A method for sample tracing based on bacterial community structure

Abstract

The present invention provides a method for tracing the source of samples based on flora structure, and belongs to the technical field of microbial analysis. The present invention obtains the flora structure of a large number of samples through 16S sequence amplification, metagenomics and big data, determines the flora map of a certain source by judging the Shannon index of the sample to be tested and the similarity of the flora structure, and traces the source of the sample based on the flora map. The sample tracing method of the present invention is efficient, accurate, fast, and easy to operate.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial analysis, and in particular to a method for tracing the origin of a sample based on bacterial community structure. Background Art

[0002] Microbial source tracking (MST) is a process that uses the indicators of individual or group microorganisms to establish a distinction and identification of the source of microorganisms. In recent years, MST technology has been increasingly used in the detection and identification of pathogenic microorganisms in water. Microbial source tracking research helps people to have a deeper understanding of the origin and evolution of pathogen population structure and its diversity, and provides important information and scientific basis for the quarantine, epidemic monitoring, and integrated prevention and control of pathogens. In the research on pathogenic microorganism source tracking technology, biological methods, phage sensitivity, multi-site enzyme electrophoresis, and other phenotypic analysis methods and molecular methods such as amplified fragment length polymorphism, ribosome typing, repetitive sequence PCR, simple repeat sequence, single nucleotide polymorphism, multi-site variable tandem repeat sequence, and multi-site sequence analysis have all played an important role.

[0003] Microbial traceability analysis is relatively difficult, and the phylogenetic relationships of microorganisms are relatively complex. Describing the evolutionary relationships between species or molecules by constructing phylogenetic trees is an important application of bioinformatics methods in traceability analysis in the field of microorganisms. For example, when researchers are studying microorganisms, if they discover a new virus, they need to sequence the entire genome of the virus, and then find similar viruses through gene comparison to conduct phylogenetic analysis and establish a phylogenetic tree. Through the phylogenetic tree, you can find viruses that are homologous to the virus, analyze and compare them, and quickly find the roots of microbial evolution and how to apply them.

[0004] Patent CN108841942A discloses a method for rapid analysis and risk assessment of the composition source of PM2.5 bacterial communities, which includes PM2.5 sampling, microbial genome extraction, PCR amplification, product purification, fluorescence quantification, high-throughput sequencing, and bacterial diversity and abundance analysis, so as to accurately determine the bacterial source of PM2.5. Patent CN111122689A discloses a method for tracing the source of microbial contamination, which obtains a single colony by culturing a sample microorganism, and uses a matrix-assisted laser desorption ionization-time of flight mass spectrometer to perform mass spectrometry analysis on the single colony to obtain the spectrum data and species information of the single microbial colony, so as to determine the source of the microorganisms in the contaminated product. In the prior art, the traceability of microbial samples is highly professional, and professional equipment needs to be used for analysis, which has high requirements for technical personnel.

[0005] Therefore, it is urgent to develop an accurate, fast, efficient and easy-to-operate method for sample traceability. Summary of the invention

[0006] In view of the above shortcomings, the present invention provides a method for sample tracing based on flora structure. The present invention obtains the flora structure of a large number of samples through 16S sequences, metagenomes and big data, determines the flora map of a certain source by judging the Shannon index of the sample to be tested and the similarity of the flora structure, and traces the source of the sample based on the flora map. The method described in the present invention can efficiently, accurately and quickly realize the traceability of the sample to be tested.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides a method for tracing the origin of a sample based on bacterial flora structure, the method comprising the following steps:

[0009] (1) Determine the strain clustering threshold and obtain the bacterial community structure of known samples through 16S sequence amplification, metagenomics, and big data analysis;

[0010] (2) Analyze the bacterial flora structure of the sample to be tested;

[0011] (3) Compare the bacterial flora structure of the known sample in step (1) and the sample to be tested in step (2), determine the bacterial flora map of the sample to be tested, and trace the source of the sample.

[0012] Specifically, the strain clustering threshold in step (1) is 97-100%, preferably 99%.

[0013] Specifically, the 16S sequence described in step (1) is the V3-V4 region, and the 16S sequence amplification primer is SEQ ID NO: 1: F: 5′-AATGATACGGCGACCACC-3′; SEQ ID NO: 2: R: 5′-CAAGCAGAAGACGGCATA-3′.

[0014] Specifically, the big data analysis described in step (1) includes the following steps: removing primer adapters to obtain the true sequence of the sample; removing redundant sequences to speed up the analysis progress and reduce the requirements for the hardware of the analysis equipment; clustering according to similarity and comparing with the database to obtain annotation information of each sequence; comparing the sequence without primer adapters with the clustering results to obtain the specific sequence number, relative abundance and Shannon index of each bacterial community in each sample.

[0015] Specifically, the bacterial community structure described in step (1) includes bacterial species type, bacterial species ratio and Shannon index parameter.

[0016] More specifically, the bacterial species include dominant bacterial populations and inferior bacterial populations.

[0017] More specifically, the comparison of the bacterial flora structure of the known sample in step (1) and the sample to be tested in step (2) in step (3) includes the following steps: determining whether the Shannon index parameter of the sample to be tested is within the range of the Shannon index parameter of the known sample; and determining whether the types and proportions of the dominant and inferior bacterial flora of the sample to be tested are 75-100% similar to those of the known sample.

[0018] On the other hand, the present invention provides an application of a bacterial community structure in sample traceability, wherein the bacterial community structure includes bacterial species type, bacterial species ratio and Shannon index parameter.

[0019] Specifically, the bacterial species include dominant bacterial populations and inferior bacterial populations.

[0020] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0021] The present invention provides a method for sample tracing based on bacterial community structure. By comparing the bacterial community structure of the sample to be tested with that of a known sample, including the bacterial species type, the proportion of each bacteria (dominant bacterial community and inferior bacterial community) and Shannon index parameters, the sample to be tested can be traced efficiently, accurately and quickly, and the operation is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the Shannon index parameter result diagram.

[0023] Figure 2 This is the sample traceability test result diagram. DETAILED DESCRIPTION

[0024] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0026] Example 1 Sample tracing

[0027] (1) Using 97-100% as the strain clustering threshold, the bacterial community structure of known samples, including the types, proportions and Shannon index parameters of dominant and inferior bacterial communities, was obtained through 16S sequencing, metagenomics and big data analysis (removing primer adapters to obtain the true sequence of the sample; removing redundant sequences to speed up the analysis and reduce the requirements for analytical equipment hardware; clustering according to similarity and comparing with the database to obtain annotation information for each sequence; comparing the sequences without primer adapters with the clustering results to obtain the specific sequence number, relative abundance and Shannon index of each bacterial community in each sample).

[0028] The primers for 16S sequence amplification are:

[0029] SEQ ID NO: 1: F: 5′-AATGATACGGCGACCACC-3′;

[0030] SEQ ID NO: 2: R: 5'-CAAGCAGAAGACGGCATA-3'.

[0031] (2) Analyze the bacterial flora structure of the sample to be tested, including the types, proportions and Shannon index parameters of the dominant and inferior flora.

[0032] (3) Compare the bacterial flora structure of known samples and samples to be tested, determine the bacterial flora profile of a certain source by judging the Shannon index of the samples and the similarity of the bacterial flora structure, and trace the source of the samples based on the bacterial flora profile.

[0033] Experimental Example 1 Accuracy Test

[0034] Taking soil samples as an example, the accuracy of the method of the present invention is tested.

[0035] According to the representative of 110 soil samples in Beijing, the bacterial species maps at the genus level and the species level were statistically analyzed, and the top 50 samples with higher content and the bottom 50 samples with lower content were selected as the bacterial species maps and the corresponding Shannon index parameters. The statistical results are shown in Table 1 below.

[0036] Table 1 Representative statistical results of soil samples in Beijing

[0037]

[0038]

[0039]

[0040]

[0041]

[0042] o__, f__, g__ represent any bacteria whose capsular antigens are o, f, or g antigens.

[0043] When the Shannon index parameter of the traced sample is within the range shown in Table 1, and the types and relative contents of the dominant and inferior bacterial communities are 85% similar to those shown in Table 1, the sample to be traced can be considered to be a Beijing soil sample.

[0044] The method of Example 1 and Experimental Example 1 of the present application was used to trace the origin of 440 samples, including 40 Beijing soil samples and 400 Northeast soil samples. The Shannon index parameters are as follows: Figure 1 As shown, the test results are Figure 2 As shown, the results show that by using the sample tracing method based on microbial structure described in this application, 440 soil samples were traced, and 40 Beijing soil samples and 400 Northeast soil samples could be accurately identified. The operation is simple and the accuracy is high.

[0045] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. Sequence Listing <110> Beijing Institute of Genomics, Chinese Academy of Sciences (National Center for Bioinformatics) <120> A method for sample tracing based on bacterial community structure <130> 20210114 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial sequence <400> 1 aatgatacgg cgaccacc 18 <210> 2 <211> 18 <212> DNA <213> Artificial sequence <400> 2 caaccagaag acggcata 18

Claims

1. A method for sample tracing based on bacterial community structure, characterized in that: The method comprises the following steps: (1) The bacterial species clustering threshold is determined to be 97%-100%, and the bacterial community structure of the known sample is obtained by 16S sequence amplification, metagenomics and big data analysis; the 16S sequence is the V3-V4 region, and the 16S sequence amplification primer is SEQ ID NO: 1: F: 5'-AATGATACGGCGACCACC-3'; SEQ ID NO: 2: R: 5'-CAAGCAGAAGACGGCATA-3'; (2) Analyze the bacterial flora structure of the sample to be tested; (3) Compare the bacterial flora structure of the known sample in step (1) and the sample to be tested in step (2), determine whether the Shannon index parameter is within the Shannon index parameter range of the known sample and whether the types and proportions of the dominant and inferior bacterial flora of the sample to be tested are 75-100% similar to those of the known sample, and trace the source of the sample.

2. The sample traceability method according to claim 1, characterized in that: The strain clustering threshold described in step (1) is 99%.

3. The sample traceability method according to claim 2, characterized in that: Big data analysis described in step (1) The method comprises the following steps: removing the primer adapter to obtain the true sequence of the sample; Eliminate redundant sequences, speed up analysis and reduce the requirements on analytical equipment hardware; Clustering was performed based on similarity and compared with the database to obtain annotation information for each sequence; the sequence after removing the primer linker was compared with the clustering result to obtain the specific sequence number, relative abundance and Shannon index of each bacterial community in each sample.

4. An application of a bacterial community structure in the sample traceability as claimed in claim 1, characterized in that: The bacterial community structure includes the types of dominant bacterial communities and inferior bacterial communities, bacterial species ratios and Shannon index parameters.

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