Reagent combination, kit and method for detecting organic sulfur cycle metabolism related genes

Abstract

The invention 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 provided by the invention have very high sensitivity, specificity and coverage in detection of organic sulfur cycle metabolism related genes. The method disclosed by the invention is based on HT-qPCR, can simultaneously perform quantitative detection on a plurality of environmental samples, can well distinguish different organic sulfur cycle metabolism genes in different environments while reducing the detection cost, performs absolute quantitative and relative quantitative detection on the abundance of the metabolism genes, provides a high-throughput molecular tool for related research of organic sulfur cycle, and has a wide application prospect. The defects that qPCR detection flux is low and absolute quantification cannot be achieved in metagenome sequencing are effectively overcome, and powerful technical support is provided for ecology research.

Description

Technical Field

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

[0002] The organic sulfur cycle is a process in nature where various organic sulfur molecules are transformed or converted from an organic form to an inorganic form, and it is an important part of the global sulfur cycle. Important metabolites in the organic sulfur cycle include dimethylsulfoniopropionate (DMSP), dimethyl sulfide (DMS), methanethiol (MeSH), dimethyl sulfoxide (DMSO), etc. Among them, DMSP is one of the most abundant organic sulfur molecules on Earth and can serve as an osmoprotectant, stress protectant, antioxidant, cryoprotectant, predator defense agent, etc. for phytoplankton and various algae. DMS is a "greenhouse gas" produced by the catabolism of DMSP. After DMS enters the atmosphere, it undergoes an oxidation reaction, and the oxidation products coagulate to promote the formation of clouds as cloud condensation nuclei, enhancing the reflectivity of sunlight, thereby having a negative regulatory effect on global warming. MeSH is an important intermediate metabolite in the conversion between 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 osmoprotectant and antioxidant in microorganisms, DMSO can also be reduced to DMS, affecting climate change. Therefore, studying the metabolic process of organic sulfur can not only help understand the growth and metabolic characteristics of microorganisms and algae and the structural functions of biological communities, but also contribute to evaluating the contribution of the ocean to climate regulation and predicting potential trends in climate change, providing rich basic data for biogeochemistry and environmental science.

[0003] In the prior art, the research on the organic sulfur cycle is mainly carried out by methods such as metagenomic sequencing, amplicon sequencing, and real-time fluorescence quantitative PCR (qPCR). However, metagenomic sequencing has high costs, long sequencing times, and relatively complex result data processing; amplicon sequencing cannot perform absolute quantification of functional genes; although the qPCR method reduces the amount of information and can quickly perform absolute quantification on various environmental samples, it has high costs and is difficult to correct between different batches of multiple samples.

[0004] High-throughput qPCR technology (HT-qPCR) can simultaneously perform quantitative detection on dozens of functional genes, and the abundance information of multiple genes can be obtained through only one experiment. It has many advantages such as high speed, high throughput, and low cost, providing a more convenient and efficient detection method for the research of key metabolic genes in the organic sulfur cycle. Therefore, in order to accelerate the research progress of organic sulfur metabolism, it is urgent to develop a high-throughput qPCR detection chip for organic sulfur cycle metabolism genes. Summary of the Invention

[0005] To solve at least one of the above technical problems, the technical solutions adopted in this application are as follows.

[0006] In the first aspect of this application, a reagent combination for detecting genes related to the organic sulfur cycle metabolism is provided. The reagent combination includes a primer pair combination for specifically amplifying genes related to the organic sulfur cycle metabolism, and the genes related to the organic sulfur cycle 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 feasible embodiments of this application, the primer pair combination includes:

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

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

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

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

[0012] (5) A primer pair composed of SEQ ID No. 11 and SEQ ID No. 12, and a primer pair composed of SEQ ID No. 13 and SEQ ID No. 14, which are used to specifically amplify at least a partial fragment of the megL gene;

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

[0014] The primer pairs consisting of SEQ ID No. 17 and SEQ ID No. 18, and the primer pairs consisting of SEQ ID No. 19 and SEQ ID No. 20 are used for specifically amplifying at least partial fragments of the dddX gene;

[0015] (8)The primer pairs consisting of SEQ ID No. 21 and SEQ ID No. 22, and the primer pairs consisting of SEQ ID No. 23 and SEQ ID No. 24 are used for specifically amplifying at least partial fragments of the dddL gene;

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

[0017] (10)The primer pairs consisting of SEQ ID No. 27 and SEQ ID No. 28, the primer pairs consisting of SEQ ID No. 29 and SEQ ID No. 30, and the primer pairs consisting of SEQ ID No. 31 and SEQ ID No. 32 are used for specifically amplifying at least partial fragments of the dddQ gene;

[0018] (11)The primer pairs consisting of SEQ ID No. 33 and SEQ ID No. 34, and the primer pairs consisting of SEQ ID No. 35 and SEQ ID No. 36 are used for specifically amplifying at least partial fragments of the dddW gene;

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

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

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

[0022] (15)The primer pairs consisting of SEQ ID No. 43 and SEQ ID No. 44, and the primer pairs consisting of SEQ ID No. 45 and SEQ ID No. 46 are used for specifically amplifying at least partial fragments of the acuH gene;

[0023] (16)The primer pairs consisting of SEQ ID No. 47 and SEQ ID No. 48, and the primer pairs consisting of SEQ ID No. 49 and SEQ ID No. 50, are used for specifically amplifying at least partial fragments of the dmdA gene;

[0024] (17)The primer pairs consisting of SEQ ID No. 51 and SEQ ID No. 52, the primer pairs consisting of SEQ ID No. 53 and SEQ ID No. 54, and the primer pairs consisting of SEQ ID No. 55 and SEQ ID No. 57, are used for specifically amplifying at least partial fragments of the dmdB gene;

[0025] (18)The primer pairs consisting of SEQ ID No. 57 and SEQ ID No. 58, and the primer pairs consisting of SEQ ID No. 59 and SEQ ID No. 60, are used for specifically amplifying at least partial fragments of the dmdC gene;

[0026] (19)The primer pairs consisting of SEQ ID No. 61 and SEQ ID No. 62, and the primer pairs consisting of SEQ ID No. 63 and SEQ ID No. 64, are used for specifically amplifying at least partial fragments of the dmdD gene;

[0027] (20)The primer pairs consisting of SEQ ID No. 65 and SEQ ID No. 66, and the primer pairs consisting of SEQ ID No. 67 and SEQ ID No. 68, are used for specifically amplifying at least partial fragments of the mtoX gene;

[0028] (21)The primer pairs consisting of SEQ ID No. 69 and SEQ ID No. 70, the primer pairs consisting of SEQ ID No. 71 and SEQ ID No. 72, and the primer pairs consisting of SEQ ID No. 73 and SEQ ID No. 74, are used for specifically amplifying at least partial fragments of the mddA gene;

[0029] (22)The primer pairs consisting of SEQ ID No. 75 and SEQ ID No. 76, and the primer pairs consisting of SEQ ID No. 77 and SEQ ID No. 78, are used for specifically amplifying at least partial fragments of the mddH gene;

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

[0031] (24)A primer pair consisting of SEQ ID No. 81 and SEQ ID No. 82, which is used for specifically amplifying at least a partial fragment of the dmoA gene;

[0032] (25)A primer pair consisting of SEQ ID No. 83 and SEQ ID No. 84, which is used for specifically amplifying at least a partial fragment of the ddhA gene;

[0033] (26)A primer pair consisting of SEQ ID No. 85 and SEQ ID No. 86, and a primer pair consisting of SEQ ID No. 87 and SEQ ID No. 88, which are used for specifically amplifying at least a partial fragment of the dsoB gene;

[0034] (27)A primer pair consisting of SEQ ID No. 89 and SEQ ID No. 90, which is used for specifically amplifying at least a partial fragment of the tmm gene;

[0035] (28)A primer pair consisting of SEQ ID No. 91 and SEQ ID No. 92, which is used for specifically amplifying at least a partial fragment of the dmsA gene;

[0036] (29)A primer pair consisting of SEQ ID No. 93 and SEQ ID No. 94, which is used for specifically amplifying at least a partial fragment of the dorA gene.

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

[0038] A primer pair consisting of SEQ ID No. 95 and SEQ ID No. 96, which is used for specifically amplifying at least a partial fragment of the 16S rRNA gene.

[0039] The second aspect of the present application provides a gene chip, which includes any of the reagent combinations described in the first aspect of the present application.

[0040] The third aspect of the present application provides a kit, which includes any of the reagent combinations described in the first aspect of the present application or the gene chip described in the second aspect of the present application.

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

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

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

[0044] S1, obtaining the metagenomic DNA of the marine sample to be tested;

[0045] S2, performing qPCR analysis on the metagenomic DNA by using any of the reagent combinations described in the first aspect of the present application, or the gene chip described in the second aspect of the present application, or any of the reagent kits described in the third aspect of the present application;

[0046] S3, obtaining the abundance of the genes related to the organic sulfur cycle metabolism based on the results of the gene qPCR analysis.

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

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

[0049] Compared with the prior art, the present application has the following beneficial effects:

[0050] Through a rigorous and meticulous degenerate primer design process, the present application designs degenerate primers for 29 currently known genes related to the organic sulfur cycle metabolism, and verifies the degenerate primers through a series of technical means such as computer verification and experimental verification, and obtains a set of primer combinations with very high sensitivity, specificity and coverage. Further combined with the high-throughput qPCR (HT-qPCR) technology, a gene chip capable of detecting multiple genes related to the organic sulfur cycle metabolism is constructed and successfully applied to the research of various environmental samples.

[0051] The gene chip of the present application can perform 5184 quantitative PCR reactions in one detection, can simultaneously perform quantitative detection of multiple environmental samples, while reducing the detection cost, can well distinguish different genes related to the organic sulfur cycle metabolism in different environments, and perform absolute and relative quantitative detection of their abundances, providing a high-throughput molecular tool for the related research of the organic sulfur cycle, effectively complementing the disadvantages of low detection throughput of qPCR and inability to perform absolute quantification of metagenomic sequencing, and providing strong technical support for the research of ecology.

[0052] It should be understood that the content described in this part is not intended to identify the key or important features of the embodiments of the present application, nor is it used to limit the scope of the present application. Other features of the present application will become easily understood through the following description. Description of the Drawings

[0053] By reading the following detailed description with reference to the accompanying drawings, the above and other objects, features, and advantages of the exemplary embodiments of the present application will become readily understandable. In the drawings, several embodiments of the present application are shown in an exemplary rather than restrictive manner, where:

[0054] Figure 1 Shows the phylogenetic tree of 29 organic sulfur metabolism genes in Example 1 of the present application.

[0055] Figure 2 Shows the amplification results of the primer pairs corresponding to the dmdB gene in the PCR verification in Example 2 of the present application. A: Experimental group. B: Negative control group.

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

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

[0058] Figure 5 Shows the amplification efficiency of 53 primer pairs in Example 2 of the present application.

[0059] Figure 6 Shows the standard curve of the mixed plasmid standard product under 10-fold gradient dilution in Example 2 of the present application.

[0060] Figure 7 Shows the absolute abundances of various organic sulfur cycle metabolism genes obtained by analyzing submarine sediment samples using the OSMG chip in Example 3 of the present application.

[0061] Figure 8 Shows the relative abundances of various organic sulfur cycle metabolism genes obtained by analyzing submarine sediment samples using the OSMG chip in Example 3 of the present application.

[0062] Figure 9 Shows the absolute abundances of various organic sulfur cycle metabolism genes obtained by analyzing seawater samples using the OSMG chip in Example 3 of the present application.

[0063] Figure 10 Shows the relative abundances of various organic sulfur cycle metabolism genes obtained by analyzing seawater samples using the OSMG chip in Example 3 of the present application. Detailed implementation manners

[0064] Unless otherwise indicated, implied by the context, or conventional in the art, all parts and percentages in this application are based on weight, and the test and characterization methods used are contemporaneous with the filing date of this application. To the extent applicable, any patents, patent applications, or publications referred to in this application are hereby incorporated by reference in their entirety, and their equivalent family patents are also incorporated by reference, particularly the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0065] In order to make the technical problems, technical solutions, and beneficial effects solved by this application more clearly understood, the following further details this application in conjunction with embodiments.

[0066] The following examples are used herein to demonstrate the preferred embodiments of this application. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques that the inventors have found can be used to implement this application and can therefore be regarded as preferred embodiments for implementing 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 and still obtain 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 belongs. All references cited herein and the materials they cite will be incorporated by reference.

[0068] Those skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the inventions described herein through routine experimentation. Such equivalents will be included in the claims.

[0069] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instrumentation used in the following examples is all conventional laboratory instrumentation unless otherwise specified; the test materials used in the following examples are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0070] Example 1 Chip Design

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

[0072] Search the literature related to the organic sulfur cycle metabolism, collect all the protein sequences and gene sequences with related functions involved in the literature, and finally download 144 amino acid sequences and 132 nucleotide sequences for 29 genes.

[0073] Among them, the functional information of 29 genes is shown in Table 1.

[0074] Table 1 Genes related to the metabolism of organic sulfur cycle and their functions

[0075]

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

[0077] Retrieve homologous proteins through the public databases NCBI (https: / / www.ncbi.nlm.nih.gov) and Uniprot (https: / / www.ncbi.nlm.nih.gov) and download the sequence files. The retrieval is carried out in two steps:

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

[0079] In the second step, use BLASTp to further screen out protein sequences with a similarity greater than 40% and an e-value less than 10 -30 .

[0080] Finally, a total of 5477 amino acid sequences and 5439 nucleotide sequences were downloaded for the 29 genes.

[0081] 3. Constructing a phylogenetic tree

[0082] Construct a phylogenetic tree for the homologous protein sequences of each functional gene. Use the MAFFT program for sequence alignment and IQ-TREE2 v2.2.2.7 to construct the phylogenetic tree, with the bootstrap value set to 1000. Thus, the phylogenetic trees of 29 organic sulfur metabolism genes are obtained, as Figure 1 shown.

[0083] 4. Primer design

[0084] Design primers for each cluster of the phylogenetic tree, using the following 3 methods:

[0085] (A) Use the j-CODEHOP program in the Base-By-Base software to design degenerate primers. The parameter settings are: primer length between 16 and 40 bases, GC content 40% - 60%, annealing temperature 60 °C, amplified sequence length 80 - 400 bases, maximum degeneracy of the core region 256, and at least 3 consecutive amino acid conserved regions. 66 pairs of primers were designed through this method.

[0086] (B) Degenerate primer design was performed using Geneious Primer software with the following parameter settings: primer length between 16 and 35 bases, GC content of 40% - 60%, annealing temperature of 60 °C, amplified sequence length of 80 - 400 bases, and maximum degeneracy of 256. A total of 537 primer pairs were obtained by 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, and it was reverse-translated into a nucleotide sequence according to the codon bias of the species containing the gene. The following parameters were used to select a suitable region: primer length between 16 and 35 bases, GC content of 40% - 60%, annealing temperature of 60 °C, amplified sequence length of 80 - 400 bases, and maximum degeneracy of 256. A total of 7 primer pairs were obtained by this method.

[0088] A total of 610 new primer pairs were obtained by the above methods. At the same time, 2 primer pairs that had been applied to environmental samples were collected from the literature, and a total of 612 primer pairs were used for subsequent chip verification. The statistics are shown in Table 2:

[0089] Table 2 Statistical table of the number of primer pairs designed for each gene using three methods

[0090]

[0091] Taking dmdB and dddX as examples, the designed primers 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 verification and screening

[0100] The primers are judged to meet the expectations by the size of the products amplified by PCR. Using the DNA in each environmental sample as a template, the DNA was extracted using the DNeay PowerSoil Pro Kit (QIANGEN) kit, and then purified using the DNeasyPowerClean Pro Clean Kit (QIANGEN) kit. The quality of the DNA was detected using a NanoDrop ND-2000 ultra-micro spectrophotometer. A qualified quality is indicated by an OD260 / 280 value range between 1.6 and 2.0; the concentration of the DNA was measured using Qubit 3.0, and a concentration range above 10 ng / μL indicates that the concentration meets the detection requirements, and the total DNA quality requirement is above 500 ng.

[0101] The PCR system was 50 μL: containing 25 μL of 2×rTaq Mix, 0.5 μM primers, and 1 ng / μL of DNA template.

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

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

[0104] The agarose gel electrophoresis results of the amplification products of some primer pairs designed for the dmdB gene are as Figure 2 shown. As can be seen from Figure 2 , primer dimer bands appeared in the three pairs of primers numbered 25, 29, and 31, so they were excluded; non-specific amplification occurred in the two pairs of primers numbered 17 and 22, so they were excluded; the two pairs of primers numbered 19 and 26 conformed to the rules of PCR verification, so they were retained. Finally, three pairs of primers numbered 19, 26, and 30 were screened out and renamed dmdB1.1, dmdB1.2, and dmdB1.3.

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

[0106] Through PCR screening, 523 pairs of primers were excluded from 612 pairs of primers, and 89 pairs of primers with correct PCR verification were subjected to subsequent verification.

[0107] 2. Amplicon sequencing verification and screening

[0108] Select the PCR-verified primer pairs and the above DNA as templates, perform PCR amplification again, cut and recover the bands of the correct size by gel electrophoresis, construct a library according to the standard procedure of the NEB Next Ultra DNA library Prep Kit for Illumina, and perform PE250 sequencing on the constructed amplicon library using the Illumina platform. Analysis of the amplicon data showed that 94.4% of the amplicons had more than 80% similarity to the corresponding targets. Through amplicon sequencing verification, 5 pairs of primers were excluded from 89 pairs of primers, and 84 pairs of primers with correct verification were subjected to subsequent verification.

[0109] 3. qPCR Verification

[0110] Use real-time fluorescence quantitative PCR (qPCR) to detect the amplification efficiency of the above primer pairs. First, prepare plasmid standards, extract plasmid DNA containing the target gene, and dilute the plasmid to an initial copy number of approximately 10 9 copies for each target gene, and then perform 10-fold serial dilution to 10 2 copies. Use a qPCR instrument for detection to obtain a standard curve.

[0111] The qPCR system is 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 copy number of each gene.

[0112] The qPCR program is: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 60°C for 30 s, extension at 72°C for 10 s, for 35 cycles, and finally increase the temperature from 72°C to 97°C at a rate of 4°C / s, and perform a melting curve determination with a measurement point of 0.4°C.

[0113] All qPCR reactions contain 3 technical replicates. Samples that are successfully amplified in at least two technical replicates are considered positive detections and used for further analysis. The melting curve is used to determine whether there are dimers in the primer pairs, which is automatically generated by the ABI software. The standard curve fitting uses linear regression analysis of R 4.0.3, and the amplification efficiency is calculated according to the slope of the standard curve. The formula is Eff = 10(-1 / slope)-1 to obtain the amplification efficiency of each pair of primers. The ideal amplification efficiency is between 80% and 120%.

[0114] Take Figure 3 megL-A2H1 in

[0115] TakeFigure 4 Taking burB-A10 in [reference] as an example, since the amplification efficiency of this primer pair is less than 80%, it was excluded.

[0116] As Figure 5 shown, the amplification efficiencies of 53 primer pairs were between 80% and 120%, with an average of 97.78%, and the R 2 -squared values were between 0.983 and 0.999, with an average of 0.991. Since primers were designed separately for each cluster after phylogenetic tree construction for each gene, only 1 primer pair was retained for each cluster, resulting in 47 primer pairs for subsequent verification. Information on the 47 primer pairs is shown in Table 5.

[0117] Table 5 Information on 47 primer pairs

[0118]

[0119] Table 5 (continued) Information on 47 primer pairs

[0120]

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

[0122] 4. HT-qPCR verification

[0123] The SmartChip Real-time PCR system (Thermo Fisher) was used to test the sensitivity of the primer pairs for detecting the OSMG chip. The standards were the same as the samples in the qPCR verification step. The qPCR amplification reaction system was prepared with a total volume of 100 nL in each 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: pre-denaturation at 95°C for 10 min; pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 60°C for 30 s, extension at 72°C for 10 s, for 35 cycles, and finally heating from 72°C to 97°C at a rate of 4°C / s, with a melting curve measured at 0.4°C intervals.

[0124] Obtain the amplification results and conduct data analysis; determine the optimal cycle CT cut-off value according to the standard curve. Evaluate the sensitivity of the chip through the Limit of Detection (LoD) and the Limit of Quantitation (LoQ) (Table 4). The LoD is the lowest copy number of the target nucleic acid that can be detected. The LoQ is the lowest copy number of the target nucleic acid that can be accurately quantified, and the basis for evaluating the precision is the Coefficient of Variation (CV). When CV ≤ 35%, the data is considered acceptable. The calculation method of the coefficient of variation is:

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

[0126] According to Figure 6 It can be seen that when the copy number is 10 2 , stable Ct values can already be obtained, and the LoD and LoQ calculated according to the standard curve are shown in Table 6.

[0127] Table 6 CT values, detection limits, and quantification limits of the OSMG chip

[0128]

[0129] As can be seen from Table 6, the LoD range of the OSMG chip is between 1 - 13,674 copies / reaction, with an average value of 634 copies / reaction. The LoQ range is between 64 - 57,074 copies / reaction, with an average value of 2,918 copies / reaction. The above results indicate that the OSMG chip has high sensitivity.

[0130] 5. Computer verification

[0131] To test the coverage and specificity of the OSMG chip, computer simulation was used to test the coverage and specificity of the primer pairs in the OSMG chip. By retrieving the homologous protein sequences of the organosulfur metabolism genes from the NCBI and Uniprot databases, a total of 29 primer evaluation databases were constructed. Each pair of primers was aligned with the primer evaluation database to evaluate the coverage and specificity of each primer pair. The maximum allowable number of mismatched bases was 2. The primer pair coverage is the percentage of the sequences simultaneously matched by the forward primer and the reverse primer in the total number of sequences in each primer evaluation database; the specificity is the proportion of the retrieved sequences belonging to the target gene database; the false positive rate is the percentage of the number of false positive sequences observed in the alignment divided by the total number of sequences in the primer evaluation database.

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

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

[0134]

[0135] Note: The coverage is calculated as the sum of the coverages of all primer pairs for each gene.

[0136] As can be seen from Table 7, the coverage of the primers involved in the OSMG chip ranges from 10% to 100%, the average coverage is 36.6%, and the number of sequences with 100% specificity is greater than 95% of the total. The above results indicate that the OSMG chip has high specificity and coverage.

[0137] Example 3 Application of OSMG Chip

[0138] 1. Sample collection and preparation

[0139] The environmental samples include two types: seawater samples and seafloor sediment samples. The seawater samples include 1 surface seawater sample from the coast of Qingdao and 20 surface and deep seawater samples from the western Pacific Ocean. 1 L of seawater is filtered through 3 μm and 0.2 μm filters, and then DNA is extracted. The seafloor sediment samples include 1 sediment sample from the South China Sea, 7 hydrothermal sediment samples, and 6 trench sediment samples, a total of 34 samples. The obtained environmental samples are used to extract metagenomic DNA using the DNA extraction method in the PCR verification step, and the quality and concentration are detected. The results are shown in Table 8, and the qualified DNA is used as the test sample for subsequent detection.

[0140] Table 8 DNA concentration and quality of environmental samples

[0141]

[0142] Introduce primer pairs specific for amplifying the 16S rRNA gene:

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

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

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

[0146] (1) Use 48 primer pairs with restriction enzyme sites to perform PCR on environmental DNA. After agarose gel electrophoresis, gel extraction is performed, and it is ligated to the pET28a plasmid vector, transformed into Escherichia coli DH5a cells, and after enrichment culture, the plasmid containing the target fragment is extracted and used as a standard for subsequent experiments.

[0147] (2) PCR was performed on pure bacteria containing the target gene using 48 primers with restriction sites. The fragment was recovered after agarose gel electrophoresis, connected to the pET28a plasmid vector, and transformed into Escherichia coli DH5a cells. After enrichment culture, the plasmid containing the target fragment was extracted and used as a standard for subsequent experiments.

[0148] (3) The target gene is synthesized, connected to the pET28a plasmid vector, and transformed into Escherichia coli DH5a cells. After enrichment culture, the plasmid containing the target fragment is extracted and used as a standard for subsequent experiments.

[0149] In this example, the method (1) was used to prepare the standard sample as the positive control. 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] According to the arrangement principle in the SmartChip multi-sample nanodispenser instrument instructions, 48 ​​primer pairs and 36 samples (including 34 samples to be tested, 1 positive control and 1 negative control) were added to the 384-well plate, and then the primers and samples were evenly distributed on an OSMG chip according to the operating instructions of the multi-sample nanodispenser. The reaction system and reaction procedure were consistent with the conditions in the HT-qPCR validation in the chip validation link. All qPCR reactions contained 3 technical replicates, and the amplification of the CT value within plus or minus 1 CT value in the three technical replicates was considered positive. At the same time, samples that were successfully amplified in at least two technical replicates were considered positive detection 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, set the optimal CT cutoff value of the cycle to 33, perform data screening, and then calculate the abundance. Gene abundance includes relative abundance (Relative Abundance, RA), comparative absolute abundance (Comparative Absolute Abundance, CAA) and absolute abundance (Absolute Abundance, AA).

[0155] The relative abundance is calculated as:

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

[0157] RA OSG =CG OSG / CG16s

[0158] Among them, CG is the gene abundance, and CG OSG is the abundance of the organic sulfur metabolism gene, and CG 16S is the abundance of the 16S rRNA gene.

[0159] The calculation method of the comparative absolute abundance is as follows:

[0160] CAA OSG= RA OSG ×AA 16S

[0161] Among them, RA OSG is the relative abundance of the organic sulfur metabolism gene, and AA 16S is the absolute abundance of the 16S rRNA gene, and AA 16S is calculated according to the standard curve formula of the 16S rRNA gene.

[0162] The absolute abundance is calculated according to the standard curve formula of each gene to obtain the corresponding gene absolute abundance.

[0163] 4. Result analysis of the OSMG chip

[0164] Using the OSMG chip to detect environmental samples, a total of 25 genes were detected. After data analysis, it was found that ( Figures 7 to 10 ), in the seawater at different stations, a total of 25 types of metabolic genes were detected, but there were significant differences in gene abundances. The gene abundances and richness in the coastal seawater were both higher than those in the open ocean seawater, and the absolute abundances ranged from 1221 copies / L to 13,9857,9197 copies / L. In the seawater at the same station and different depths, the richness of metabolic genes generally showed a decreasing trend, but the changes in the absolute abundances of different genes were not consistent. According to the detection results in sediments, the abundances and richness of the organic sulfur metabolism gene generally decreased with the increase in depth. A total of 8 types of metabolic genes were detected in the trench sediments, and the absolute abundances ranged from 4354 copies / g to 13,4743 copies / g. A total of 14 metabolic genes were detected in the hydrothermal surface sediments, and the absolute abundances ranged from 9582 copies / g to 150,3298 copies / g.

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

[0166] All documents mentioned in this application are incorporated herein by reference as if each individual document was specifically and individually incorporated by reference. In addition, it should be understood that after reading the above teachings of this application, those skilled in the art can make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A reagent combination for detecting genes related to the organic sulfur cycle metabolism, characterized in that, The reagent combination includes a primer pair combination for specifically amplifying genes related to the organic sulfur cycle metabolism, and the genes related to the organic sulfur cycle 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.

2. The reagent combination according to claim 1, wherein The primer pair combination includes: (1) A primer pair composed of SEQ ID No. 1 and SEQ ID No. 2, and a primer pair composed of SEQ ID No. 3 and SEQ ID No. 4, for specifically amplifying at least a partial fragment of the mmtN gene; (2) A primer pair composed of SEQ ID No. 5 and SEQ ID No. 6, for specifically amplifying at least a partial fragment of the burB gene; (3) A primer pair composed of SEQ ID No. 7 and SEQ ID No. 8, for specifically amplifying at least a partial fragment of the dsyB gene; (4) A primer pair composed of SEQ ID No. 9 and SEQ ID No. 10, for specifically amplifying at least a partial fragment of the dsyGD gene; (5) A primer pair composed of SEQ ID No. 11 and SEQ ID No. 12, and a primer pair composed of SEQ ID No. 13 and SEQ ID No. 14, for specifically amplifying at least a partial fragment of the megL gene; (6) A primer pair composed of SEQ ID No. 15 and SEQ ID No. 16, for specifically amplifying at least a partial fragment of the dddD gene; (7) A primer pair composed of SEQ ID No. 17 and SEQ ID No. 18, and a primer pair composed of SEQ ID No. 19 and SEQ ID No. 20, for specifically amplifying at least a partial fragment of the dddX gene; (8) A primer pair composed of SEQ ID No. 21 and SEQ ID No. 22, and a primer pair composed of SEQ ID No. 23 and SEQ ID No. 24, for specifically amplifying at least a partial fragment of the dddL gene; (9) A primer pair composed of SEQ ID No. 25 and SEQ ID No. 26, for specifically amplifying at least a partial fragment of the dddP gene; (10) A primer pair composed of SEQ ID No. 27 and SEQ ID No. 28, a primer pair composed of SEQ ID No. 29 and SEQ ID No. 30, and a primer pair composed of SEQ ID No. 31 and SEQ ID No. 32, for specifically amplifying at least a partial fragment of the dddQ gene; (11)The primer pair consisting of SEQ ID No. 33 and SEQ ID No. 34, and the primer pair consisting of SEQ ID No. 35 and SEQ ID No. 36, which are used for specifically amplifying at least a partial fragment of the dddW gene; (12)The primer pair consisting of SEQ ID No. 37 and SEQ ID No. 38, which is used for specifically amplifying at least a partial fragment of the dddY gene; (13)The primer pair consisting of SEQ ID No. 39 and SEQ ID No. 40, which is used for specifically amplifying at least a partial fragment of the dddK gene; (14)The primer pair consisting of SEQ ID No. 41 and SEQ ID No. 42, which is used for specifically amplifying at least a partial fragment of the dddU gene; (15)The primer pair consisting of SEQ ID No. 43 and SEQ ID No. 44, and the primer pair consisting of SEQ ID No. 45 and SEQ ID No. 46, which are used for specifically amplifying at least a partial fragment of the acuH gene; (16)The primer pair consisting of SEQ ID No. 47 and SEQ ID No. 48, and the primer pair consisting of SEQ ID No. 49 and SEQ ID No. 50, which are used for specifically amplifying at least a partial fragment of the dmdA gene; (17)The primer pair consisting of SEQ ID No. 51 and SEQ ID No. 52, the primer pair consisting of SEQ ID No. 53 and SEQ ID No. 54, and the primer pair consisting of SEQ ID No. 55 and SEQ ID No. 57, which are used for specifically amplifying at least a partial fragment of the dmdB gene; (18)The primer pair consisting of SEQ ID No. 57 and SEQ ID No. 58, and the primer pair consisting of SEQ ID No. 59 and SEQ ID No. 60, which are used for specifically amplifying at least a partial fragment of the dmdC gene; (19)The primer pair consisting of SEQ ID No. 61 and SEQ ID No. 62, and the primer pair consisting of SEQ ID No. 63 and SEQ ID No. 64, which are used for specifically amplifying at least a partial fragment of the dmdD gene; (20)The primer pair consisting of SEQ ID No. 65 and SEQ ID No. 66, and the primer pair consisting of SEQ ID No. 67 and SEQ ID No. 68, which are used for specifically amplifying at least a partial fragment of the mtoX gene; (21)The primer pair consisting of SEQ ID No. 69 and SEQ ID No. 70, the primer pair consisting of SEQ ID No. 71 and SEQ ID No. 72, and the primer pair consisting of SEQ ID No. 73 and SEQ ID No. 74, which are used for specifically amplifying at least a partial fragment of the mddA gene; The primer pairs consisting of SEQ ID No. 75 and SEQ ID No. 76, and the primer pairs consisting of SEQ ID No. 77 and SEQ ID No. 78 are used to specifically amplify at least partial fragments of the mddH gene; The primer pair consisting of SEQ ID No. 79 and SEQ ID No. 80 is used to specifically amplify at least partial fragments of the mtsA gene; The primer pair consisting of SEQ ID No. 81 and SEQ ID No. 82 is used to specifically amplify at least partial fragments of the dmoA gene; The primer pair consisting of SEQ ID No. 83 and SEQ ID No. 84 is used to specifically amplify at least partial fragments of the ddhA gene; The primer pairs consisting of SEQ ID No. 85 and SEQ ID No. 86, and the primer pairs consisting of SEQ ID No. 87 and SEQ ID No. 88 are used to specifically amplify at least partial fragments of the dsoB gene; The primer pair consisting of SEQ ID No. 89 and SEQ ID No. 90 is used to specifically amplify at least partial fragments of the tmm gene; The primer pair consisting of SEQ ID No. 91 and SEQ ID No. 92 is used to specifically amplify at least partial fragments of the dmsA gene; The primer pair consisting of SEQ ID No. 93 and SEQ ID No. 94 is used to specifically amplify at least partial fragments of the dorA gene.

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

4. A gene chip, characterized in that, The gene chip includes the reagent combination according to any one of claims 1 to 3.

5. A kit, characterized in that, The kit includes the reagent combination according to any one of claims 1 to 3 or the gene chip according to claim 4.

6. The kit according to claim 5, characterized in that, The kit further includes a reagent for extracting genomic DNA of a sample to be tested, a DNA purification reagent, and / or a PCR amplification buffer.

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

8. A method for detecting genes related to the organic sulfur cycle metabolism in marine samples, characterized in that, It includes the following steps: S1, obtaining the metagenomic DNA of the marine sample to be tested; S2, performing qPCR analysis on the metagenomic DNA by using the reagent combination according to any one of claims 1 to 2, or the gene chip according to claim 3, or the kit according to any one of claims 4 to 6; S3, obtaining the abundances of the genes related to the organic sulfur cycle metabolism from the results of the gene qPCR analysis.

9. The method according to claim 8, characterized in that, The marine sample is seawater or trench sediment.

10. The method according to claim 7 or 8, characterized in that, In step S2, the qPCR analysis is performed by using the high-throughput qPCR technology.

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