A primer set for inferring the drowning ground of a corpse in water, a preparation method and application thereof

Abstract

This application relates to the field of biotechnology, and in particular to a primer set, preparation method, and application for inferring the location of drowning of a body in water. The primer set includes six primer pairs as shown in SEQ ID NO:1-12. By designing primers targeting the genome in the chloroplasts of diatoms, and ensuring that the primer pairs do not conflict with each other, multiple detection results can be obtained through a single high-throughput sequencing and analysis in the subsequent detection stage. Correlation analysis of the detection results can then accurately infer the location of drowning of the body in water. Since the primer set is designed for multiple marker sites in diatom chloroplasts, it avoids missed detections of single marker sites and misjudgments based solely on morphology. It also reduces the tedious workload of single marker detection and morphological observation. Therefore, it not only improves the efficiency and specificity of the detection process but also enhances the accuracy of the detection.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a primer set for inferring the location of drowning of a body in water, its preparation method, and its application. Background Technology

[0002] Aquatic corpses are a common type of body found in forensic practice. However, because the location where a body is found is often not the actual point of drowning (i.e., the location is not the drowning site), it is not conducive to identifying the source of the body in actual investigations. Therefore, at present, inferring the drowning location of a body in water generally requires the combination of multiple detection techniques. Among them, diatom testing is an important means for forensic medicine to infer the drowning location in the examination of bodies in water. Diatoms are a type of single-celled algae widely distributed in water, with numerous species, currently estimated at 20,000 to 2,000,000. The cell walls of most diatoms are highly silicified, making them highly resistant to destruction. Even after using concentrated sulfuric acid or concentrated nitric acid to digest the organic matter of tissues and organs, their cell walls can still be observed. Therefore, by analyzing the types of diatoms in the tissues and organs of a body in water and in suspected drowning fluids, the location of drowning can be inferred.

[0003] Currently, the main research methods for diatom examination in forensic medicine are divided into two categories: morphological methods and molecular biological methods. Morphological methods involve separating diatoms from drowning or tissues using methods such as nitric acid digestion and microwave digestion. Then, optical or electron microscopy is used to observe the morphological characteristics of the diatoms to determine the species and quantity of diatoms in the tested sample. This is the standard method for diatom examination in forensic practice. However, morphological examination of diatoms has its limitations. For example, scanning electron microscopes are expensive, and the examination process requires manual interpretation of a large number of diatom species in the photographs, which is costly, time-consuming, and labor-intensive. Furthermore, the wide variety of diatom species and morphologies demands a high level of experience in diatom classification from the examiners, requiring years of training and practice to become competent. Compared to morphological methods, molecular biological methods have the following advantages:

[0004] ① It has high detection specificity and can be used to detect diatoms whose morphology cannot be identified by a microscope;

[0005] ② It has high sensitivity and requires a significantly smaller sample size than the traditional diatom detection method;

[0006] ③ It can be used to deduce the location of drowning;

[0007] However, current molecular biology methods generally only use single primer pairs to detect diatom marker sites. Due to the wide variety of diatoms, this can lead to missed detections or false positives, affecting accuracy. When using multiple primer pairs, they are prone to interference, which also affects the accuracy of the detection. Therefore, how to provide a primer set for inferring the location of drowning in water bodies to improve the accuracy of diatom testing for water bodies is a technical problem that urgently needs to be solved. Summary of the Invention

[0008] This application provides a primer set, preparation method, and application for inferring the location of drowning of a body in water, in order to solve the technical problem that morphological and molecular biological methods are difficult to improve the accuracy of diatom detection of bodies in water.

[0009] In a first aspect, this application provides a primer set for inferring the location of a drowning in water, the primer set including at least one of a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, and a sixth primer pair; wherein the nucleotide sequence of the forward primer of the first primer pair is shown in SEQ ID NO:1, and the nucleotide sequence of the reverse primer of the first primer pair is shown in SEQ ID NO:2;

[0010] The nucleotide sequence of the forward primer of the second primer pair is shown in SEQ ID NO:3, and the nucleotide sequence of the reverse primer of the second primer pair is shown in SEQ ID NO:4;

[0011] The nucleotide sequence of the forward primer of the third primer pair is shown in SEQ ID NO:5, and the nucleotide sequence of the reverse primer of the third primer pair is shown in SEQ ID NO:6.

[0012] The nucleotide sequence of the forward primer of the fourth primer pair is shown in SEQ ID NO:7, and the nucleotide sequence of the reverse primer of the fourth primer pair is shown in SEQ ID NO:8.

[0013] The nucleotide sequence of the forward primer of the fifth primer pair is shown in SEQ ID NO:9, and the nucleotide sequence of the reverse primer of the fifth primer pair is shown in SEQ ID NO:10.

[0014] The nucleotide sequence of the forward primer of the sixth primer pair is shown in SEQ ID NO:11, and the nucleotide sequence of the reverse primer of the sixth primer pair is shown in SEQ ID NO:12.

[0015] Optionally, the GC content of the primer set is 30% to 80%.

[0016] Optionally, the length of each primer in the primer set is 18bp to 35bp.

[0017] Optionally, the primer set amplifies fragments with a length of 150bp to 275bp.

[0018] Secondly, this application provides a method for preparing the primer set described in the first aspect, the method comprising:

[0019] Download the genome sequence of diatom chloroplasts;

[0020] Based on the sequence of the diatom chloroplast genome, conserved sequences were identified to obtain conserved regions;

[0021] Based on the conserved regions, the conserved region combinations of the diatom chloroplast genome are screened to obtain polymorphic molecular marker sites that meet the preset conditions.

[0022] Based on the polymorphic molecular marker sites, multiple primers were designed to obtain primer sets.

[0023] Optionally, the step of finding conserved sequences and obtaining conserved regions based on the sequence of the diatom chloroplast genome specifically includes:

[0024] Based on the sequence of the diatom chloroplast genome, a sliding window translation method was used to search for conserved sequences and obtain conserved regions.

[0025] Thirdly, this application provides a kit for inferring the location of a body drowning in water, the kit comprising the primer set and multiplex PCR premix described in the first aspect.

[0026] Fourthly, this application provides an application of a primer set for inferring the location of drowning of a body in water, the application comprising using the primer set described in the first aspect in a reagent for inferring and / or assisting in inferring the location of drowning of a body in water.

[0027] Optionally, the specific process for inferring and / or assisting in inferring the location of drowning of a body in water includes:

[0028] Diatom DNA was extracted from water samples from multiple different locations and from diatom DNA samples from carcasses in the water.

[0029] Design the primer set described in the first aspect;

[0030] Using the diatom DNA and the diatom DNA to be tested as templates, respectively, and employing the primer set, an amplification reaction was carried out to obtain the amplification product and the amplification product to be tested.

[0031] High-throughput libraries were constructed from the amplification product and the amplification product to be tested, respectively, to obtain high-throughput libraries;

[0032] The high-throughput library was screened and then sequenced to obtain high-throughput sequencing data;

[0033] Gene sequence analysis was performed on the high-throughput sequencing data to obtain the diatom community composition in the water sample and the diatom community composition of the dead organisms in the water.

[0034] Correlation analysis was performed on the diatom community composition and the diatom community composition to be tested to obtain characteristic data of the diatom community composition and untested characteristic data of the diatom community composition to be tested, respectively.

[0035] By comparing the correlation coefficients between the feature matrix and the feature matrix to be tested, the location of drowning of the body in the water can be determined;

[0036] The feature data includes feature vectors or feature matrices.

[0037] Optionally, the step of screening the high-throughput library and then performing sequencing to obtain high-throughput sequencing data specifically includes:

[0038] The actual concentration of the high-throughput library was obtained;

[0039] Based on the actual concentration and the standard concentration of the high-throughput library, determine whether the high-throughput library is qualified;

[0040] If the actual concentration is greater than or equal to the standard concentration, a high-throughput library is output, followed by sequencing to obtain high-throughput sequencing data.

[0041] If the actual concentration is less than the standard concentration, then diatom DNA from multiple water samples at different locations or diatom DNA from carcasses in the water should be extracted again, or the primer set should be redesigned.

[0042] The standard concentration of the high-throughput library is ≥2 ng / μL.

[0043] The technical solutions provided in this application have the following advantages compared with the prior art:

[0044] This application provides a primer set for inferring the location of drowning in water. Designed to target the genome in diatom chloroplasts, the primer pairs are mutually non-conflicting, allowing for multiple detection results obtained with a single high-throughput sequencing and analysis in the subsequent detection stage. Correlation analysis of these results then accurately infers the location of the drowning. Since the primer set is designed for multiple marker sites in diatom chloroplasts, it avoids missed detections of single marker sites and misjudgments based solely on morphology. It also reduces the tedious workload of individual marker detection and morphological observation. Therefore, it not only improves the efficiency and specificity of the detection process but also enhances the accuracy of the detection. Attached Figure Description

[0045] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0046] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 This is a schematic diagram of the process for preparing primer sets provided in the embodiments of this application;

[0048] Figure 2 A detailed flowchart illustrating the preparation of primer sets is provided for embodiments of this application.

[0049] Figure 3 A schematic diagram illustrating the process of inferring and / or assisting in inferring the location of drowning of a body in water, as provided in the embodiments of this application;

[0050] Figure 4 A detailed flowchart illustrating the process of inferring and / or assisting in inferring the location of drowning of a body in water, as provided in the embodiments of this application;

[0051] Figure 5 A graph illustrating the analysis of the detection efficiency of multiple primers provided in the embodiments of this application;

[0052] Figure 6 A box plot of the correlation coefficient of mantel.test between the diatom populations in Triangle Lake and the Yangtze River provided in this application embodiment;

[0053] Figure 7 A diagram illustrating the types of genotypes detected using multiple primers, as provided in the embodiments of this application. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0055] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0056] This application provides a primer set for inferring the location of a drowning in water. The primer set includes at least one of a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, and a sixth primer pair. The nucleotide sequence of the forward primer of the first primer pair is shown in SEQ ID NO:1, and the nucleotide sequence of the reverse primer of the first primer pair is shown in SEQ ID NO:2.

[0057] The nucleotide sequence of the forward primer of the second primer pair is shown in SEQ ID NO:3, and the nucleotide sequence of the reverse primer of the second primer pair is shown in SEQ ID NO:4;

[0058] The nucleotide sequence of the forward primer of the third primer pair is shown in SEQ ID NO:5, and the nucleotide sequence of the reverse primer of the third primer pair is shown in SEQ ID NO:6.

[0059] The nucleotide sequence of the forward primer of the fourth primer pair is shown in SEQ ID NO:7, and the nucleotide sequence of the reverse primer of the fourth primer pair is shown in SEQ ID NO:8.

[0060] The nucleotide sequence of the forward primer of the fifth primer pair is shown in SEQ ID NO:9, and the nucleotide sequence of the reverse primer of the fifth primer pair is shown in SEQ ID NO:10.

[0061] The nucleotide sequence of the forward primer of the sixth primer pair is shown in SEQ ID NO:11, and the nucleotide sequence of the reverse primer of the sixth primer pair is shown in SEQ ID NO:12.

[0062] In some optional embodiments, the GC content of the primer set is 30% to 80%.

[0063] In this embodiment of the application, limiting the GC content in the primer set has the positive effect of ensuring that the primer pairs in the primer set react smoothly in the amplification reaction, and ensuring that the target sequence can be effectively amplified.

[0064] In some alternative implementations, the length of each primer in the primer set is 18bp to 35bp.

[0065] In this embodiment of the application, the specific length of each primer in the primer set is limited, which can ensure that each primer pair covers the target gene with high density, thereby ensuring the amplification of the target product and reducing the output of non-target sequences.

[0066] In some optional embodiments, the primer set amplifies fragments of 150 bp to 275 bp in length.

[0067] In this embodiment of the application, the specific length of the fragment amplified by the primer set is limited, which can ensure that there are enough polymorphic regions in the amplification product, thereby facilitating subsequent genotyping and differentiation of diatom populations.

[0068] like Figure 1 As shown, based on a general inventive concept, this application provides a method for preparing the primer set, the method comprising:

[0069] S1. Download the genome sequence of diatom chloroplasts;

[0070] S2. Based on the sequence of the diatom chloroplast genome, find conserved sequences to obtain conserved regions;

[0071] S3. Based on the conserved regions, the conserved region combinations of the diatom chloroplast genome are screened to obtain polymorphic molecular marker sites that meet the preset conditions;

[0072] S4. Based on the polymorphic molecular marker sites, design multiple primers to obtain primer sets.

[0073] In this embodiment of the application, by analyzing the existing diatom chloroplast genome and screening and combining its conserved regions, it is possible to ensure that the primer pairs in the designed primer set do not affect each other, thus ensuring the accuracy of the amplification products and ensuring that a sufficient amount of target sequences are obtained subsequently.

[0074] This method is for the preparation of the primer set described above. The specific composition and sequence of the primer set can be referred to in the above embodiments. Since this method adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0075] like Figure 2 As shown, in some optional embodiments, the step of finding conserved sequences and obtaining conserved regions based on the sequence of the diatom chloroplast genome specifically includes:

[0076] S201. Based on the sequence of the diatom chloroplast genome, a sliding window translation method is used to search for conserved sequences and obtain conserved regions.

[0077] In this embodiment of the application, the specific method for finding conserved sequences is defined, which can accurately filter out the expected conserved regions by sliding the window, thereby facilitating the design of multiple primers.

[0078] Based on a general inventive concept, embodiments of this application provide a kit for inferring the location of a body drowning in water, the kit comprising the test primer set and a multiplex PCR premix.

[0079] This kit is based on the primer set described above. The specific composition and sequence of the primer set can be referred to in the above embodiments. Since this kit adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0080] Based on a general inventive concept, embodiments of this application provide an application of a primer set for inferring the location of drowning of a body in water, the application including using the primer set in reagents for inferring and / or assisting in inferring the location of drowning of a body in water.

[0081] This application is based on the primer set described above. The specific composition and sequence of the primer set can be referred to in the above embodiments. Since the kit adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0082] like Figure 3 As shown, in some optional embodiments, the specific process for inferring and / or assisting in inferring the location of drowning of a body in water includes:

[0083] S1. Extract diatom DNA from water samples from multiple different locations and diatom DNA from carcasses in the water for testing;

[0084] S2. Design the primer set;

[0085] S3. Using the diatom DNA and the diatom DNA to be tested as templates respectively, and using the primer set, an amplification reaction is carried out to obtain the amplification product and the amplification product to be tested;

[0086] S4. Construct high-throughput libraries for the amplification product and the amplification product to be tested, respectively, to obtain high-throughput libraries;

[0087] S5. The high-throughput library is screened and then sequenced to obtain high-throughput sequencing data;

[0088] S6. Analyze the gene sequences of the high-throughput sequencing data to obtain the diatom community composition in the water sample and the diatom community composition of the dead organisms in the water.

[0089] S7. Perform correlation analysis on the diatom community composition and the diatom community composition to be tested to obtain the characteristic data of the diatom community composition and the characteristic data to be tested of the diatom community composition to be tested, respectively.

[0090] S7. By comparing the correlation coefficients of the feature matrix and the feature matrix to be tested, the location of the drowning of the body in the water is determined;

[0091] The feature data includes feature vectors or feature matrices.

[0092] In this embodiment, a specific process for inferring and / or assisting in inferring the drowning location of a body in water is defined. This involves extracting DNA from diatoms at different locations and from the body in water, amplifying the DNA using a designed primer set, and performing high-throughput sequencing to obtain an accurate high-throughput library. Finally, analysis of the high-throughput library determines the characteristic data of the diatom community composition and the characteristic data of the diatom community composition to be tested. By comparing correlation coefficients, the source of the diatoms in the body in water can be determined, thereby identifying the drowning location. Therefore, by using a designed primer set in conjunction with a single high-throughput sequencing run, the exact drowning location of a body in water can be accurately determined, thus improving the accuracy of the detection.

[0093] like Figure 4 As shown, in some optional embodiments, the step of screening the high-throughput library and then performing sequencing to obtain high-throughput sequencing data specifically includes:

[0094] S501. Obtain the actual concentration of the high-throughput library;

[0095] S502. Based on the actual concentration and the standard concentration of the high-throughput library, determine whether the high-throughput library is qualified;

[0096] If the actual concentration is greater than or equal to the standard concentration, a high-throughput library is output, followed by sequencing to obtain high-throughput sequencing data.

[0097] If the actual concentration is less than the standard concentration, then diatom DNA from multiple water samples at different locations or diatom DNA from carcasses in the water should be extracted again, or the primer set should be redesigned.

[0098] The standard concentration of the high-throughput library is ≥2 ng / μL.

[0099] In this embodiment of the application, by judging the constructed high-throughput library, the accuracy of sequencing can be improved, and the insufficient sequencing data or sequencing failure caused by low-concentration library can be avoided.

[0100] The standard concentration of high-throughput libraries was set at ≥2 ng / μL to ensure that the concentration of the constructed high-throughput libraries met the sequencing requirements, thus eliminating the problem of insufficient sequencing volume or sequencing failure due to insufficient library concentration.

[0101] In some optional embodiments, the amplification reaction includes pre-denaturation, a first amplification reaction, and a second amplification reaction, wherein the denaturation temperature is 94°C and the denaturation time is 15 min;

[0102] The procedure for the first amplification reaction includes: denaturation at 94°C for 20 seconds, annealing and extension at 65°C to 57°C for 60 seconds, and 10 falling cycles, wherein the final temperature of the falling cycle is 0.8°C.

[0103] The procedure for the second amplification reaction includes: denaturation at 94°C for 20 seconds, followed by annealing and extension at 57°C for 60 seconds.

[0104] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. If there is no corresponding national standard, then general international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0105] Example 1

[0106] Developing molecular marker sites for diatom chloroplasts to infer the location of drowning in bodies in water:

[0107] 1. Download all known diatom chloroplast genome sequences from the NCBI website (https: / / www.ncbi.nlm.nih.gov / );

[0108] 2. Conserved regions on the genome were obtained from the downloaded diatom chloroplast genome using a sliding window method of 20bp to 50bp. The specific steps were as follows: the sliding window was set to 30bp, and the shift was 10bp at a time. Window regions meeting these conditions were identified as conserved regions if their frequency of occurrence in the downloaded sequence was greater than 60%, and if a 10% variation within the sliding window size was allowed.

[0109] 3. Based on the conserved regions, combinations of these conserved regions are screened from the genome to obtain molecular marker sites with high polymorphism that meet the criteria. Specifically, the sequence length between any two conserved regions must be greater than 100 bp and less than 275 bp; and the fragment (molecular marker site) formed by these two conserved regions must have a discrimination ratio of 20% among the downloaded sequences. Such a fragment is considered a molecular marker site with high polymorphism. Each downloaded genome is treated as a sample, and the discrimination ratio of each molecular marker site is the percentage of sample pairs that the marker site can effectively distinguish out of all sample pairs.

[0110] 4. Based on the conserved regions at both ends of these highly polymorphic sites, primers were designed using Primer3. These designed primers are the multiplex primers used in our subsequent experiments. The amplification primers must meet the following conditions:

[0111] (1) Primer length is designed to be between 18bp and 35bp, providing high-density coverage of the target region (i.e., 80% coverage of each genome), and the amplified fragment length is between 150bp and 275bp, resulting in high polymorphism in the amplified region;

[0112] (2) Avoid SSR and N regions when designing primers;

[0113] (3) Calculate the GC content of all primer sequences, with the GC content ranging from 30% to 80%;

[0114] (4) Calculate the hairpin structure and dimer of all primers.

[0115] Finally, the primers that met the requirements were synthesized at Wuhan Qingke Biotechnology Co., Ltd., and the synthesized primers are shown in Table 1:

[0116] Table 1. Sequence information of each primer pair in the primer set.

[0117]

[0118]

[0119] Example 2

[0120] Comparing Example 2 with Example 1, the difference between Example 2 and Example 1 is as follows:

[0121] Application of primer sets for detecting diatom diversity in water: The primer sets were used to detect diatom diversity in Triangle Lake.

[0122] 1. Experimental materials: Three water samples (S1, S2 and S3) came from Sanjiaohu Lake in Wuhan Economic and Technological Development Zone. These three points are geographically some distance apart.

[0123] 2. DNA template preparation: Genomic DNA was extracted from water samples using the OMEGA (USA) Water Sample Genomic DNA Extraction Kit (D5525-02). Simultaneously, DNA from the test samples extracted using the OMEGA Water Sample Genomic DNA Extraction Kit was also used.

[0124] 3. PCR amplification, library construction, and sequencing:

[0125] Genomic DNA from samples was amplified using six sets of multiplex PCR primer pairs. The amplification products from each sample were ligated with sequencing adapters and specific sample DNA barcodes and then mixed to form a high-throughput sequencing library. The high-throughput sequencing library was then detected and sequenced using a high-throughput sequencing platform. The high-throughput sequencing data required quality control using FASTQC.

[0126] 4. Results Analysis and Interpretation:

[0127] (1) Each primer pair is analyzed using a method similar to that used for microbial 16S rDNA OTUs (Operational Taxonomic Units), and the abundance of each OTU is calculated. Specifically, the sequences are first clustered according to a certain degree of similarity, and each cluster is called an OTU. The difference between sequences in an OTU cannot exceed the specified similarity. The similarity used in this application is 99%. After analysis using vsearch software, the abundance of each OUT is calculated (expressed as the percentage of the number of sequences in each OUT to the total number of sequences). OTUs with a relative abundance of less than 5% are filtered out.

[0128] (2) In a sample, each pair of primers forms an abundance vector, and multiple pairs of primers form a matrix.

[0129] (3) This matrix serves as the diatom community composition feature matrix for each sample, representing the molecular characteristics of the diatom community composition of each sample. A total of 3 samples were analyzed, forming 3 vector matrices, one of which is shown in Table 2.

[0130] Table 2. Diversity analysis results of diatom species in a sample from Triangle Lake.

[0131]

[0132]

[0133] As shown in Table 2, only one library preparation and sequencing analysis is needed to detect six molecular marker sites, avoiding missed detections when diatom content in water is low. Especially when using a microscope for observation and analysis, it is difficult to observe diatoms when the sample content is very low. The use of PCR significantly improves the sensitivity of the detection, and the use of multiple markers will also greatly improve the accuracy of the detection.

[0134] Example 3

[0135] Comparing Example 3 and Example 2, the differences between Example 3 and Example 2 are as follows:

[0136] Application of primer combinations for detecting diatom diversity in water: Primer sets containing primers are used to analyze the repeatability of detection results, and the detection efficiency of multiple primers is also analyzed.

[0137] 1. Experimental materials: Three water samples were taken from three sampling points (S1, S2 and S3) in Sanjiaohu, Wuhan Economic and Technological Development Zone. These three sampling points were geographically distant. Three biological replicates were taken from each point, for a total of 9 samples, which were named S1-1, S1-2, S1-3, S2-1, S2-2, S2-3, S3-1, S3-2 and S3-3 respectively.

[0138] 2. The DNA extraction, library construction and sequencing process is similar to that in Example 2.

[0139] 3. The molecular feature matrix of each sample was obtained based on the sequencing data in the same manner as in Example 2.

[0140] 4. Repeatability analysis:

[0141] Diatom species analysis was performed on three biological replicates from the same sampling point, and the mantel.test correlation coefficient of diatom population composition among the three biological replicates was calculated. The analysis results showed that the average correlation coefficient of diatom population composition among the biological replicates was close to 0.98, indicating the reliability and accuracy of using multiple molecular markers to analyze the diatom population composition of a single sample. Moreover, the results of multiple samples can be compared and analyzed in one library construction and sequencing analysis, which greatly saves the detection time.

[0142] 5. Detection efficiency analysis of multiple primers:

[0143] The detection efficiency of multiplex primers is expressed as the percentage of the target sequence amplified by each primer pair relative to the total number of reads from all single-primer sequencing reads. The results are as follows: Figure 5 As shown, the multiple primer pair diatom4 has a high amplification efficiency. Therefore, if you want to use only one primer pair to analyze the composition of diatom populations later, you can choose this primer pair.

[0144] Example 4

[0145] Comparing Example 4 with Example 3, the differences between Example 4 and Example 3 are as follows:

[0146] Application of primer combinations for detecting diatom diversity in water: The reagents were used to detect the diversity of diatom species in the Yangtze River, analyze the differences between the diatom populations in the Yangtze River and the Delta Lake, and analyze the types of genotypes detected by each amplification primer.

[0147] 1. Experimental materials: Samples were taken from three experimental sites (C1, C2, and C3) in the Wuhan section of the Yangtze River, which were geographically distant from each other. One site was located under the Yangsigang Yangtze River Bridge, another under the Baishazhou Bridge, and the third about 2 kilometers downstream of Baishazhou. Three biological replicates were taken from each site, all from the left bank of the Yangtze River, for a total of 9 samples, which were named C1-1, C1-2, C1-3, C2-1, C2-2, C2-3, C3-1, C3-2, and C3-3, respectively. In addition, three experimental sites were taken from the same Sanjiaohu Lake as in Example 3.

[0148] 2. The DNA extraction, library construction and sequencing process is similar to that in Example 2.

[0149] 3. The feature matrix of each sample was obtained based on the sequencing data, which is consistent with the process in Example 2.

[0150] 4. Analyze the differences in diatom populations between samples from Triangle Lake and the upper reaches of the Yangtze River:

[0151] First, the diatom population composition feature matrix of each sample was calculated based on the sequencing data. Then, the correlation coefficient of the pairwise sample feature matrices was calculated using the Python package mantel.test. The correlation coefficient of diatom population composition between replicates of Yangtze River samples is represented by c-in, the correlation coefficient of diatom population composition between replicates of samples from Triangle Lake is represented by s-in, the correlation coefficient of diatom population composition between replicates of non-sample organisms in Yangtze River is represented by c-outer, the correlation coefficient of diatom population composition between replicates of non-sample organisms in Triangle Lake is represented by s-outer, and the correlation coefficient of diatom population composition between Triangle Lake and Yangtze River samples is represented by cs.

[0152] The correlation coefficients of algal populations between any two combinations of 18 samples were calculated using the mantel.test method. The results are presented as box plots. Figure 6 As shown in the figure, the Mantel.test correlation coefficients of algal community composition among the three biological replicates at the same sampling point are relatively close. Among them, the Mantel.test correlation coefficients of algal community composition among the biological replicates in the Yangtze River are the closest. This may be because the water in the Yangtze River is flowing and the distances are relatively close, so the diatom communities at the three sampling points in the Yangtze River are more similar. However, the three water samples in the Triangle Lake are stagnant water, and the diatoms in the water are greatly affected by the sampling location. A certain area may mainly grow a few 1-3 species of diatoms, unlike the rich diatom communities in the Yangtze River. In addition, the average Mantel.test correlation coefficient between the diatom communities in the Triangle Lake and the diatom communities in the Yangtze River is less than 0.8, indicating that there are significant differences between the diatom communities in these two places.

[0153] The most abundant sequences corresponding to the first primer pair in two random samples, C1-1 and S3-1, were analyzed. It was found that the most abundant sequence (9.24%) in sample C1-1 was identified as Stephanodiscus after online BLAST analysis by NCBI, while the most abundant sequence (44.38%) in sample S3-1 was identified as Discostella woltereckii. This further proves that the diatom species in these two locations are indeed different.

[0154] 5. Evaluate the types of diatoms detected by the designed multiplex primers:

[0155] We evaluated the differences in the identification of diatom species using 18 samples from three sampling sites in the Yangtze River Delta and three sampling sites in the Yangtze River Delta.

[0156] The specific evaluation steps are as follows: For each sampling point, the OTUs (number of genotypes) with an abundance greater than 0.09% identified by each primer pair are considered as the diatom species identified by that primer pair. Then, the diatom species identified by each primer pair in each sample are analyzed, and the results are as follows: Figure 7 As shown in the figure, the fourth primer pair identified the most diatom species. Therefore, the fourth primer pair is the best choice in terms of both amplification efficiency and the number of diatom species identified. If you want to use only one primer pair to analyze the composition of diatom populations in the future, you can choose this primer pair.

[0157] Example 5

[0158] Comparing Example 5 and Example 4, the differences between Example 5 and Example 4 are as follows:

[0159] Application of primer combinations for detecting diatom diversity in samples: using reagents to infer the location of drowning of bodies in water.

[0160] 1. Experimental materials: The bodies (H) that drowned in the Yangtze River (Wuhan section) came from 5 experimental sites (CL1, CL2, CL3, CL4 and CL5) in the Yangtze River basin in Wuhan. The body retrieval locations were CL4, 5 km upstream (CL3), 10 km upstream (CL2), 15 km upstream (CL1) and 5 km downstream (CL5).

[0161] 2. The diatom DNA to be tested in the corpse was extracted from the lung tissue, and the diatom DNA in the water sample was extracted from the diatoms at the corresponding retrieval location. The construction and sequencing process of the Chinese database in the water and the corpse was similar to that in Example 2.

[0162] 3. The feature matrix of each sample was obtained based on the sequencing data, which is consistent with the process in Example 2.

[0163] 4. Determine the location where the body drowned in the water:

[0164] The feature matrix of diatom population composition for each sample was calculated based on the sequencing data, and the mantel.test correlation coefficient between the carcasses in the water and the diatom population composition at four sampling points in the Yangtze River was calculated. The results are shown in Table 3.

[0165] Table 3. Correlation analysis between underwater carcasses and diatom populations at each sampling point.

[0166] H CL1 0.66 Salvage 15 kilometers upstream H CL2 0.89 Salvage 10 kilometers upstream H CL3 0.78 Salvage 5 kilometers upstream H CL4 0.59 Salvage site H CL5 0.43 Salvage 5 kilometers downstream

[0167] As shown in Table 3, the correlation coefficient of the mantel.test between the diatom population of the carcass in the water and the diatom population of the CL2 sample was the highest, reaching 0.89. Based on this result, it can be inferred that the drowning site of the carcass in the water may be about 10 kilometers upstream of its retrieval site, rather than at the retrieval site itself. It is possible that the carcass drifted to the retrieval site with the water after death. This result fully demonstrates that the method of using molecular markers of diatom chloroplasts to infer the drowning site of carcasses in the water is feasible.

[0168] One or more technical solutions in the embodiments of this application have at least the following technical effects or advantages:

[0169] (1) The primer set provided in this application for inferring the location of drowning of a body in water uses primers with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.12. The amplification products can be subjected to high-throughput sequencing and analysis in one go, obtaining multiple detection results including multiple suspected drowning locations and diatom species in the body. Based on the correlation analysis between each suspected drowning location and the diatom species in the body, the drowning location of the body in water can be determined. Using primer pairs designed for multiple marker sites of diatoms avoids missed detection of single marker sites and misjudgment based on morphology alone. At the same time, it reduces the tedious workload of single marker detection and morphological observation, greatly improves the efficiency, sensitivity and accuracy of detection, saves costs, and is easy to apply and promote.

[0170] (2) The application of a primer set provided in this application for inferring the location of drowning of a body in water has the effects of high throughput, high accuracy, high specificity and high sensitivity in inferring and / or assisting in inferring the location of drowning of a body in water.

[0171] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0172] In the description of this application, the terms "comprising," "including," etc., mean "including but not limited to." In this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. In this document, "and / or" describes the relationship between related objects, indicating that three relationships can exist; for example, A and / or B can represent: A alone, A and B simultaneously, or B alone. A and B can be singular or plural. In this document, "at least one" means one or more, and "more than" means two or more. "At least one," "at least one of the following," or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c" or "at least one of a, b, and c" can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be a single or multiple.

[0173] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A primer set for inferring a drowning ground of a cadaver in water, characterized by, The primer set includes a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, and a sixth primer pair; wherein the nucleotide sequence of the forward primer of the first primer pair is shown in SEQ ID NO:1, and the nucleotide sequence of the reverse primer of the first primer pair is shown in SEQ ID NO:2; The nucleotide sequence of the forward primer of the second primer pair is shown in SEQ ID NO:3, and the nucleotide sequence of the reverse primer of the second primer pair is shown in SEQ ID NO:4; The nucleotide sequence of the forward primer of the third primer pair is shown in SEQ ID NO:5, and the nucleotide sequence of the reverse primer of the third primer pair is shown in SEQ ID NO:

6. The nucleotide sequence of the forward primer of the fourth primer pair is shown in SEQ ID NO:7, and the nucleotide sequence of the reverse primer of the fourth primer pair is shown in SEQ ID NO:

8. The nucleotide sequence of the forward primer of the fifth primer pair is shown in SEQ ID NO:9, and the nucleotide sequence of the reverse primer of the fifth primer pair is shown in SEQ ID NO:

10. The nucleotide sequence of the forward primer of the sixth primer pair is shown in SEQ ID NO:11, and the nucleotide sequence of the reverse primer of the sixth primer pair is shown in SEQ ID NO:

12. The GC content of the primer set is 30%–80%; The primers in the primer set are 18 bp to 35 bp in length; The primer set amplifies fragments ranging from 150 bp to 275 bp in length.

2. A reagent kit for determining the location of drowning of a body in water, characterized in that, The kit includes the primer set and multiplex PCR premix as described in claim 1.

3. An application of a primer set for inferring the location of drowning of a body in water, characterized in that, The application includes using the primer set as described in claim 1 in reagents for inferring and / or assisting in inferring the location of drowning of a body in water.

4. The application according to claim 3, characterized in that, The specific procedures for inferring and / or assisting in inferring the location of drowning of a body in water include: Diatom DNA was extracted from water samples from multiple different locations and from diatom DNA samples from dead organisms in the water. Design the primer set as described in claim 1; Using the diatom DNA and the diatom DNA to be tested as templates, respectively, and employing the primer set, an amplification reaction was carried out to obtain the amplification product and the amplification product to be tested. High-throughput libraries were constructed from the amplification product and the amplification product to be tested, respectively, to obtain high-throughput libraries; The high-throughput library was screened and then sequenced to obtain high-throughput sequencing data; Gene sequence analysis was performed on the high-throughput sequencing data to obtain the diatom community composition in the water sample and the diatom community composition of the dead organisms in the water. Correlation analysis was performed on the diatom community composition and the diatom community composition to be tested to obtain characteristic data of the diatom community composition and untested characteristic data of the diatom community composition to be tested, respectively. By comparing the correlation coefficients between the feature matrix and the feature matrix to be tested, the location of drowning of the body in the water can be determined; The feature data includes feature vectors or feature matrices.

5. The application according to claim 4, characterized in that, The process of screening the high-throughput library and then performing sequencing to obtain high-throughput sequencing data specifically includes: The actual concentration of the high-throughput library was obtained; Based on the actual concentration and the standard concentration of the high-throughput library, determine whether the high-throughput library is qualified; If the actual concentration is greater than or equal to the standard concentration, a high-throughput library is output, followed by sequencing to obtain high-throughput sequencing data. If the actual concentration is less than the standard concentration, then diatom DNA from multiple water samples at different locations or diatom DNA from carcasses in the water should be extracted again, or the primer set should be redesigned. The standard concentration of the high-throughput library is ≥2 ng / μL.

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