Primer combination, kit and application for detecting cadmium-resistant helper metabolic genes in soil phage virus community

Abstract

The application provides a primer combination, a kit and an application for detecting cadmium-resistant helper metabolic genes in a soil phage virus community; the primer combination has phage specificity, cadmium specificity and classification according to cadmium resistance mechanisms, fills the technical gap of a special detection tool for phage helper metabolic genes and a special cadmium deep detection, and has important significance for analyzing the abundance and diversity of various cadmium-resistant helper metabolic genes in the soil phage virus community, analyzing the phage-mediated cadmium resistance transmission mechanism and establishing a cadmium pollution accurate evaluation system based on the phage community.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbial detection, and in particular relates to a primer combination, kit, and application for detecting cadmium resistance auxiliary metabolic genes in soil bacteriophage virus communities. Background Technology

[0002] Cadmium is characterized by its high mobility, biotoxicity, and tendency to accumulate in the food chain, posing a serious threat to soil ecological security and agricultural product quality. Soil microbial communities are a core biological component of cadmium pollution response; bacteria can acquire cadmium resistance through mechanisms such as efflux pumps, metal-binding proteins, and reductases, and related research is relatively mature. However, the ecological function of bacteriophages (bacterial viruses) under heavy metal stress has been severely underestimated. Under cadmium pollution stress, lysogenic bacteriophages carrying auxiliary metabolic genes (AMGs) significantly accumulate in the soil. These AMGs carried by bacteriophages can integrate into the host bacterial genome through lysogenic transformation, reprogramming host metabolism and significantly improving the survival and adaptation of bacteria under cadmium stress.

[0003] Patent document CN114525276A describes a high-throughput qPCR chip and its application in detecting microbial heavy metal resistance genes. It designs a primer set of 57 resistance genes for 9 heavy metals in microorganisms (mainly bacteria). However, the target of this patent document is total soil DNA (i.e., metagenomic DNA), which cannot distinguish whether the resistance genes originate from the bacterial genome or the bacteriophage genome. Since bacteriophage auxiliary metabolic genes have significant sequence differences from the homologous genes of host bacteria and have unique evolutionary paths, directly applying bacterial resistance gene primers cannot effectively cover the bacteriophage community. Therefore, this patent document focuses on bacteria as the main target and cannot specifically detect bacteriophage auxiliary metabolic genes.

[0004] Moreover, existing research lacks in-depth detection schemes for specific heavy metals. Most existing studies pursue "broad spectrum coverage" (simultaneous detection of multiple heavy metals), but lack systematic classification and detection capabilities for different resistance mechanisms (efflux, binding, reduction, etc.) of a single heavy metal, cadmium. This makes it impossible to provide high-resolution molecular tools for accurately assessing cadmium pollution levels and microbial community response patterns.

[0005] Therefore, it is necessary to provide a primer combination, kit, and application for detecting cadmium resistance auxiliary metabolic genes in soil bacteriophage virus communities, in order to solve the technical problem of how to accurately analyze the abundance and diversity of various cadmium resistance auxiliary metabolic genes in soil bacteriophage virus communities. Summary of the Invention

[0006] The main objective of this invention is to provide a primer combination, kit, and application for detecting cadmium resistance auxiliary metabolic genes in soil bacteriophage virus communities, in order to solve the technical problem of how to accurately analyze the abundance and diversity of various cadmium resistance auxiliary metabolic genes in soil bacteriophage virus communities.

[0007] To achieve the above objectives, the present invention provides a primer combination for detecting cadmium resistance auxiliary metabolic genes in soil bacteriophage virus communities, the primer combination comprising nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.74.

[0008] Furthermore, the primer combination targets cadmium resistance accessory metabolism genes including: acrD (efflux pump mechanism), cadA (efflux pump mechanism), cadB (efflux pump mechanism), cadC (regulatory system mechanism), cadR (regulatory system mechanism), cadX (regulatory system mechanism), cysH (reduction / transformation mechanism), cysI (reduction / transformation mechanism), cysJ (reduction / transformation mechanism), cysM (reduction / transformation mechanism), czcA (efflux pump mechanism), czcB (efflux pump mechanism), czcR (regulatory system mechanism), czrA (regulatory system mechanism), czrB (efflux pump mechanism), dsbA (reduction / transformation mechanism), mdtD (efflux pump mechanism), and efflux pump mechanism. The following are the mechanisms and their corresponding names: mntH (external pump mechanism), nccA (external pump mechanism), pbrA (external pump mechanism), perR (regulation system mechanism), smtB (regulation system mechanism), ziaR (regulation system mechanism), cadD (external pump mechanism), cdh (reduction / conversion mechanism), crnA (external pump mechanism), cysK (reduction / conversion mechanism), czcC (external pump mechanism), czcD (external pump mechanism), czcS (regulation system mechanism), dsbB (reduction / conversion mechanism), fieF (external pump mechanism), mdtA (external pump mechanism), merR (regulation system mechanism), smtA (binding / isolation mechanism), sseA (reduction / conversion mechanism), and ydaE (binding / isolation mechanism).

[0009] The present invention also provides the application of primer combinations as described above in the preparation of a kit for detecting cadmium resistance auxiliary metabolic genes in soil bacteriophage virus communities.

[0010] The present invention also provides a kit for detecting cadmium resistance co-metabolic genes in soil bacteriophage virus communities, the kit comprising any of the primer combinations described above.

[0011] Furthermore, the kit also includes multiplex PCR reagents other than the primer combination.

[0012] Furthermore, the kit also includes at least one of library construction reagents and sequencing reagents.

[0013] The present invention also provides the application of the kit described above in detecting cadmium resistance co-metabolic genes in soil bacteriophage virus communities.

[0014] Furthermore, the application includes the following steps: S1, which enriches virus particles in the soil; S2, using the DNA of the viral particles as a template, multiplex PCR amplification is performed using the primer combination to obtain PCR products; S3, after purifying the PCR product, library construction and high-throughput sequencing are performed; S4. After analyzing the sequencing data, the abundance and diversity of cadmium resistance auxiliary metabolic genes in the soil bacteriophage virus community were obtained.

[0015] Furthermore, before analyzing the data obtained after sequencing, primer sequence trimming and noise reduction are performed on the sequencing data.

[0016] Furthermore, during the analysis of the sequencing data, the sequencing data was compared and annotated with the phage cadmium resistance reference sequence database.

[0017] The beneficial effects of the present invention include at least the following: This invention develops a primer set that specifically distinguishes between bacteriophage and bacterial origins and classifies and detects them according to cadmium resistance mechanisms. This primer set is used to detect cadmium resistance auxiliary metabolic genes in soil bacteriophage virus communities. It can accurately analyze the abundance and diversity of various cadmium resistance auxiliary metabolic genes in soil bacteriophage virus communities, which is of great significance for elucidating the cadmium resistance transmission mechanism mediated by bacteriophages and establishing a precise assessment system for cadmium pollution based on bacteriophage communities.

[0018] The primer combination in this invention possesses technical characteristics such as phage specificity, cadmium specificity, and the ability to distinguish four major resistance mechanisms, filling the technological gap in dedicated detection tools for phage auxiliary metabolic genes and in-depth cadmium detection. Based on the primer combination provided by this invention, combined with detection methods such as virus particle enrichment, multiplex PCR amplification, and high-throughput sequencing (next-generation sequencing), this invention can also eliminate bacterial DNA interference at the source, ensuring that the detection signal truly originates from the phage community. Ultimately, it achieves high-throughput, high-specificity, and sequence-level single-base resolution detection, supporting research on soil cadmium pollution risk assessment, remediation effect monitoring, and phage ecological function.

[0019] This invention features a fundamental innovation in the detection target: For the first time, this invention combines virus particle enrichment with auxiliary metabolic gene detection, fundamentally solving the problem that the soil total DNA extraction method cannot distinguish between bacterial and bacteriophage-derived resistance genes, ensuring that the detection signal truly reflects the cadmium resistance potential of the bacteriophage community; this technical approach fills a long-standing technical gap in the field of environmental viral ecology.

[0020] This invention features a significant upgrade in detection dimensions: it abandons the existing approach of "broad coverage of all heavy metals" and focuses on the single heavy metal cadmium. It systematically classifies the target auxiliary metabolic genes according to four types of resistance mechanisms: efflux pump, binding / isolation, reduction / conversion, and regulation. The detection results of this invention can not only answer "whether there are cadmium resistance genes" but also "what mechanism they respond to cadmium stress," providing high-resolution molecular evidence for accurately assessing cadmium pollution levels and microbial community response patterns.

[0021] This invention represents a breakthrough in both detection throughput and information content: It employs multiplex PCR combined with high-throughput sequencing technology, which, compared to qPCR, not only allows for the simultaneous detection of multiple targets in a single tube reaction but also enables the acquisition of complete sequence information of the amplified products. This achieves the dual functions of accurate quantification of known genes and discovery of unknown sequence variations. Combined with multi-sample index pooled sequencing, it significantly improves detection throughput and cost-effectiveness.

[0022] This invention provides dual phage specificity: During primer design, a rigorous strategy of comparing positive sequences (phage helper metabolic genes) and negative sequences (bacterial homologous genes) ensures that the primer target region is unique to the phage genome. Furthermore, the invention enriches viral particles during detection, providing dual specificity at both the sequence and nucleic acid levels. Moreover, through a strategy of prioritizing specificity and then backing down to generality, this invention ensures comprehensive gene symbol coverage for the four major mechanisms of cadmium resistance, avoiding detection blind spots caused by limitations of existing databases, and specifically labels primers for this type.

[0023] This invention has broad application prospects: It can be widely applied to fields such as risk assessment of cadmium pollution in farmland soil, monitoring of soil remediation effects in mining areas, soil health assessment, and research on the ecological functions of bacteriophages, providing core technical support for elucidating the mechanism of heavy metal resistance transmission mediated by bacteriophages and establishing a pollution early warning system based on bacteriophage communities. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0025] Figure 1 This is a flowchart of the overall technical route of the present invention; in the figure, (a) corresponds to the sample pretreatment process; (b) corresponds to the primer design process; and (c) corresponds to the detection and analysis process. Figure 2 This is a phylogenetic tree of representative helper metabolic gene target sequences of the efflux pump system in Embodiment 1 of the present invention, showing the branching differences between bacteriophage sequences and bacterial homologous sequences; Figure 3 This is a phylogenetic tree of representative helper metabolic gene target sequences in the reduction / transformation system of Embodiment 1 of the present invention, showing the branching differences between bacteriophage sequences and bacterial homologous sequences; Figure 4 This is a phylogenetic tree of representative auxiliary metabolic gene target sequences of the regulatory system in Embodiment 1 of the present invention, showing the branching differences between bacteriophage sequences and bacterial homologous sequences; Figure 5 This is a phylogenetic tree of representative helper metabolic gene target sequences of the binding / isolation system in Embodiment 1 of the present invention, showing the branching differences between bacteriophage sequences and bacterial homologous sequences; Figure 6 This is an agarose gel electrophoresis image of the multiplex PCR products in Example 2 of the present invention.

[0026] The realization of the objective, functional characteristics and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0028] This invention provides a primer set for detecting cadmium resistance auxiliary metabolic genes in soil bacteriophage virus communities, the primer set comprising nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.74.

[0029] As a further explanation of the primer combination, the primer combination targets the following cadmium resistance auxiliary metabolic genes: efflux pump mechanism (acrD), efflux pump mechanism (cadA), efflux pump mechanism (cadB), regulatory system mechanism (cadC), regulatory system mechanism (cadR), regulatory system mechanism (cadX), reduction / transformation mechanism (cysH), reduction / transformation mechanism (cysI), reduction / transformation mechanism (cysJ), reduction / transformation mechanism (cysM), efflux pump mechanism (czcA), efflux pump mechanism (czcB), regulatory system mechanism (czcR), regulatory system mechanism (czrA), efflux pump mechanism (czrB), reduction / transformation mechanism (dsbA), and efflux pump mechanism (mdt). D, mntH of the external pump mechanism, nccA of the external pump mechanism, pbrA of the external pump mechanism, perR of the control system mechanism, smtB of the control system mechanism, ziaR of the control system mechanism, cadD of the external pump mechanism, cdh of the reduction / conversion mechanism, crnA of the external pump mechanism, cysK of the reduction / conversion mechanism, czcC of the external pump mechanism, czcD of the external pump mechanism, czcS of the control system mechanism, dsbB of the reduction / conversion mechanism, fieF of the external pump mechanism, mdtA of the external pump mechanism, merR of the control system mechanism, smtA of the binding / isolation mechanism, sseA of the reduction / conversion mechanism, ydaE of the binding / isolation mechanism.

[0030] The present invention also provides the application of primer combinations as described above in the preparation of a kit for detecting cadmium resistance auxiliary metabolic genes in soil bacteriophage virus communities.

[0031] The present invention also provides a kit for detecting cadmium resistance co-metabolic genes in soil bacteriophage virus communities, the kit comprising any of the primer combinations described above.

[0032] In some embodiments, the kit may also include multiplex PCR reagents other than the primer combination.

[0033] In some embodiments, the kit also includes at least one of library construction reagents and sequencing reagents.

[0034] The present invention also provides the application of the kit described above in detecting cadmium resistance co-metabolic genes in soil bacteriophage virus communities.

[0035] As an illustration of the application, the application includes the following steps: S1, enriching viral particles in the soil; wherein, enriching viral particles in the soil includes: viral particle elution, centrifugation to remove bacteria and impurities, filtration to remove bacteria, viral particle concentration, removal of free DNA, and extraction of viral DNA.

[0036] S2, using the DNA of the viral particles as a template, multiplex PCR amplification is performed using the primer combination to obtain PCR products; the multiplex PCR uses two reaction chambers, namely reaction chamber 1 and reaction chamber 2, reaction chamber 1 uses SEQ ID NO.1~SEQ ID NO.46 of the primer combination, and reaction chamber 2 uses SEQ ID NO.47~SEQ ID NO.74 of the primer combination.

[0037] S3, after purifying the PCR product, library construction and high-throughput sequencing are performed; wherein, after PCR product purification, sequencing libraries are prepared using a library construction kit compatible with Illumina or MGI platform, and index tags are added to different samples; sequencing is performed using the PE250 sequencing strategy.

[0038] S4. After analyzing the sequencing data, the abundance and diversity of cadmium resistance auxiliary metabolic genes in the soil bacteriophage virus community were obtained.

[0039] In some preferred embodiments, primer sequence trimming and noise reduction are performed on the sequencing data before analyzing the data obtained after sequencing.

[0040] As an explanation of the analysis process, this invention compares and annotates the data obtained after sequencing with the phage cadmium resistance reference sequence database during the analysis of the data obtained after sequencing.

[0041] The phage cadmium resistance reference sequence database mainly includes representative sequences of cadmium resistance-related auxiliary metabolic genes screened from multiple sources such as the BACMET2 database, RefSeq viral protein database, Pfam HMM model search, and keyword search. It covers representative reference sequences of 37 core target genes in four types of mechanisms: efflux pump system (acrD, cadA, cadB, cadD, cnrA, czcA, czcB, czcC, czcD, czrB, fieF, mdtA, mdtD, mntH, nccA, pbrA), binding / isolation system (smtA, ydaE), reduction / transformation system (cdh, cysH, cysI, cysJ, cysK, cysM, dsbA, dsbB, sseA), and regulatory system (cadC, cadR, cadX, czcR, czcS, czrA, merR, perR, smtB, ziaR).

[0042] The following is a further description of the present invention: See Figure 1To understand this invention, the core technical solution mainly consists of three inseparable parts: virus particle enrichment pretreatment, primer combination design classified according to cadmium resistance mechanism, and a detection system combining multiplex PCR and high-throughput sequencing, as detailed below: Firstly, sample pretreatment: This invention pre-enriches viral particles to ensure phage specificity.

[0043] Existing technologies typically employ direct extraction of total soil DNA (metagenomic DNA). However, since bacteriophage genomes are extremely small, usually 1 / 100th the size of a bacterial genome or even less, over 99% of the obtained DNA originates from bacteria and fungi, with bacteriophage DNA accounting for a very low percentage. Consequently, the majority of subsequent PCR amplification signals originate from bacterial resistance genes, failing to accurately reflect the abundance and diversity of auxiliary metabolic genes (AMGs) in the bacteriophage community.

[0044] This invention enriches viral particles by separating bacteriophage particles from the soil matrix and most bacterial cells before DNA extraction. The specific steps are as follows: (1) Virus particle elution: Take a fresh soil sample, add SM buffer (100mM NaCl, 8mM MgSO4·7H2O, 50mM Tris-HCl, pH 7.5), and vortex to elute the virus particles adsorbed on the surface of the soil particles. (2) Centrifugation to remove bacteria and impurities: Centrifuge at low speed (4℃, 3000×g, 10min) to remove soil particles and most bacterial cells, and collect the supernatant; (3) Filtration and sterilization: The supernatant is passed through 0.45μm and 0.22μm polyethersulfone (PES) filter membranes in sequence to further remove residual bacterial cells and collect the filtrate containing virus particles; (4) Virus particle concentration: The virus particles in the filtrate are concentrated by polyethylene glycol (PEG 8000) precipitation or ultracentrifugation. (5) Removal of free DNA: Add DNase I to the virus concentrate to digest and remove non-viral DNA adsorbed on the surface of virus particles or free in the solution; (6) Viral DNA extraction: Viral DNA was extracted and purified using a commercially available viral DNA extraction kit (such as QIAamp MinElute VirusSpin Kit) and used as a template for subsequent PCR amplification.

[0045] Through the above pretreatment process, bacterial DNA interference can be largely eliminated, ensuring that the detection signal specifically originates from the bacteriophage community.

[0046] Secondly, primer design: This invention designs primer combinations according to the classification of cadmium resistance mechanisms.

[0047] This invention does not simply design primers for "cadmium resistance genes," but rather, based on the molecular mechanisms of cadmium resistance, classifies target helper metabolic genes into four functional categories according to different resistance molecular mechanisms: efflux pump systems, binding / isolation systems, reduction / conversion systems, and regulatory systems, as shown in Tables 1-4 below, to achieve mechanism-specific detection. A three-tiered regression strategy is used to design primers for 37 core target genes.

[0048] In this invention, the efflux pump system refers to the system that actively transports Cd into cells. 2+ Protein systems that pump Cd out of the cell to reduce intracellular toxic concentrations; binding / isolation systems refer to the chelation of intracellular free Cd by cysteine-rich metallothioneins or other binding proteins. 2+ Protein systems that reduce the bioavailability of Cd, and reduction / conversion systems refer to those that promote Cd metabolism via sulfur metabolism pathways. 2+ Enzyme systems that form insoluble CdS precipitates or regulate redox states to alleviate Cd stress; regulatory systems refer to protein systems that regulate the expression of downstream resistance genes through metal-sensing proteins to determine the response time, intensity, and specificity of the Cd resistance system; the genes in Tables 1-4 refer to the set of core target genes used in the detection system of this invention, which were identified through literature review and bioinformatics analysis among the above four types of mechanisms.

[0049] Table 1. Information on the functional mechanism of cadmium resistance genes (efflux pump system) Table 2. Information on the functional mechanisms of cadmium resistance genes (binding / isolation system) Table 3. Information on the functional mechanism of cadmium resistance genes (reduction / transformation system) Table 4. Information on the functional mechanisms of cadmium resistance genes (regulatory system) The primer combinations of this invention are obtained through the following multi-source fusion and multi-level design strategies: (1) Multi-source acquisition and systematic classification of target gene sequences: First, keywords related to cadmium resistance were identified, and preliminary target protein seed sequences were obtained by searching public databases such as BACMET2 and RefSeq. Simultaneously, based on the HMM model of the Pfam database, virus-related proteins were back-referenced from RefSeq to supplement unannotated or distantly related homologs with significant sequence differences. After redundancy removal from multiple levels of candidate seed sequences using CD-HIT, a high-quality set of positive seed sequences was obtained.

[0050] Secondly, Diamond was used to perform large-scale sequence alignment of the RefSeq bacterial and viral libraries to obtain all homologous protein sequences of the target gene in bacteriophages and bacteria. Combined with the functional annotation of the seed sequence, each sequence was classified into a category of "mechanism / efflux pump, binding isolation, reduction transformation, regulatory system / bacteriophage or bacteria" according to the four major resistance mechanisms, thus establishing a systematic homologous sequence set of the target gene.

[0051] (2) Three-stage backtracking primer design strategy: For each target gene symbol, this invention establishes a three-level backtracking primer design strategy to ensure that high-quality detection primers can be obtained for all target genes: Level 1 (Preferred Strategy): Design based on conserved regions of phage homologous sequences. When there are ≥2 phage sequences for the target gene, MAFFT is used for multiple sequence alignment and conserved regions are extracted. Regions highly similar to bacteria are quickly screened out by k-mer frequency, retaining phage-specific regions. The selected conserved protein regions are mapped to a viral genome nucleic acid library using tblastn. After obtaining the coding DNA sequence, primers are designed using Primer3.

[0052] Level 2 (Retreat Strategy 1): Traverse all homologous protein sequences of the phage. If the number of phage sequences for the target gene is less than 2, or if the Level 1 strategy fails to produce a qualified primer, then retreat to traversing every sequence of the gene in the phage protein set, performing tblastn mapping and primer design sequentially, until successful.

[0053] Level 3 (Rollback Strategy 2): Supplementing the design using bacterial core protein sequences. If the target gene has no homologous sequence in the existing viral database, or if the above two strategies fail, then first use CD-HIT to remove redundancy from the bacterial protein sequence, and then design primers by traversing representative sequences. Although this strategy uses bacterial homologous sequences as templates, combined with the virus particle enrichment pretreatment steps of this invention, it can still ensure that the detection signal specifically originates from the phage community.

[0054] (3) Optimization of pooling for multiplex PCR compatibility: All candidate primers underwent 3' end dimer risk assessment (complementarity of the last four bases). A greedy algorithm was used to assign conflict-free primer pairs to the same reaction pool, while conflicting primer pairs were assigned to new pools. Ultimately, all primer pairs covering 37 core target genes across four major Cd resistance mechanisms were efficiently assigned to just two multiplex PCR reaction pools (Pool 1 / Pool 2), demonstrating extremely high multiplex detection efficiency and practical value.

[0055] Thirdly, detection and analysis: This invention uses a detection system combining multiplex PCR and high-throughput sequencing.

[0056] Compared to traditional qPCR methods, multiplex PCR combined with next-generation sequencing (NGS) can achieve the following technological breakthroughs: (1) From “qualitative / semi-quantitative” to “sequence-level precision”: Sequencing technology can not only determine the “presence” or “absence” of target genes, but also obtain precise sequence information of amplicon and discover new sequence variations and gene subtypes.

[0057] (2) From “known detection” to “unknown discovery”: By comparing sequencing data with reference databases, new AMG variants that were not covered during primer design but have highly similar sequences can be identified.

[0058] (3) Leap in throughput and efficiency: By adding unique index sequences to different samples, the amplification products of hundreds of soil samples can be mixed and sequenced in a single run. Combined with bioinformatics split analysis, true high-throughput detection can be achieved.

[0059] The detection method of the present invention is as follows: (1) Using the viral DNA extracted after enrichment of the above-mentioned viral particles as a template, multiplex PCR amplification was performed using the primer combination of the present invention. The PCR reaction system included: viral DNA template, mixture of primer pairs, high-fidelity DNA polymerase, dNTPs, and PCR buffer. The reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 30-35 cycles; 72℃ final extension for 5 min.

[0060] (2) After PCR product purification, high-throughput sequencing libraries were prepared using a library construction kit compatible with Illumina or MGI platform, and unique index tags were added to different samples.

[0061] (3) Mixed library sequencing was performed using the PE250 sequencing strategy.

[0062] (4) After quality control, primer sequence trimming, OTU clustering or ASV noise reduction, the data from the machine were compared and annotated with the phage cadmium resistance auxiliary metabolism gene reference database, and the abundance and diversity of auxiliary metabolism genes of each mechanism category in each sample were statistically analyzed.

[0063] This invention isolates soil bacteriophages through viral particle enrichment, performs multiplex PCR using a designed primer combination, and then combines this with high-throughput sequencing to detect cadmium resistance-related auxiliary metabolic genes in the bacteriophage community through different mechanisms. This invention develops a set of primer combinations and detection methods capable of specifically distinguishing between bacteriophage / bacterial origins and classifying detection according to cadmium resistance mechanisms. This has significant scientific value and urgent application needs for elucidating the bacteriophage-mediated cadmium resistance transmission mechanism and establishing a precise cadmium pollution assessment system based on bacteriophage communities.

[0064] The following are specific examples of the present invention: Example 1 Primer design, screening, and final selection: This embodiment details the design method of primer combinations for detecting Cd-AMG (helper metabolic gene) in soil bacteriophage communities and the final high-quality primers determined in this invention.

[0065] I. Obtaining the target seed gene sequence: Keywords related to cadmium resistance genes (cadA | zntA | cadD | czcA | czcB | czcC | czcD | czrB | cnrA | nccA | fieF | mdtA | mdtD | metallothionein | smtA | bmtA | cysteine ​​synthase | cysK | cysM | PAPS reductase | cysH | cysJ | cysI | sulfite reductase | cadC | cadR | czcR | czcS | czrA | smtB | ziaR | cadmium resistance | cobalt-zinc-cadmium | heavy metal ATPase | cadmiumefflux | cadmium transport) were collected through literature review. Preliminary target protein seed sequences were obtained by searching the BACMET2 and RefSeq virus databases. Simultaneously, based on the Pfam database, HMM models including PF00122, PF00702, PF00873, PF01545, PF01439, PF01507, PF00291, PF01022, PF12840, PF00376, PF02069, and NF041288 were retrieved to backtrack virus-related proteins from the RefSeq database. The candidate target protein seed sequences obtained from multiple layers were then used for redundancy removal via cd-hit (-c0.95 -n 5 -M 16000 -T 32), ultimately yielding 557 high-quality positive seed sequences.

[0066] II. Large-scale database seed sequence alignment: Diamond was used to perform large-scale alignments of the RefSeq bacterial and viral libraries based on high-quality seed sequences (viruses: --max-target-seqs 200, --evalue 1e-3; bacteria: --max-target-seqs 500, --evalue 1e-5). A total of 258,547 bacterial protein sequences and 2,635 viral protein sequences were obtained. Combined with the excellent annotations of the seed sequences, the obtained sequences were categorized into mechanism / {efflux, sequestration, reduction, regulation} / {phage, bacteria}.

[0067] III. Construction of the core sequence set of target genes and multi-level primer design strategy: For each target gene symbol, based on the availability of its upstream and downstream homologous sequences, this invention establishes a multi-level primer design strategy to maximize gene coverage. See also Figures 2-5 As shown, the phylogenetic tree of representative helper metabolic gene target sequences for each mechanism demonstrates the branching differences between bacteriophage sequences and bacterial homologous sequences.

[0068] (1) Priority strategy: Design based on conserved regions of phage homologous sequences.

[0069] When the target gene has ≥2 sequences in the phage protein set, the following procedure should be used preferentially: Sequence alignment and conserved region extraction: MAFFT software (--auto --thread 30) was used to perform multiple sequence alignment on the phage protein sequence and extract conserved regions with a conservation rate of ≥70% and a length of ≥30 amino acids.

[0070] Specific screening: For each extracted candidate conserved region, the similarity to the bacterial homologous sequence set was quickly assessed using the k-mer frequency (k=6). If the proportion of shared k-mers exceeded 70%, it was considered a non-specific region and excluded; highly specific regions unique to the phage community were preferentially retained.

[0071] Nucleic acid template mapping: Selected conserved protein regions are mapped to the viral genome nucleic acid library using tblastn (E-value 1e-3), and the corresponding coding DNA sequences are extracted as templates for primer design.

[0072] Primer design: Primers were designed using Primer3 software on the obtained DNA templates. The parameters were as follows: primer length 18-24 bp, annealing temperature 55.0-65.0℃, GC content 40%-60%, product length 100-400 bp. A maximum of 3 candidate primer pairs were returned for each template.

[0073] If this strategy successfully designs primers, they are labeled as mafft_specific (specific conserved region) or mafft_universal (universal conserved region).

[0074] (2) Backtracking strategy 1: Traverse all homologous protein sequences of bacteriophage.

[0075] If the number of phage sequences for the target gene is less than two, or if the aforementioned optimization strategy fails to produce qualified primers, the process reverts to iterating through every sequence of the gene in the phage protein set. tblastn mapping and Primer3 design are performed sequentially for each sequence until at least one primer pair is successfully obtained. This strategy ensures that even with only a few or distantly related phage protein sequences, corresponding detection primers can still be designed. A successful implementation of this strategy is marked as a fallback phage.

[0076] (3) Regression Strategy 2: Use bacterial core protein sequences for supplementary design.

[0077] If the target gene has no homologous sequence in the existing viral database, or if both of the above strategies fail, then the final bacterial regression strategy is initiated: Bacterial sequence redundancy removal: CD-HIT (-c 0.90) was used to remove redundancy from bacterial protein sequences, retaining a representative core sequence set to reduce computational cost.

[0078] Sequence mapping and design: Redundancy-free representative bacterial sequences are iterated and mapped to a bacterial genomic nucleic acid library using tblastn. After obtaining the coding DNA template, primers are designed using the same Primer3 parameters until successful. Successful primers are marked as fallback_bacteria.

[0079] Through the three-level strategy of "specific design → phage regression → bacterial regression" described above, this embodiment ensures that high-quality detection primers can be obtained for all target genes.

[0080] (4) Multiple compatibility evaluation and pooling of candidate primers.

[0081] To meet the needs of high-throughput multiplex PCR detection, multiplex compatibility pooling was performed on all successfully designed candidate primers: 3' dimer assessment: Calculate the 3' (last 4 bases) complementarity among all candidate primers. If the 3' end of one primer pair is complementary to the 3' or 5' end of another primer pair for 4 consecutive bases, a serious risk of cross-dimerization is identified, and they cannot be placed in the same reaction chamber.

[0082] Greedy primer pooling: A greedy algorithm is used to traverse all target genes and select the optimal pair of candidate primers for each gene, prioritizing their placement in existing, conflict-free reaction pools; if conflicts exist, a new reaction pool is created. Ultimately, primers for all genes are efficiently allocated to the reaction pools with the fewest possible combinations.

[0083] Testing showed that the primer pairs designed for 37 core target genes covering four major Cd resistance mechanisms were successfully assigned to only two multiplex PCR reaction pools, demonstrating extremely high multiplex detection efficiency.

[0084] (5) A table of final primer combinations.

[0085] After comprehensive screening as described above, and employing a backsliding design strategy for some genes, a set of primer combinations specifically targeting the four major Cd resistance mechanisms was finally determined. Multiple compatibility verification showed that all 37 primer pairs could be efficiently allocated to only two multiplex PCR reaction pools, demonstrating extremely high multiplex detection efficiency. Tables 5-6 provide detailed information on the core primer sequences included in the two reaction pools ultimately determined in this invention, and are used in the detection process of this invention.

[0086] Table 5 Primer list for multiplex PCR reaction pool 1 Table 6 Primer list for multiplex PCR reaction pool 2 Example 2 1. For a cadmium-contaminated soil sample (total cadmium content 1.5 mg / kg), the soil sample was pretreated for virus particle enrichment and DNA extraction, thereby extracting viral DNA.

[0087] In this embodiment, the process of enriching virus particles and extracting DNA from soil samples is as follows: (1) Take 5 g of fresh soil sample, add 10 mL of SM buffer, vortex for 5 min to fully wash out virus particles.

[0088] (2) Centrifuge at 3000×g for 10 min at 4℃ and collect the supernatant.

[0089] (3) The supernatant was passed through 0.45μm and 0.22μm PES filter membranes in sequence, and the filtrate was collected.

[0090] (4) Add PEG 8000 to a final concentration of 10% (w / v) and precipitate overnight at 4°C; or concentrate the virus particles by ultracentrifugation (100000×g, 2h, 4°C).

[0091] (5) The precipitate was resuspended in SM buffer, and DNase I was added to a final concentration of 10 U / mL. The precipitate was digested at 37°C for 30 min to remove free DNA.

[0092] (6) Use the QIAamp MinElute Virus Spin Kit to extract viral DNA, and follow the instructions in the manual.

[0093] 2. PCR amplification was performed using the two multiplex PCR reaction pools determined in Example 1 (reaction pool 1 contains 23 target genes and reaction pool 2 contains 14 target genes) to verify the amplification effect. The two reaction pools used the same reaction system and thermal cycling program. The products were mixed in equal amounts, libraries were constructed, and sequencing was performed.

[0094] In this embodiment, the overall process of PCR amplification, product mixing in equal volumes, library construction, and sequencing is as follows: (1) Multiplex PCR reaction system (25μL): 12.5μL of 2× high-fidelity PCR Master Mix, 5μL of primer mixture (final concentration of each primer is 0.2μM), 2μL of viral DNA template (in this example, the concentration of DNA template is 4.8 ng / μL), and nuclease-free water to make up to 25μL.

[0095] (2) PCR reaction program: 95℃ for 3 min; [95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s] × 32 cycles; 72℃ for 5 min.

[0096] (3) After the PCR products were purified by 1.8× magnetic beads, index PCR was performed using the Nextera XT Index Kit to add different sample tags to the amplification products of the two reaction chambers.

[0097] (4) After the libraries in the two reaction chambers were mixed in equimolar amounts, they were sequenced using the Illumina NovaSeq platform PE250.

[0098] (5) The primer sequences were removed using Cutadapt, and the Amplicon Sequence Variant (ASV) was denoised and its abundance was counted using QIIME2. The phage cadmium resistance reference sequence database was used for BLAST annotation (threshold: identity≥90%, coverage≥90%). ASV represents the deredundant sequence of the gene.

[0099] The phage cadmium resistance reference sequence database mainly includes representative sequences of cadmium resistance-related auxiliary metabolic genes screened from multiple sources such as the BACMET2 database, RefSeq viral protein database, Pfam HMM model search, and keyword search. It covers representative reference sequences of 37 core target genes in four types of mechanisms: efflux pump system (acrD, cadA, cadB, cadD, cnrA, czcA, czcB, czcC, czcD, czrB, fieF, mdtA, mdtD, mntH, nccA, pbrA), binding / isolation system (smtA, ydaE), reduction / transformation system (cdh, cysH, cysI, cysJ, cysK, cysM, dsbA, dsbB, sseA), and regulatory system (cadC, cadR, cadX, czcR, czcS, czrA, merR, perR, smtB, ziaR).

[0100] 3. Test Results: In this embodiment, the agarose gel electrophoresis image of the multiplex PCR products is shown below. Figure 6 As shown in Table 7, after completing the entire process of PCR amplification, product mixing, library construction and sequencing, the phage Cd-AMG target gene was successfully detected in the sample after BLAST annotation.

[0101] Table 7 Results of Detection of Cd-AMG Target Genes in Bacteriophages The above results demonstrate that the multiplex PCR detection system of this invention can effectively capture cadmium resistance auxiliary metabolic genes of various mechanisms in soil bacteriophage communities, verifying the feasibility of "mechanism-specific detection". Among them, efflux pump and reductive transformation AMGs were detected at high abundance, suggesting that bacteriophages in this contaminated soil mainly assist the host in coping with cadmium stress by mediating active efflux and sulfur metabolism transformation pathways.

[0102] Example 3 Detection of soil samples with different levels of cadmium contamination: This embodiment aims to verify the effectiveness of pretreatment for virus particle enrichment and the amplification specificity of multiplex PCR primer combinations by simulating soil samples with different cadmium pollution gradients.

[0103] (1) Experimental design: In this embodiment, farmland soil with minimal cadmium pollution (total Cd content 0.27 mg / kg) was collected. Plant roots and other non-soil materials were removed, and the samples were thoroughly mixed. Consistently weighed soil samples were placed in a test chamber, and cadmium nitrate solution was added to the soil samples to reduce the Cd content. 2+The concentrations are shown in Table 8. The samples were placed in an artificial climate chamber to simulate the temperature, humidity, and light conditions of the sampling site and cultured for 30 days. This embodiment included one control group and four cadmium treatment groups, each with three independent samples, as detailed in Table 8.

[0104] The pretreatment methods in Table 8 refer to the treatments performed before PCR amplification; among them, the virus particle enrichment process is the same as the virus particle enrichment and DNA extraction process of the soil samples in Example 2 (only the DNA template concentration changes); in this example, the concentration of DNA template after virus particle enrichment and direct extraction of total DNA is controlled within 1-5 ng / μL, and this concentration range does not affect the final effect.

[0105] In this embodiment, CK-1 / 2 / 3 corresponds to CK-1, CK-2, and CK-3; Cd-L-1 / 2 / 3 corresponds to Cd-L-1, Cd-L-2, and Cd-L-3; Cd-M-1 / 2 / 3 corresponds to Cd-M-1, Cd-M-2, and Cd-M-3; Cd-H-1 / 2 / 3-VF corresponds to Cd-H-1-VF, Cd-H-2-VF, and Cd-H-3-VF; and Cd-H-1 / 2 / 3-TD corresponds to Cd-H-1-TD, Cd-H-2-TD, and Cd-H-3-TD.

[0106] Table 8. Description of each sample After processing all samples according to the pretreatment method in Table 8, the same process as in Example 2 was performed, including multiplex PCR amplification, product mixing in equal amounts, library construction, and sequencing. The data after sequencing were quality controlled and primer sequences were removed before being used for subsequent analysis.

[0107] (2) Verification of virus particle enrichment effect: For high-concentration cadmium treatment, the effects of two pretreatment methods—virus particle enrichment and direct extraction of total DNA—on the proportion of viral sequences in the sequencing data were compared. Kraken2 combined with CheckV was used to perform taxonomic annotation of the effective sequences for each sample (database: NCBI RefSeq Virus and Bacterial Complete Genomes). Sequencing data statistics for Cd-H-1-VF, Cd-H-2-VF, Cd-H-3-VF, and Cd-H-1-TD, Cd-H-2-TD, Cd-H-3-TD are shown in Table 9.

[0108] Table 9. Sequencing data statistics for Cd-H-1 / 2 / 3-VF and Cd-H-1 / 2 / 3-TD In samples from which total DNA was directly extracted, viral sequences accounted for only 0.67% to 1.17%, while bacterial sequences were absolutely dominant (>87%). Their PCR amplification signals mainly originated from the bacterial genome and could not be used for the specific detection of bacteriophage auxiliary metabolic genes.

[0109] After pretreatment for virus particle enrichment, the viral sequence content increased to 38.52%–47.51%, a 40.6–57.5-fold increase compared to direct extraction, achieving highly efficient enrichment of phage DNA. Although approximately 50% of the bacterial sequence remained (possibly from a small amount of unfiltered bacteria or bacterial DNA released from the lysis of lysogenic phages within the host bacteria), the absolute amount of viral sequence was sufficient for subsequent specific detection and analysis. This result demonstrates that the virus particle enrichment step of this invention can significantly eliminate bacterial DNA interference, ensuring that the detection signal primarily originates from the phage community.

[0110] (3) Verification of the specificity of the target region amplified by primers: To evaluate the amplification specificity of the primer combination of the present invention in multiplex PCR, the pooled library construction and sequencing data of all 12 samples (CK-1 / 2 / 3, Cd-L-1 / 2 / 3, Cd-M-1 / 2 / 3, Cd-H-1 / 2 / 3-VF) that underwent viral particle enrichment were analyzed, as shown in Table 10.

[0111] Table 10 Analysis of merged library preparation and sequencing data The 12 samples corresponding to virus particle enrichment showed a highly consistent matching rate for the viral target region, averaging 86.65% (SD=1.39%), indicating that the primer combination exhibited stable and efficient amplification specificity under different soil conditions and cadmium concentrations. Other sequences failed to match the expected target region, mainly due to primer dimers (which could be partially removed through length screening), a very small number of non-specific amplifications (usually showing some similarity to the target region but not reaching the threshold), and error sequences introduced by sequencing. This level is excellent for two-reaction-pool multiplex PCR, superior to previously reported similar environmental microbial multiplex targeted sequencing methods.

[0112] The results directly demonstrate that the primer combination of the present invention can highly specifically enrich the target cadmium resistance auxiliary metabolic gene fragment in the context of complex soil viral DNA templates, providing a high-quality data foundation for subsequent quantitative and diversity analyses.

[0113] (4) Summary: This embodiment, through comparative experiments and sequencing data analysis, systematically verifies the effectiveness of the core steps of the present invention: Viral particle enrichment increases the proportion of viral sequences from about 1% to over 40%, greatly alleviating the difficulty of capturing viral data caused by bacterial DNA from the source.

[0114] The primer combination achieved a target region matching rate of 86.65% in real soil samples, demonstrating the high accuracy of multiplex PCR amplification.

[0115] The above results fully support the ability of this invention to specifically detect cadmium-resistant auxiliary metabolic genes in soil bacteriophage virus communities, laying a reliable technical foundation for subsequent studies on abundance and diversity under different pollution levels.

[0116] The above technical solutions of the present invention are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made under the technical concept of the present invention using the contents of the present invention specification and drawings, or direct / indirect applications in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A primer combination for detecting cadmium resistance accessory metabolic genes in soil bacteriophage virus communities, characterized in that, The primer combination comprises nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.

74.

2. The primer combination according to claim 1, characterized in that, The primer combination targets the following cadmium resistance accessory metabolism genes: efflux pump mechanism (acrD), efflux pump mechanism (cadA), efflux pump mechanism (cadB), regulatory system mechanism (cadC), regulatory system mechanism (cadR), regulatory system mechanism (cadX), reduction / transformation mechanism (cysH), reduction / transformation mechanism (cysI), reduction / transformation mechanism (cysJ), reduction / transformation mechanism (cysM), efflux pump mechanism (czcA), efflux pump mechanism (czcB), regulatory system mechanism (czcR), regulatory system mechanism (czrA), efflux pump mechanism (czrB), reduction / transformation mechanism (dsbA), efflux pump mechanism (mdtD), and efflux pump mechanism (mdtD). mntH, nccA for effluent pump mechanism, pbrA for effluent pump mechanism, perR for control system mechanism, smtB for control system mechanism, ziaR for control system mechanism, cadD for effluent pump mechanism, cdh for reduction / conversion mechanism, crnA for effluent pump mechanism, cysK for reduction / conversion mechanism, czcC for effluent pump mechanism, czcD for effluent pump mechanism, czcS for control system mechanism, dsbB for reduction / conversion mechanism, fieF for effluent pump mechanism, mdtA for effluent pump mechanism, merR for control system mechanism, smtA for binding / isolation mechanism, sseA for reduction / conversion mechanism, ydaE for binding / isolation mechanism.

3. The use of a primer combination as described in claim 1 or 2 in the preparation of a kit for detecting cadmium resistance auxiliary metabolic genes in soil bacteriophage virus communities.

4. A kit for detecting cadmium resistance co-metabolic genes in soil bacteriophage virus communities, characterized in that, The kit includes the primer combination as described in claim 1 or 2.

5. The kit for detecting cadmium resistance co-metabolic genes in soil bacteriophage virus communities according to claim 4, characterized in that, The kit also includes multiplex PCR reagents other than the primer combination.

6. The kit for detecting cadmium resistance co-metabolic genes in soil bacteriophage virus communities according to claim 4, characterized in that, The kit also includes at least one of library construction reagents and sequencing reagents.

7. The use of a kit as described in any one of claims 4-6 in detecting cadmium resistance co-metabolic genes in soil bacteriophage virus communities.

8. The application according to claim 7, characterized in that, The application includes the following steps: S1, which enriches virus particles in the soil; S2, using the DNA of the viral particles as a template, multiplex PCR amplification is performed using the primer combination to obtain PCR products; S3, after purifying the PCR product, library construction and high-throughput sequencing are performed; S4. After analyzing the sequencing data, the abundance and diversity of cadmium resistance auxiliary metabolic genes in the soil bacteriophage virus community were obtained.

9. The application according to claim 8, characterized in that, Before analyzing the data obtained after sequencing, primer sequence trimming and noise reduction are performed on the sequencing data.

10. The application according to claim 8, characterized in that, During the analysis of the sequencing data, the data obtained after sequencing were compared and annotated with the phage cadmium resistance reference sequence database.

Images