiple SNP sites. Dictyostelium caveatum Using the full-length 18S rRNA sequence (GenBank: AM168077) as a reference, the assembly sequences of each genus were compared using Mega software to screen for conserved and variable regions.

[0055] Based on the read length of second-generation high-throughput sequencing, a *Dendrobium*-specific primer set was designed using Primer Premier 5 software near alignment sites 481 and 958. The PCR detection primers consisted of upstream primer 18SF1, downstream primer 18SR1; upstream primer 18SF2, downstream primers 18SR2, 18SRa, 18SRb, 18SRc; and upstream primer 18SF3, downstream primer 18SR3. The nucleotide sequences of each primer are shown in Table 1.

[0056] Table 1. Primer combinations for high-throughput sequencing of *Slimecium styli*.

[0057] A primer set for *Dendrobium* PCR that can be applied to amplicon high-throughput sequencing includes nucleotide sequences as described above. The PCR detection primers consist of upstream primer 18SF1, downstream primer 18SR1; upstream primer 18SF2, downstream primers 18SR2, 18SRa, 18SRb, 18SRc; upstream primer 18SF3, downstream primer 18SR3.

[0058] The PCR kit for detecting *Dendrolimus* includes the primer combination listed in Table 1, Premix Taq enzyme, and ddH2O.

[0059] The method for detecting *Dendrolimus* in environmental samples using the above primers via PCR and amplicon high-throughput sequencing is as follows: (1) Extract genomic DNA from the test sample, which includes environmental samples such as biofilms, water bodies, and soil.

[0060] (2) Using the above genome as a template, PCR amplification was performed using the *Dendrolimus* detection kit described above. The amplification conditions were: 95℃ for 3 min; (95℃ for 30 s, 55℃ for 30 s, 72℃ for 45 s), 20 cycles; 72℃ for 10 min. The amplification reaction system included the following reagents and quantities: Premix Taq (Ex Taq) TM 25 µL of (Version 2.0 plus dye) primer solution, 1 µL of primer solution (each primer concentration is 10 μM), 1 µL of template DNA, and the remaining volume is made up with ddH2O, for a total of 30 µL; replace 1 µL of template DNA with 1 µL of ddH2O as a blank control.

[0061] (3) Amplicon high-throughput sequencing was performed using PCR products as templates.

[0062] The results of agarose gel electrophoresis of extracted genomic DNA and PCR amplification products are as follows: Figure 1 As shown, the PCR amplification products of different analytes all showed bands ranging from 254 bp to 510 bp in agarose gel electrophoresis, while the blank control showed no reaction band. Sequencing and BLAST comparison of the PCR amplification fragments confirmed that all amplified fragments were fragments of the *Dermatophyte* 18S rRNA target gene. This demonstrates that the primer set and kit provided by this invention for detecting *Dermatophyte* resources in environmental samples can clearly detect *Dermatophyte* resources in environmental samples, exhibiting high specificity and the advantage of not producing false positive results. Furthermore, there is no cross-amplification reaction with bacteria or fungi in the environment.

[0063] Example 2: Sample Detection The primer amplification efficiency was tested in 35 species of *Dendrobium* belonging to 5 genera and 4 families. Using the primer combinations and detection method described in Example 1, 32 species of *Dendrobium* belonging to 5 genera and 4 families were amplified. The results are shown in Table 2. The amplification efficiency of the 18SF1 / 18SR1 primer combination was 77%, while the 18SF2 / 18SRc and 18SF3 / 18SR3 combinations were above 65%. The other three combinations could further supplement the insufficient amplification caused by SNP polymorphism, indicating that the detection primers provided by this invention have high specificity and good detection effect.

[0064] Table 2. PCR amplification results of 35 *Dendrophyllus* samples with different primer combinations.

[0065] Note: "+" indicates that it was detected, and "-" indicates that it was not detected.

[0066] Example 3: Sample Detection In this embodiment, amplicon high-throughput sequencing was performed on *Dendrolimus* in the environment, following the primer set and detection method provided in Example 1.

[0067] Primers were applied to 16 samples (see Table 3) including soil, canopy soil, and humus from tropical, temperate, and northern forests. The specific method was as follows: 50g of topsoil and attached humus samples were collected from a depth of 5cm and placed in a resealable bag. 10g of soil was mixed with 90mL of sterile distilled water in a beaker and placed in a constant-temperature shaker at 23℃ and 280rpm / min for 2min. After shaking, the mixture was allowed to stand for 1h. The supernatant was separated from the soil samples, and genomic DNA was extracted using the DNeasyPowerMax Soil Kit. Nested PCR was performed using total soil DNA as a template (see schematic diagram of the amplification protocol). Figure 2 The first round of amplification was performed using primers 18SF-A (SEQ ID NO.10: 5'-AACCTGGTTGATCCTGCCAG-3') and 18SR-B (SEQ ID NO.11: 5'-TGATCCTTCTGCAGGTTCAC-3') (the PCR reaction system and procedure were the same as in Example 1). The first round amplification product was used as a template for the second round of PCR amplification using the detection primers in Table 1. The second round amplification product was used as a high-throughput sequencing template. Sequencing primers were synthesized according to the above primer sequences, and amplicon high-throughput sequencing was performed. The sequencing results were compared with ASV sequences and analyzed by bioinformatics in databases such as Genbank and Silva to obtain the relative abundance and diversity of *Dendrocalamus* in this soil sample.

[0068] Table 3 Soil samples and collection sites used for the detection primers

[0069] The results showed that amplicon sequencing of the collected soil samples yielded a total of 886,555 reads, with a minimum of 11,746 reads and a maximum of 27,071 reads per sample. Therefore, the detection limit for *Dystrophus* in the tested samples using this method is 11,746 reads. Through comparison and annotation with Amoebozoa data in the NCBI GenBank database, 274,224 entries of 1390 ASVs were obtained, including *Dystrophus*, *Myxobacteria*, and other amoebae. *Dystrophus* accounted for 136,341 entries, representing 137 ASVs, or 49.7% of the total obtained ASVs. The results for these 137 ASVs are as follows: Figure 3 and Figure 4 As shown, it includes 2 orders of stylomycetes: Stylomycetes and Stylomycetes; 4 families: Stylomycetes, Stylomycetes, Stylomycetes, and Stylomycetes; and 8 genera: Stylomycetes, Stylomycetes, Stylomycetes, Stylomycetes, Stylomycetes, Stylomycetes, and Stylomycetes.

[0070] Alpha-dilution curves were prepared based on *ASVs* of *Dendrocalamus*, and the results are as follows: Figure 5 As shown, the curve tends to flatten out when the sequencing depth is 339, and the α-diversity Shannon index analysis (see...) Figure 6 This indicates that the sequencing volume is sufficient to reflect the species diversity of *Dendrolimus* in the sample.

[0071] The PCR primers and amplicon high-throughput detection method provided by this invention clearly demonstrates that trace amounts of DNA in soil samples can be detected, ensuring detection accuracy while greatly shortening the time required to obtain results using traditional separation methods, improving the detection efficiency of *Dendrolimus* in the environment and reducing detection costs.

[0072] Comparative Example 1: Experiments with Other Primers Following the method in Example 3, the primer sets shown in Table 1 were replaced with the primer sets shown in Table 4 before the experiment was conducted.

[0073] Table 4 Primer Description

[0074] The results showed that primer combinations F1 and R1, based on Primer-blast results, could only be used for amplification. Acytostelium It belongs to the genus *Dendrolimus*; primer combination F2, R2, Ra, Rb, and Rc can amplify... Rostrostelium, Dictyostelium It belongs to the genus *Dendrolimus*, and primer combination F3 and R3 can amplify... Speleostelium The species are *Dystoidea*. Compared to the primer set provided in this invention (see Table 1), the replaced primer set can amplify fewer *Dystoidea* species, making it difficult to cover most *Dystoidea* species. Furthermore, the presence of numerous SNPs in the sequence fragments directly affects the amplification efficiency.

[0075] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A primer set for detecting slime molds with reticulate stems, characterized in that, The primer set comprises SEQ ID NO. 1-9.

2. The primer set according to claim 1, characterized in that, The primer set also includes SEQ ID NO.10 and SEQ ID NO.

11.

3. A method for detecting reticulate slime mold cells, characterized in that, The detection was performed using the primer set described in claim 1 or 2.

4. The method according to claim 3, characterized in that, Includes the following steps: Genomic DNA was extracted from the sample to be tested, and the genomic DNA was amplified by PCR using the primer set to obtain PCR products. Sequencing was performed using the PCR products as templates.

5. The method according to claim 4, characterized in that, The sample to be tested is selected from one or more of biofilm samples, water samples, and soil samples.

6. The method according to claim 4, characterized in that, The PCR amplification of genomic DNA using the primer set includes the following steps: first, the genomic DNA is amplified for the first time using SEQ ID NO.10 and SEQ ID NO.11 to obtain the first amplification product, and then the first amplification product is amplified using SEQ ID NO.1-9 to obtain the PCR product.

7. The method according to claim 4, characterized in that, The PCR amplification reaction program included: 95℃ for 3 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 45 s, 20 cycles; 72℃ for 10 min.

8. A kit for microbial detection, characterized in that, The kit contains the primer set as described in claim 1 or 2.

9. The reagent kit according to claim 8, characterized in that, The kit also includes DNA polymerase and DNA extraction reagents.

10. The use of the primer set according to any one of claims 1-2, the method according to any one of claims 3-7, or the kit according to any one of claims 8-9 in microbial detection.

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