A reagent combination, kit and method for detecting genes related to organic sulfur cycle metabolism

Abstract

The application provides a reagent combination, a kit and a method for detecting organic sulfur cycle metabolism related genes, and belongs to the field of organic sulfur cycle metabolism related gene detection. The reagent combination comprises a primer pair combination for specifically amplifying organic sulfur cycle metabolism related genes. The reagent combination, the gene chip and the kit of the application have very high sensitivity, specificity and coverage for detecting organic sulfur cycle metabolism related genes. The method of the application is based on HT-qPCR, can simultaneously quantitatively detect multiple environmental samples, can well distinguish different organic sulfur cycle metabolism genes in different environments, and can absolutely quantitatively and relatively quantitatively detect the abundance, provides a high-throughput molecular tool for related research of organic sulfur cycle, effectively supplements the defects of low qPCR detection throughput and inability of absolute quantification of metagenome sequencing, and provides strong technical support for ecological research.

Description

Technical Field

[0001] This application relates to the field of detection of genes related to organic sulfur cycle metabolism, and in particular to a reagent combination, kit and method for detecting genes related to organic sulfur cycle metabolism. Background Technology

[0002] The organic sulfur cycle is a process in nature involving the transformation of various organic sulfur molecules, or their conversion from organic to inorganic forms. It is an important component of the global sulfur cycle. Key metabolites in the organic sulfur cycle include dimethyl mercaptopropionate (DMSP), methyl sulfide (DMS), methanethiol (MeSH), and dimethyl sulfoxide (DMSO). DMSP is one of the most abundant organic sulfur molecules on Earth, serving as an osmotic protectant, pressure protectant, antioxidant, cryoprotectant, and predation defense agent for phytoplankton and various algae. DMS is a "cold chamber gas" produced by the decomposition and metabolism of DMSP. Once in the atmosphere, DMS undergoes oxidation, and its oxidation products condense, acting as cloud nuclei to promote cloud formation and enhance solar reflectivity, thus negatively impacting global warming. MeSH is an important intermediate metabolite in the transformation of various organic sulfur molecules and can be used as a sulfur source or electron donor by certain microorganisms. In addition to its roles as an osmotic protectant and antioxidant in microorganisms, DMSO can also be reduced to DMS, influencing climate change. Therefore, studying the metabolic processes of organic sulfur can not only help us understand the growth and metabolic characteristics of microorganisms and algae and the structure and function of biological communities, but also help us assess the ocean's contribution to climate regulation and predict potential trends in climate change, providing rich basic data for biogeochemistry and environmental science.

[0003] In existing technologies, the study of organic sulfur cycling is mainly carried out through metagenomic sequencing, amplicon sequencing, and real-time quantitative PCR (qPCR). However, metagenomic sequencing is costly, time-consuming, and complex to process; amplicon sequencing cannot perform absolute quantification of functional genes; although qPCR reduces the amount of information and can quickly perform absolute quantification of various environmental samples, it is costly and difficult to perform calibration between batches of multiple samples.

[0004] High-throughput qPCR (HT-qPCR) technology can simultaneously quantify dozens of functional genes, obtaining abundance information for multiple genes in a single experiment. It offers numerous advantages, including high speed, high throughput, and low cost, providing a more convenient and efficient detection method for studying key metabolic genes in the organic sulfur cycle. Therefore, to accelerate research progress in organic sulfur metabolism, there is an urgent need to develop a high-throughput qPCR detection chip for organic sulfur cycle metabolic genes. Summary of the Invention

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

[0006] The first aspect of this application provides a reagent combination for detecting genes related to organosulfur cycling metabolism. The reagent combination includes a primer pair combination for specifically amplifying genes related to organosulfur cycling metabolism, wherein the genes related to organosulfur cycling metabolism include mmtN, burB, dsyB, dsyGD, megL, dddD, dddX, dddL, dddP, dddQ, dddW, dddY, dddK, dddU, dmdA, dmdB, dmdC, dmdD, acuH, mtoX, mtsA, mddA, mddH, dmoA, ddhA, dsoB, Tmm, dmsA, and dorA.

[0007] In some possible embodiments of this application, the primer pair combination includes:

[0008] (1) The primer pair consisting of SEQ ID No. 1 and SEQ ID No. 2, and the primer pair consisting of SEQ ID No. 3 and SEQ ID No. 4, are used to specifically amplify at least a partial fragment of the mmtN gene;

[0009] (2) The primer pair consisting of SEQ ID No. 5 and SEQ ID No. 6 is used to specifically amplify at least a partial fragment of the burB gene;

[0010] (3) The primer pair consisting of SEQ ID No. 7 and SEQ ID No. 8 is used to specifically amplify at least a partial fragment of the dsyB gene;

[0011] (4) The primer pair consisting of SEQ ID No. 9 and SEQ ID No. 10 is used to specifically amplify at least a partial fragment of the dsyGD gene;

[0012] (5) Primer pair consisting of SEQ ID No. 11 and SEQ ID No. 12, and primer pair consisting of SEQ ID No. 13 and SEQ ID No. 14, are used to specifically amplify at least a portion of the megL gene;

[0013] (6) The primer pair consisting of SEQ ID No. 15 and SEQ ID No. 16 is used to specifically amplify at least a partial fragment of the dddD gene;

[0014] (7) The primer pair consisting of SEQ ID No. 17 and SEQ ID No. 18, and the primer pair consisting of SEQ ID No. 19 and SEQ ID No. 20, are used to specifically amplify at least a portion of the dddX gene;

[0015] (8) The primer pair consisting of SEQ ID No. 21 and SEQ ID No. 22, and the primer pair consisting of SEQ ID No. 23 and SEQ ID No. 24, are used to specifically amplify at least a portion of the dddL gene;

[0016] (9) The primer pair consisting of SEQ ID No. 25 and SEQ ID No. 26 is used to specifically amplify at least a partial fragment of the dddP gene;

[0017] (10) Primer pairs consisting of SEQ ID No. 27 and SEQ ID No. 28, primer pairs consisting of SEQ ID No. 29 and SEQ ID No. 30, and primer pairs consisting of SEQ ID No. 31 and SEQ ID No. 32, for specifically amplifying at least a portion of the dddQ gene;

[0018] (11) Primer pair consisting of SEQ ID No. 33 and SEQ ID No. 34, and primer pair consisting of SEQ ID No. 35 and SEQ ID No. 36, are used to specifically amplify at least a portion of the dddW gene;

[0019] (12) The primer pair consisting of SEQ ID No. 37 and SEQ ID No. 38 is used to specifically amplify at least a partial fragment of the dddY gene;

[0020] (13) The primer pair consisting of SEQ ID No. 39 and SEQ ID No. 40 is used to specifically amplify at least a partial fragment of the dddK gene;

[0021] (14) The primer pair consisting of SEQ ID No. 41 and SEQ ID No. 42 is used to specifically amplify at least a partial fragment of the dddU gene;

[0022] (15) Primer pair consisting of SEQ ID No. 43 and SEQ ID No. 44, and primer pair consisting of SEQ ID No. 45 and SEQ ID No. 46, are used to specifically amplify at least a portion of the acuH gene;

[0023] (16) Primer pairs consisting of SEQ ID No. 47 and SEQ ID No. 48, and primer pairs consisting of SEQ ID No. 49 and SEQ ID No. 50, are used to specifically amplify at least a portion of the dmdA gene;

[0024] (17) Primer pairs consisting of SEQ ID No. 51 and SEQ ID No. 52, primer pairs consisting of SEQ ID No. 53 and SEQ ID No. 54, and primer pairs consisting of SEQ ID No. 55 and SEQ ID No. 57, for the specific amplification of at least a portion of the dmdB gene;

[0025] (18) Primer pair consisting of SEQ ID No. 57 and SEQ ID No. 58, and primer pair consisting of SEQ ID No. 59 and SEQ ID No. 60, for specifically amplifying at least a portion of the dmdC gene;

[0026] (19) Primer pair consisting of SEQ ID No. 61 and SEQ ID No. 62, and primer pair consisting of SEQ ID No. 63 and SEQ ID No. 64, are used to specifically amplify at least a portion of the dmdD gene;

[0027] (20) Primer pair consisting of SEQ ID No. 65 and SEQ ID No. 66, and primer pair consisting of SEQ ID No. 67 and SEQ ID No. 68, for specifically amplifying at least a portion of the mtoX gene;

[0028] (21) Primer pairs consisting of SEQ ID No. 69 and SEQ ID No. 70, primer pairs consisting of SEQ ID No. 71 and SEQ ID No. 72, and primer pairs consisting of SEQ ID No. 73 and SEQ ID No. 74, are used to specifically amplify at least a portion of the mddA gene;

[0029] (22) Primer pair consisting of SEQ ID No. 75 and SEQ ID No. 76, and primer pair consisting of SEQ ID No. 77 and SEQ ID No. 78, are used to specifically amplify at least a portion of the mddH gene;

[0030] (23) The primer pair consisting of SEQ ID No. 79 and SEQ ID No. 80 is used to specifically amplify at least a partial fragment of the mtsA gene;

[0031] (24) The primer pair consisting of SEQ ID No. 81 and SEQ ID No. 82 is used to specifically amplify at least a partial fragment of the dmoA gene;

[0032] (25) The primer pair consisting of SEQ ID No. 83 and SEQ ID No. 84 is used to specifically amplify at least a partial fragment of the ddhA gene;

[0033] (26) Primer pairs consisting of SEQ ID No. 85 and SEQ ID No. 86, and primer pairs consisting of SEQ ID No. 87 and SEQ ID No. 88, are used to specifically amplify at least a portion of the dsoB gene;

[0034] (27) The primer pair consisting of SEQ ID No. 89 and SEQ ID No. 90 is used to specifically amplify at least a partial fragment of the tmm gene;

[0035] (28) The primer pair consisting of SEQ ID No. 91 and SEQ ID No. 92 is used to specifically amplify at least a partial fragment of the dmsA gene;

[0036] (29) The primer pair consisting of SEQ ID No. 93 and SEQ ID No. 94 is used to specifically amplify at least a portion of the dorA gene.

[0037] In some specific embodiments of this application, the primer pair combination further includes:

[0038] The primer pair consisting of SEQ ID No. 95 and SEQ ID No. 96 is used to specifically amplify at least a partial fragment of the 16S rRNA gene.

[0039] A second aspect of this application provides a gene chip, the gene chip comprising any of the reagent combinations described in the first aspect of this application.

[0040] A third aspect of this application provides a reagent kit comprising any of the reagent combinations described in the first aspect of this application or the gene chip described in the second aspect of this application.

[0041] In some technical solutions of this application, the kit also includes genomic DNA extraction reagent for the sample to be tested, DNA purification reagent and / or PCR amplification buffer.

[0042] In some technical solutions of this application, the kit further includes a positive control and / or a negative control, wherein the positive control is a mixed sample composed of plasmid standards corresponding to the primer pair combination.

[0043] The fourth aspect of this application provides a method for detecting genes related to organic sulfur cycle metabolism in marine samples, comprising the following steps:

[0044] S1, Obtain metagenomic DNA from the marine sample to be tested;

[0045] S2, perform qPCR analysis on the metagenomic DNA using any of the reagent combinations described in the first aspect of this application, or the gene chip described in the second aspect of this application, or the kit described in any of the third aspect of this application;

[0046] S3, the abundance of the genes related to the organic sulfur cycle metabolism was obtained from the gene qPCR analysis results.

[0047] In some embodiments of this application, the marine sample is seawater or trench sediment.

[0048] In some possible embodiments of this application, in step S2, the qPCR analysis is performed using high-throughput qPCR technology.

[0049] Compared with the prior art, the invention title of this application has the following beneficial effects:

[0050] This application employs a rigorous and meticulous degenerate primer design process to design degenerate primers for 29 currently known organosulfur cycling metabolism genes. The degenerate primers were validated through a series of techniques, including computer verification and experimental verification, resulting in a primer combination with very high sensitivity, specificity, and coverage. Furthermore, by combining high-throughput qPCR (HT-qPCR) technology, a gene chip capable of detecting multiple organosulfur cycling metabolism genes was constructed and successfully applied to the study of various environmental samples.

[0051] The gene chip described in this application can perform 5184 quantitative PCR reactions in a single detection, enabling simultaneous quantitative detection of multiple environmental samples. While reducing detection costs, it can effectively distinguish different organic sulfur cycle metabolic genes under different environments and perform absolute and relative quantitative detection of their abundance. This provides a high-throughput molecular tool for research related to the organic sulfur cycle, effectively compensating for the shortcomings of low throughput in qPCR detection and the inability of metagenomic sequencing to perform absolute quantification, and providing strong technical support for ecological research.

[0052] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0053] The above and other objects, features, and advantages of exemplary embodiments of this application will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings. Several embodiments of this application are illustrated in the drawings by way of example and not limitation, in which:

[0054] Figure 1 A phylogenetic tree of 29 organic sulfur metabolism genes in Example 1 of this application is shown.

[0055] Figure 2 The amplification results of the primer pair corresponding to the dmdB gene in PCR verification in Example 2 of this application are shown. A: Experimental group. B: Negative control group.

[0056] Figure 3 The amplification curve of the megL-A2H1 primer pair in qPCR verification in Example 2 of this application is shown, showing the presence of primer dimers.

[0057] Figure 4 This demonstrates that the amplification efficiency of the burB-A10 primer pair in the qPCR verification of Example 2 of this application is less than 70%.

[0058] Figure 5 The amplification efficiency of 53 primer pairs in Example 2 of this application is shown.

[0059] Figure 6 The standard curves of the mixed plasmid standard under 10-fold serial dilution in Example 2 of this application are shown.

[0060] Figure 7 The absolute abundance of each organic sulfur cycle metabolic gene obtained by analyzing seabed sediment samples using the OSMG chip in Example 3 of this application is shown.

[0061] Figure 8 The relative abundance of each organic sulfur cycle metabolic gene obtained by analyzing seabed sediment samples using the OSMG chip in Example 3 of this application is shown.

[0062] Figure 9 The absolute abundance of each organic sulfur cycle metabolic gene obtained by analyzing seawater samples using the OSMG chip in Example 3 of this application is shown.

[0063] Figure 10 The relative abundance of each organic sulfur cycle metabolic gene obtained by analyzing seawater samples using the OSMG chip in Example 3 of this application is shown. Detailed Implementation

[0064] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, particularly the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0065] To make the technical problems, technical solutions and beneficial effects solved by this application clearer, the following detailed description is provided in conjunction with embodiments.

[0066] The following examples are used to illustrate preferred embodiments of this application. Those skilled in the art will understand that the techniques disclosed in the examples represent technologies discovered by the inventors that can be used to implement this application, and therefore can be considered preferred embodiments of this application. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of this application.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and all materials cited herein and referenced by them are incorporated herein by reference.

[0068] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0070] Example 1: Chip Design

[0071] 1. Obtaining protein and gene sequences related to the organic sulfur cycle.

[0072] Literature related to organic sulfur cycle metabolism was retrieved, and protein and gene sequences with relevant functions were collected from all literature. Finally, 144 amino acid sequences and 132 nucleotide sequences were downloaded from 29 genes.

[0073] The functional information of 29 genes is shown in Table 1.

[0074] Table 1. Genes related to organic sulfur cycling metabolism and their functions

[0075]

[0076] 2. Obtaining homologous protein sequences and gene sequences of 29 genes.

[0077] Homologous proteins were retrieved and sequence files downloaded from the public databases NCBI (https: / / www.ncbi.nlm.nih.gov) and Uniprot (https: / / www.ncbi.nlm.nih.gov). The retrieval was performed in two steps:

[0078] The first step was to construct HMM models for all 29 genes using hmmbuild of HMMER (Hidden Markov Models) v3.4, and to screen all homologous proteins using the models.

[0079] The second step is to use BLASTp to further filter for samples with a similarity greater than 40% and an e-value less than 10. -30 The protein sequence.

[0080] In the end, a total of 5,477 amino acid sequences and 5,439 nucleotide sequences were downloaded from 29 genes.

[0081] 3. Construct a phylogenetic tree

[0082] A phylogenetic tree was constructed using homologous protein sequences of each functional gene. Sequence alignment was performed using MAFFT, and the phylogenetic tree was constructed using IQ-TREE2 v2.2.2.7 with a bootstrap value set to 1000. This yielded a phylogenetic tree for 29 organosulfur metabolism genes, as follows: Figure 1 As shown.

[0083] 4. Primer design

[0084] Primers were designed for each cluster in the phylogenetic tree, using the following three methods:

[0085] (A) Degenerate primers were designed using the j-CODEHOP program in the Base-By-Base software. The parameters were set as follows: primer length between 16 and 40 bases, GC content 40%-60%, annealing temperature 60℃, amplified sequence length between 80 and 400 bases, a maximum core degeneracy of 256, and at least three consecutive conserved amino acid regions. Sixty-six primer pairs were designed using this method.

[0086] (B) Degenerate primers were designed using Geneious Primer software with parameters set as follows: primer length between 16 and 35 bases, GC content between 40% and 60%, annealing temperature at 60°C, amplified sequence length between 80 and 400 bases, and a maximum degeneracy of 256. 537 primer pairs were obtained using this method.

[0087] (C) After sequence alignment, a conserved region with four consecutive conserved amino acid sites upstream and downstream of the sequence was identified. Reverse translation into a nucleotide sequence was then performed based on the codon bias of the species containing the gene. Suitable regions were selected according to the following parameters: primer length between 16 and 35 bases, GC content between 40% and 60%, annealing temperature of 60°C, amplified sequence length between 80 and 400 bases, and a maximum degeneracy of 256. Seven primer pairs were obtained using this method.

[0088] A total of 610 new primer pairs were obtained using the above methods. Additionally, two primer pairs already used in environmental samples were collected from the literature, bringing the total to 612 primer pairs for subsequent microarray validation. The statistics are shown in Table 2.

[0089] Table 2. Statistics on the number of primer pairs designed for each gene using three methods.

[0090]

[0091] Taking dmdB and dddX as examples, the primers designed are shown in Tables 3 and 4:

[0092] Table 3 Primers designed for the dmdB gene using three methods

[0093]

[0094] Table 4 Primers designed for the dddX gene using three methods

[0095]

[0096] Table 4 (continued) Primers designed for the dddX gene using three methods

[0097]

[0098] Example 2: Chip Verification and Screening

[0099] 1. PCR validation screening

[0100] The size of the PCR amplification product was used to determine whether the primers met expectations. DNA from various environmental samples was used as a template, extracted using the DNEAY PowerSoil Pro Kit (QIANGEN), and then purified using the DNEAY PowerClean Pro Clean Kit (QIANGEN). DNA quality was assessed using a NanoDrop ND-2000 micro-volume spectrophotometer; an OD260 / 280 value between 1.6 and 2.0 indicated acceptable quality. DNA concentration was determined using a Qubit 3.0 spectrophotometer; a concentration above 10 ng / μL met detection requirements, and the total DNA mass was required to be above 500 ng.

[0101] The PCR system consisted of 50 μL of 2×rTaq Mix, 0.5 μM primers, and 1 ng / μL DNA template.

[0102] The PCR program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 min, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 30 cycles; and finally 72℃ extension for 5 min.

[0103] The obtained DNA was subjected to 2% agarose gel electrophoresis. Primers with single, bright bands and no primer dimers were selected, while primers with two or more bands or primer dimer bands were excluded.

[0104] The agarose gel electrophoresis results of the amplification products of some primer pairs designed for the dmdB gene are as follows: Figure 2 As shown. From Figure 2 As can be seen, primer pairs numbered 25, 29, and 31 showed primer dimer bands and were therefore discarded; primer pairs numbered 17 and 22 showed non-specific amplification and were therefore discarded; primer pairs numbered 19 and 26 met the PCR validation rules and were therefore retained. Finally, primer pairs numbered 19, 26, and 30 were selected and renamed dmdB1.1, dmdB1.2, and dmdB1.3.

[0105] For the dddX gene, primer pairs numbered 75 and 65 were retained and renamed dddX1.1 and dddX1.2.

[0106] Through PCR screening, 523 primer pairs were eliminated from 612 primer pairs, and the 89 primer pairs that were correctly verified by PCR were further verified.

[0107] 2. Amplicon sequencing validation and screening

[0108] Using PCR-validated primer pairs and the aforementioned DNA as templates, PCR amplification was performed again. Bands of the correct size were recovered by gel electrophoresis. Libraries were constructed according to the standard procedure of the NEB Next Ultra DNA library Prep Kit for Illumina, and the constructed amplicon libraries were sequenced using the Illumina platform with PE250 sequencing. Analysis of the amplicon data showed that 94.4% of the amplicones had more than 80% similarity to their corresponding targets. Through amplicon sequencing validation, 5 primer pairs were removed from the 89 primer pairs, and the 84 valid primer pairs were used for further validation.

[0109] 3. qPCR validation

[0110] The amplification efficiency of the above primer pairs was detected using real-time quantitative PCR (qPCR). First, plasmid standards were prepared, and plasmid DNA containing the target gene was extracted. The plasmids were then diluted to an initial copy number of approximately 10 for each target gene. 9 Copy, then serially diluted 10-fold to 10 2 Copy. Use a qPCR instrument to perform detection and obtain a standard curve.

[0111] The qPCR system was 25 μL, containing 12.5 μL of 2×TB Green Premix Ex Taq II Fast qPCR mix (Takara), 0.4 μM primers, and 1 μL of plasmid standards of each gene copy number.

[0112] The qPCR program was as follows: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s; 60℃ annealing for 30s; 72℃ extension for 10s, for 35 cycles. Finally, the temperature was increased from 72℃ to 97℃ at a rate of 4℃ / s, and the melting curve was determined at 0.4℃ intervals.

[0113] All qPCR reactions contained three technical replicates. Samples successfully amplified in at least two technical replicates were considered positive and used for further analysis. Melting curves were used to determine the presence of dimers in primer pairs, automatically generated by ABI software. Standard curve fitting was performed using linear regression analysis with R 4.0.3. Amplification efficiency was calculated based on the slope of the standard curve using the formula Eff = 10(-1 / slope)⁻¹, yielding the amplification efficiency for each primer pair. The ideal amplification efficiency was between 80% and 120%.

[0114] by Figure 3 Taking megL-A2H1 as an example, its melting curve showed two signal peaks, so it was discarded. According to the melting curves, a total of 64 primer pairs showed a single peak in their melting curves.

[0115] by Figure 4 Taking burB-A10 as an example, it was removed because the amplification efficiency of this primer pair was less than 80%.

[0116] like Figure 5 As shown, 53 primer pairs had amplification efficiencies between 80% and 120%, with a mean of 97.78%, and R... 2 The coefficients ranged from 0.983 to 0.999, with a mean of 0.991. Since primers were designed separately for each cluster after phylogenetic analysis for each gene, only one primer pair was retained per cluster, resulting in 47 primer pairs for subsequent validation. Information on the 47 primer pairs is shown in Table 5.

[0117] Table 5. Primer information for 47 pairs

[0118]

[0119] Table 5 (continued) 47 Primer Pairs Information

[0120]

[0121] Organic sulfur cycle metabolism-related gene chip (OSMG chip) was prepared using the above 47 primer pairs.

[0122] 4. HT-qPCR Validation

[0123] The sensitivity of primer pairs for detecting the OSMG chip was tested using a SmartChip Real-time PCR system (Thermo Fisher). The standards were the same as those used in the qPCR validation step. A qPCR amplification reaction system was prepared in a total volume of 100 nL per well, including 1×TB Green Premix Ex Taq II Fast qPCR mix (Takara), 0.4 μM forward primer, 0.4 μM reverse primer, and 50 ng / μL DNA. The amplification program was: 95℃ pre-denaturation for 10 min; 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 30 s, and 72℃ extension for 10 s, for 35 cycles. Finally, the temperature was increased from 72℃ to 97℃ at a rate of 4℃ / s, and melting curves were measured at 0.4℃ intervals.

[0124] The amplification results were obtained and data analysis was performed; the optimal cyclic CT cutoff value was determined based on the standard curve. The sensitivity of the chip was evaluated using the limit of detection (LoD) and the limit of quantitation (LoQ) (Table 4). LoD is the lowest detectable copy number of the target nucleic acid. LoD is the lowest target nucleic acid copy number that can be accurately quantified. The accuracy is evaluated based on the coefficient of variation (CV). When CV ≤ 35%, the data is considered acceptable. The coefficient of variation is calculated as follows:

[0125] CV = (standard deviation / mean) × 100%.

[0126] according to Figure 6 As can be seen, at a copy number of 10 2 By then, a stable Ct value can be obtained. The LoD and LoQ calculated based on the standard curve are shown in Table 6.

[0127] Table 6. CT value, detection limit, and quantitation limit of the OSMG chip.

[0128]

[0129] Table 6 shows that the LoD range of the OSMG chip is between 1 and 13674 copies / reaction, with an average of 634 copies / reaction. The LoQ range is between 64 and 57074 copies / reaction, with an average of 2918 copies / reaction. These results indicate that the OSMG chip has high sensitivity.

[0130] 5. Computer verification

[0131] To evaluate the coverage and specificity of the OSMG microarray, computer simulations were used to test the coverage and specificity of primer pairs within the OSMG microarray. Homologous protein sequences of organosulfur metabolism genes were retrieved from the NCBI and Uniprot databases, resulting in 29 primer evaluation databases. Each primer pair was compared with these databases to assess its coverage and specificity, with a maximum permissible number of mismatched bases of 2. Primer pair coverage was defined as the percentage of sequences in each evaluation database that matched simultaneously with both the forward and reverse primers; specificity was defined as the proportion of retrieved sequences belonging to the target gene database; and the false positive rate was defined as the percentage of false positive sequences observed during the alignment divided by the total number of sequences in the primer evaluation databases.

[0132] The computer verification results are shown in Table 7.

[0133] Table 7. Computer validation shows primer pair coverage, specificity, and false positive rate.

[0134]

[0135] Note: Coverage is calculated by summing the coverage of all primer pairs for each gene.

[0136] As shown in Table 7, the primer coverage of the OSMG chip ranged from 10% to 100%, with an average coverage of 36.6%. The number of sequences with 100% specificity was greater than 95% of the total. These results indicate that the OSMG chip has high specificity and coverage.

[0137] Example 3: OSMG Chip Application

[0138] 1. Sample Collection and Preparation

[0139] Environmental samples included two types: seawater samples and seabed sediment samples. Seawater samples included one nearshore surface seawater sample from Qingdao and 20 surface and deep seawater samples from the western Pacific Ocean. One liter of seawater was filtered through 3μm and 0.2μm filter membranes before DNA extraction. Seabed sediment samples included one South China Sea sediment sample, seven hydrothermal vent sediment samples, and six trench sediment samples, totaling 34 samples. Metagenomic DNA was extracted from the obtained environmental samples using the same DNA extraction method as in the PCR validation step, and its quality and concentration were tested. The results are shown in Table 8. DNA samples that passed the tests were used as samples for further analysis.

[0140] Table 8. DNA Concentration and Mass in Environmental Samples

[0141]

[0142] Introducing primer pairs for specific amplification of the 16S rRNA gene:

[0143] Positive (5'-3'): GGGTTGCGCTCGTTGC (SEQ ID No. 95)

[0144] Reverse (5'-3'): ATGGYTGTCGTCAGCTCGTG (SEQ ID No. 96)

[0145] The positive control is a sample mixture consisting of plasmid standards corresponding to 48 primer pairs. The plasmid standards are prepared using the following three methods:

[0146] (1) PCR was performed on environmental DNA using 48 primers with restriction sites. After agarose gel electrophoresis, the DNA was recovered and ligated into the pET28a plasmid vector, which was then transformed into Escherichia coli DH5a cells. After enrichment culture, plasmids containing the target fragment were extracted and used as standards for subsequent experiments.

[0147] (2) PCR was performed on pure bacteria containing the target gene using 48 primers with enzyme restriction sites. After agarose gel electrophoresis, the bacteria were recovered and ligated into the pET28a plasmid vector, which was then transformed into Escherichia coli DH5a cells. After enrichment culture, plasmids containing the target fragment were extracted and used as standards for subsequent experiments.

[0148] (3) The target gene was synthesized, ligated into the pET28a plasmid vector, transformed into Escherichia coli DH5a cells, enriched and cultured, and then the plasmid containing the target fragment was extracted and used as a standard for subsequent experiments.

[0149] In this embodiment, the standard was prepared using method (1) as a positive control, and the concentration of each standard plasmid sample in the standard plasmid sample mixture was 10. 14 Copy / L.

[0150] The negative control was ultrapure water.

[0151] 2. High-throughput quantitative detection

[0152] Following the arrangement guidelines in the SmartChip multi-sample nanosplitter instrument manual, 48 primer pairs and 36 samples (including 34 test samples, 1 positive control, and 1 negative control) were added to 384-well plates. The primers and samples were then evenly distributed onto an OSMG chip according to the multi-sample nanosplitter operating instructions. The reaction system and procedure were consistent with the conditions in the HT-qPCR validation section of the chip validation step. All qPCR reactions contained three technical replicates. Amplification within one plus or minus 1 CT value in the three technical replicates was considered positive. Samples successfully amplified in at least two technical replicates were considered positive and used for further analysis.

[0153] 3. Data Analysis of High-Throughput Detection

[0154] After the HT-qPCR program is completed, the software will automatically analyze and export the data for processing. The optimal CT cutoff value for the cycle is set to 33 for data screening, followed by abundance calculation. Gene abundance includes relative abundance (RA), comparative absolute abundance (CAA), and absolute abundance (AA).

[0155] The method for calculating relative abundance is as follows:

[0156] CG=10 (33-CT) / (10 / 3)

[0157] RA OSG =CG OSG / CG16s

[0158] Where CG represents gene abundance, CG OSG CG represents the abundance of organic sulfur metabolism genes. 16S The abundance of 16S rRNA genes.

[0159] The method for calculating comparative absolute abundance is as follows:

[0160] CAA OSG= RA OSG ×AA 16S

[0161] Among them, RA OSG The relative abundance of organic sulfur metabolism genes, AA 16S The absolute abundance of the 16S rRNA gene, AA 16S Calculated based on the standard curve formula for the 16S rRNA gene.

[0162] Absolute abundance is calculated based on the standard curve formula for each gene.

[0163] 4. Results Analysis of the OSMG Chip

[0164] Environmental samples were analyzed using the OSMG chip, and a total of 25 genes were detected. Data analysis revealed ( Figures 7-10 A total of 25 metabolic genes were detected in seawater from different stations, but their abundance varied considerably. Nearshore seawater showed higher gene abundance and richness than open-ocean seawater, with absolute abundance ranging from 1221 copies / L to 13,9857,9197 copies / L. At the same station but at different depths, the richness of metabolic genes generally decreased, but the absolute abundance of different genes did not change consistently. Sediment analysis revealed that the abundance and richness of organic sulfur metabolic genes generally decreased with increasing depth. Eight metabolic genes were detected in trench sediments, with absolute abundance ranging from 4354 copies / g to 13,4743 copies / g. Fourteen metabolic genes were detected in hydrothermal surface sediments, with absolute abundance ranging from 9582 copies / g to 150,3298 copies / g.

[0165] The above results indicate that the OSMG chip can effectively distinguish organic sulfur metabolism genes in various environments and can detect the distribution of various metabolism genes in different environments.

[0166] All references to this application are incorporated herein by reference as if each reference were individually incorporated herein by reference. Furthermore, it should be understood that after reading the foregoing teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A reagent combination for detecting genes related to organic sulfur cycle metabolism, characterized in that, The reagent combination includes a primer pair combination for specifically amplifying genes related to organosulfur cycling metabolism. This primer pair combination is used to detect these genes using high-throughput qPCR technology. The organosulfur cycling metabolism-related genes include... mmtN, burB, dsyB, dsyGD, megL, dddD, dddX, dddL, dddP, dddQ, dddW, dddY, dddK, dddU, dmdA, dmdB, dmdC, dmdD, acuH, mtoX, mtsA, mddA, mddH, dmoA, ddhA, dsoB, tmm, dmsA and dorA The primer pair combination includes: (1) Primer pairs consisting of SEQ ID No. 1 and SEQ ID No. 2, and primer pairs consisting of SEQ ID No. 3 and SEQ ID No. 4, are used for specific amplification. mmtN At least a portion of a gene; (2) The primer pair consisting of SEQ ID No. 5 and SEQ ID No. 6 is used for specific amplification. burB At least a portion of a gene; (3) The primer pair consisting of SEQ ID No. 7 and SEQ ID No. 8 is used for specific amplification. dsyB At least a portion of a gene; (4) The primer pair consisting of SEQ ID No. 9 and SEQ ID No. 10 is used for specific amplification. dsyGD At least a portion of a gene; (5) Primer pairs consisting of SEQ ID No. 11 and SEQ ID No. 12, and primer pairs consisting of SEQ ID No. 13 and SEQ ID No. 14, are used for specific amplification. megL At least a portion of a gene; (6) The primer pair consisting of SEQ ID No. 15 and SEQ ID No. 16 is used for specific amplification. dddD At least a portion of a gene; (7) Primer pairs consisting of SEQ ID No. 17 and SEQ ID No. 18, and primer pairs consisting of SEQ ID No. 19 and SEQ ID No. 20, are used for specific amplification. dddX At least a portion of a gene; (8) Primer pairs consisting of SEQ ID No. 21 and SEQ ID No. 22, and primer pairs consisting of SEQ ID No. 23 and SEQ ID No. 24, are used for specific amplification. dddL At least a portion of a gene; (9) The primer pair consisting of SEQ ID No. 25 and SEQ ID No. 26 is used for specific amplification. dddP At least a portion of a gene; (10) Primer pairs consisting of SEQ ID No. 27 and SEQ ID No. 28, primer pairs consisting of SEQ ID No. 29 and SEQ ID No. 30, and primer pairs consisting of SEQ ID No. 31 and SEQ ID No. 32 are used for specific amplification. dddQ At least a portion of a gene; (11) Primer pairs consisting of SEQ ID No. 33 and SEQ ID No. 34, and primer pairs consisting of SEQ ID No. 35 and SEQ ID No. 36, are used for specific amplification. dddW At least a portion of a gene; (12) The primer pair consisting of SEQ ID No. 37 and SEQ ID No. 38 is used for specific amplification. dddY At least a portion of a gene; (13) The primer pair consisting of SEQ ID No. 39 and SEQ ID No. 40 is used for specific amplification. dddK At least a portion of a gene; (14) The primer pair consisting of SEQ ID No. 41 and SEQ ID No. 42 is used for specific amplification. dddU At least a portion of a gene; (15) Primer pairs consisting of SEQ ID No. 43 and SEQ ID No. 44, and primer pairs consisting of SEQ ID No. 45 and SEQ ID No. 46, are used for specific amplification. acuH At least a portion of a gene; (16) Primer pairs consisting of SEQ ID No. 47 and SEQ ID No. 48, and primer pairs consisting of SEQ ID No. 49 and SEQ ID No. 50, are used for specific amplification. dmdA At least a portion of a gene; (17) Primer pairs consisting of SEQ ID No. 51 and SEQ ID No. 52, primer pairs consisting of SEQ ID No. 53 and SEQ ID No. 54, and primer pairs consisting of SEQ ID No. 55 and SEQ ID No. 56 are used for specific amplification. dmdB At least a portion of a gene; (18) Primer pairs consisting of SEQ ID No. 57 and SEQ ID No. 58, and primer pairs consisting of SEQ ID No. 59 and SEQ ID No. 60, are used for specific amplification. dmdC At least a portion of a gene; (19) Primer pairs consisting of SEQ ID No. 61 and SEQ ID No. 62, and primer pairs consisting of SEQ ID No. 63 and SEQ ID No. 64, are used for specific amplification. dmdD At least a portion of a gene; (20) Primer pairs consisting of SEQ ID No. 65 and SEQ ID No. 66, and primer pairs consisting of SEQ ID No. 67 and SEQ ID No. 68, are used for specific amplification. mtoX At least a portion of a gene; (21) Primer pairs consisting of SEQ ID No. 69 and SEQ ID No. 70, primer pairs consisting of SEQ ID No. 71 and SEQ ID No. 72, and primer pairs consisting of SEQ ID No. 73 and SEQ ID No. 74 are used for specific amplification. mddA At least a portion of a gene; (22) Primer pairs consisting of SEQ ID No. 75 and SEQ ID No. 76, and primer pairs consisting of SEQ ID No. 77 and SEQ ID No. 78, are used for specific amplification. mddH At least a portion of a gene; (23) The primer pair consisting of SEQ ID No. 79 and SEQ ID No. 80 is used for specific amplification. mtsA At least a portion of a gene; (24) The primer pair consisting of SEQ ID No. 81 and SEQ ID No. 82 is used for specific amplification. dmoA At least a portion of a gene; (25) The primer pair consisting of SEQ ID No. 83 and SEQ ID No. 84 is used for specific amplification. ddhA At least a portion of a gene; (26) Primer pairs consisting of SEQ ID No. 85 and SEQ ID No. 86, and primer pairs consisting of SEQ ID No. 87 and SEQ ID No. 88, are used for specific amplification. dsoB At least a portion of a gene; (27) The primer pair consisting of SEQ ID No. 89 and SEQ ID No. 90 is used for specific amplification. tmm At least a portion of a gene; (28) The primer pair consisting of SEQ ID No. 91 and SEQ ID No. 92 is used for specific amplification. dmsA At least a portion of a gene; (29) The primer pair consisting of SEQ ID No. 93 and SEQ ID No. 94 is used for specific amplification. dorA At least a portion of a gene.

2. The reagent combination according to claim 1, characterized in that, The primer pair combination also includes: The primer pair consisting of SEQ ID No. 95 and SEQ ID No. 96 is used to specifically amplify at least a partial fragment of the 16S rRNA gene.

3. A gene chip, characterized in that, The gene chip comprises the reagent combination as described in claim 1 or 2.

4. A reagent kit, characterized in that, The kit comprises the reagent combination of claim 1 or 2 or the gene chip of claim 3.

5. The reagent kit according to claim 4, characterized in that, The kit also includes genomic DNA extraction reagents for the sample to be tested, DNA purification reagents, and / or PCR amplification buffers.

6. The kit according to claim 4 or 5, characterized in that, The kit also includes a positive control and / or a negative control, wherein the positive control is a mixed sample composed of plasmid standards corresponding to the primer pair combinations.

7. A method for detecting genes related to organic sulfur cycle metabolism in marine samples, characterized in that, Includes the following steps: S1, Obtain metagenomic DNA from the marine sample to be tested; S2, perform qPCR analysis on the metagenomic DNA using the reagent combination of claim 1 or 2, or the gene chip of claim 3, or the kit of any one of claims 4 to 6, and perform the qPCR analysis using high-throughput qPCR technology; S3, the abundance of the genes related to the organic sulfur cycle metabolism was obtained from the gene qPCR analysis results.

8. The method according to claim 7, characterized in that, The marine samples are seawater or trench sediments.

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