A microbial traceability method based on weighted total microbial quantity

Abstract

The present invention belongs to the field of microbial analysis technology, and specifically relates to a microbial traceability method based on weighted total microbial amount. The present invention provides a microbial traceability method, including: determining the sampling sites of the traceability source and the traceability target; collecting samples at the sampling sites of the traceability source and the traceability target respectively to obtain multiple samples; obtaining microbial sequencing information at the sampling sites; recording the sampling information of the sampling sites, and importing the sampling information of the sampling sites and the microbial sequencing information into the traceability model to obtain the original traceability result; performing weighted calculation on the original traceability result based on the total amount of microorganisms at the traceability source to obtain a weighted traceability analysis result. The present invention can more accurately calculate the microbial traceability result, and the method of correcting the microbial traceability result based on the weighted total amount of microorganisms is better than simple traceability analysis, and is more in line with actual reality.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial analysis, and in particular relates to a microbial source tracing method based on weighting of the total amount of microorganisms. Background Art

[0002] Microbial source tracking (MST) is an important topic and direction in microbial analysis technology. Microbial source tracking can solve problems such as pollution source analysis and impact analysis, and can reveal the dynamic changes in the transfer of microorganisms or substances.

[0003] Microbial traceability analysis first emerged in the 1980s. This technology determines the connection between the contaminated samples and possible contamination sources by comparing the differences in microorganisms or the presence or absence of other biomarkers between the two, thereby determining the source of contamination. Under the pressure of natural selection, microbial communities living in different regions or different hosts will develop specific environmental adaptability and integrate these characteristics into the genome and pass them on to their offspring, so that the microorganisms in this area carry specific fingerprints of their living environment, such as specific genetic characteristics, metabolites, etc. By analyzing these diverse phenotypic or genotypic characteristics, the purpose of tracing can be achieved.

[0004] Microbial traceability technology has been continuously improved and perfected with the development of biology, especially genetics and molecular biology. Early microbial traceability methods mainly include phenotypic methods and molecular methods. The phenotypic method is based on various metabolites of microorganisms, physiological and biochemical reactions of enzymes, sensitivity of bacteriophages, etc. to identify microbial species. The molecular method is based on genetic material as the research object, mainly based on PCR technology, and identifies the strain type by analyzing the polymorphic differences of strain-specific gene loci or chromosome groups. The phenotypic method has problems such as poor accuracy, long cycle, and large workload. Molecular methods require high equipment requirements due to the need for DNA extraction and identification processes, and have problems such as complex operation and high professional and technical thresholds (as shown in Table 1).

[0005] With the improvement of sequencing technology, a traceability model analysis method has been developed based on high-throughput sequencing technology. It mainly calculates the contribution of each source by importing the high-throughput sequencing results of samples collected from multiple sites into the analysis model. This method is simple to operate and highly operational during use, and is increasingly favored by scientific research staff in scientific research. There are many microbial traceability analysis models, including SourceTracker, FEAST (fast expectation-maximization for microbial source tracking) and Random Forest.

[0006] However, in actual use, we found that the results calculated using the model analysis method were quite different from the actual situation, so we need to develop a more practical microbial traceability method.

[0007] Table 1 Comparison of advantages and disadvantages of traceability analysis methods

[0008] Summary of the invention

[0009] The technical solution of the present invention is to obtain the original traceability results by analyzing the high-throughput data of multiple sites through a traceability model, and then obtain the weighted coefficient by combining the total amount of microorganisms at the traceability source (phenotypic counting method or molecular biology counting method). After calculation and analysis, a method for microbial traceability analysis with higher precision and more accurate results is obtained.

[0010] The invention provides a microorganism tracing method.

[0011] The present invention provides an application of a microbial traceability method in microbial traceability analysis and other substance traceability analysis based on microbial traceability.

[0012] In one aspect, the present invention provides a method for tracing the origin of microorganisms, comprising the following steps:

[0013] S1. Determine the sampling sites of the traceability source and traceability target;

[0014] S2. Collect samples at the sampling sites of the traceability source and the traceability target respectively to obtain multiple samples;

[0015] S3. Obtaining microbial sequencing information at the sampling site;

[0016] S4. Record the sampling information of the sampling site, and import the sampling information of the sampling site and the microbial sequencing information into the traceability model to obtain the original traceability result;

[0017] S5. Perform weighted calculation on the original traceability results based on the total amount of microorganisms at the traceability source to obtain weighted traceability analysis results.

[0018] In some embodiments, the specific steps of performing weighted calculation on the original traceability results based on the total amount of microorganisms at the traceability source described in S5 include:

[0019] S51. Counting the microorganisms in the multiple samples described in S2, and calculating the unit microbial content of the multiple samples;

[0020] S52. Calculate the unit microbial content of each traceable source according to the unit microbial content of the multiple samples described in S51, and calculate the total amount of microorganisms of each traceable source;

[0021] S53. Calculate a weighting coefficient based on the result obtained in S51 and the result obtained in S52;

[0022] S54. Based on the weighting coefficient described in S53, a weighted operation is performed on the original traceability result described in S4 to obtain a weighted traceability analysis result.

[0023] There is a big gap between the calculation results of the existing traceability model and the actual situation. The reasons for this are that, on the one hand, this method does not consider the impact of the total amount of microorganisms at each source on the traceability target during the calculation process, that is, it assumes that the total amount of microorganisms in the source samples is consistent; on the other hand, high-throughput data will not reflect the impact of the source samples on the traceability target after leveling.

[0024] The results of high-throughput sequencing are relative quantitative data and can only be used for proportion analysis. Moreover, since microorganisms cannot migrate autonomously over long distances, their spatial transfer mainly depends on the transfer of attached substances. Therefore, traceability analysis based solely on sequencing result data ignores the transfer of substances in the macroscopic system, which may lead to a large gap between the calculated results and the actual situation.

[0025] In some implementation schemes, compared with the prior art, the present invention proposes for the first time to introduce the total amount of material transfer and the total amount of microbial data it carries during the traceability analysis process, convert the two into a unified coefficient to weight the traceability analysis results, and more comprehensively and fully consider the impact of the two on microbial transfer. The present invention can more accurately calculate the microbial traceability results. The method based on the weighted total amount of microorganisms in the present invention to correct the microbial traceability results is better than simple traceability analysis and is more in line with actual reality. It can provide new traceability ideas and methods for microbial traceability analysis or other material traceability analysis based on microbial traceability, such as gene traceability, pollutant traceability, and soil traceability.

[0026] In some embodiments, the counting method in S51 adopts a dilution coating method and / or a fluorescence quantitative counting method.

[0027] In some embodiments, the counting requirement of the dilution spreading method is that the total number of colonies on the plate is 30 to 300.

[0028] In some embodiments, in the fluorescent quantitative counting method, the amplified gene fragment is selected from one or more combinations of 16S rRNA gene fragments, 18S rRNA gene fragments, and ITS gene fragments;

[0029] In some embodiments, in the fluorescent quantitative counting method, the amplified gene fragment is within the range of the 16S rRNA gene fragment.

[0030] In some embodiments, in S51, the number of counts for each sample is ≥3, and the counting results are calculated as an average value.

[0031] In some embodiments, the original traceability results described in S4 include the original percentages of each traceability source.

[0032] In some embodiments, the unit microbial content of each traceable source described in S52 is calculated by the following formula 1:

[0033] Formula 1:

[0034] In formula 1, B i is the unit microbial content of the ith traceable source; B ij is the unit microbial content of the jth sample from the i-th traceability source; k is the total number of samples collected from the i-th traceability source.

[0035] In some embodiments, the unit microbial content of the sample includes the number of microorganisms per unit mass, the number of microorganisms per unit area, and the number of microorganisms per unit volume. The unit of the unit microbial content of the sample includes CFU / g, CFU / m 2 、CFU / m 3 .

[0036] In some embodiments, the total amount of microorganisms at each traceable source described in S52 is calculated by the following formula 2:

[0037] Formula 2: Y i =A i ×B i

[0038] In formula 2, Y i is the total amount of microorganisms at the ith traceable source, in CFU; A i is the value of the ith traceable source; B i is the unit microbial content of the i-th traceable source.

[0039] In some embodiments, the unit microbial content of the traceable source includes the number of microorganisms per unit mass, the number of microorganisms per unit area, and the number of microorganisms per unit volume. The unit of the unit microbial content of the traceable source includes CFU / g, CFU / m 2 、CFU / m 3 .

[0040] In some embodiments, the measurement method of the sampling amount for tracing the source includes mass, area, and volume, and the unit of the sampling amount for tracing the source includes g, m 2 、m 3 .

[0041] In some embodiments, the weighting coefficient described in S53 is calculated by the following formula 3:

[0042] Formula 3:

[0043] In formula 3, C i is the weighting coefficient of the ith tracing source; Y i is the total amount of microorganisms at the ith traceable source, in CFU; Y min It is the minimum value of the total amount of microorganisms in all traceable sources.

[0044] In some embodiments, the traceability source also includes unknown sources. When performing weighted operation on the original traceability result described in S54, the original percentage of unknown sources in the original traceability result is kept unchanged, and weighted operation is performed on the remaining known sources.

[0045] In some embodiments, the weighted traceability analysis result described in S54 is calculated by the following formula 4:

[0046] Formula 4:

[0047] In formula 4, X' i is the weighted percentage of the ith traceable source; C i is the weighting coefficient of the ith traceability source; Xi is the original percentage of the ith traceability source; Xun is the original percentage of the unknown source; m is the total number of traceability sources excluding unknown sources; It is the sum of the original percentages of the remaining traceable sources except the unknown sources multiplied by the corresponding source weighting coefficients.

[0048] In some embodiments, obtaining the microbial sequencing information of the sampling site described in S3 includes extracting the DNA of the sample, performing PCR amplification and amplicon sequencing on specific fragments, classifying the microorganisms in each sample into species according to the sequencing results, and completing species annotation based on the species database.

[0049] In some embodiments, the specific fragment is selected from one or more combinations of 16S rRNA gene fragments, 18S rRNA gene fragments, and ITS gene fragments.

[0050] In some embodiments, the specific fragment is a 16S rRNA gene fragment.

[0051] In some embodiments, the species classification is completed at 99% similarity.

[0052] In some embodiments, samples from the same traceability source or the same traceability target use a unified high-throughput sequencing platform and species database.

[0053] In some embodiments, the microbial sequencing information described in S3 is obtained from all samples obtained from S2 in the same batch.

[0054] In some embodiments, the sampling information described in S4 includes sample grouping information, sample quantity, sampling method, and sample unit.

[0055] In some embodiments, the microbial sequencing information is flattened and then imported into the traceability model.

[0056] In some embodiments, the traceability model is selected from SourceTracker and FEAST.

[0057] In some embodiments, the number of traceability sources described in S1 is greater than or equal to 3.

[0058] In some embodiments, the sample described in S2 is packaged in a sterile sampling bag, and after collection is completed, steps S3 and S5 are immediately performed.

[0059] In some embodiments, the sample described in S2 is solid, and / or liquid, and / or gaseous.

[0060] In some embodiments, when the sample described in S2 is in liquid form, the collection further includes membrane filtration.

[0061] In some embodiments, when the sample described in S2 is in a gaseous state, the collection further comprises using an air sampler for vacuum filtration.

[0062] On the other hand, the present invention provides an application of the microbial traceability analysis method as described above in microbial traceability analysis or traceability analysis of other substances based on microbial traceability.

[0063] In some embodiments, the other substance traceability analysis based on microbial traceability includes gene traceability analysis, pollutant traceability analysis and soil traceability analysis.

[0064] In some embodiments, the microbial traceability analysis is a white wine bulk fermentation microbial traceability analysis.

[0065] In some embodiments, the liquor is a sauce-flavor liquor.

[0066] In some embodiments, the terms used in this application include the following definitions:

[0067] "Traceability source": In microbial traceability analysis, it is a general term for substances or environmental factors that can directly or indirectly affect the number or structure of traceability target microorganisms.

[0068] “Traceability target”: In microbial traceability analysis, the subject whose microbial source needs to be traced.

[0069] "Sampling volume": In this article, "sampling volume" refers to the content of the sample taken from different traceability sources in the traceability source. The unit of the sample content in the traceability source can be g, m 2 、m 3 Etc. In some embodiments, if the total amount of microorganisms in Daqu is to be analyzed, the sample at this time is Daqu, then "sampling volume" refers to the total mass of all Daqu in the traceability source of Daqu; in some embodiments, if the total amount of microorganisms in the drying hall, aisle or operating tools is to be analyzed, the sample at this time is the microorganisms on the surface of the drying hall, aisle or operating tools, then "sampling volume" refers to the total area of ​​all objects in the traceability source such as the drying hall, aisle or operating tools; in some embodiments, if the total amount of microorganisms in the air is to be analyzed, the sample at this time is air, then "sampling volume" refers to the total volume of all air in the traceability source of air.

[0070] “Total amount of microorganisms at the traceability source”: the “total amount” (also called sampling amount) of each traceability source multiplied by the unit microbial content.

[0071] "Unknown source": In microbial traceability analysis, substances or environmental factors that are not included in the traceability source but have an actual impact on the traceability target. It can also be considered as a source component that cannot be confirmed and calculated in the traceability analysis, such as changes in the microbial structure caused by the proliferation of microorganisms in the traceability target.

[0072] "Amplicon sequencing": Amplicon sequencing, also known as microbial diversity sequencing, uses the second-generation or third-generation sequencing platform to perform high-throughput sequencing of PCR products in specific regions of 16S, 18S, and ITS, breaking through the disadvantage that traditional microorganisms cannot be cultivated, and obtaining information such as the microbial community structure in environmental samples.

[0073] “99% similarity”: The samples are sequenced to obtain the original sequencing sequences. The above sequences are clustered according to the sequence similarity of 99% to obtain the OUT_table data.

[0074] “Leveling”: Due to the large difference in the number of reads corresponding to each sample in conventional amplicon sequencing, in order to avoid the deviation in analysis caused by the different sizes of sample data, each sample needs to be randomly leveled. Generally, the minimum number of reads in the sequencing sample is selected as the base, that is, the number of reads of all samples is uniformly leveled to this value.

[0075] "Original traceability result": the result output from the model after running the microbial traceability model after inputting the required parameters. In some embodiments, the result output by the model is the number corresponding to each sampling location point, which represents the contribution value of the traceability source location point to the traceability target.

[0076] "Sample grouping information": It is part of the sampling information and refers to the basis for grouping samples, such as D, S, P, A, T, sink and other information in this article. In actual applications, operators can also use different groups according to the differences of their own samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 This is a comparison chart of the original traceability model analysis results (Original_quantity) of the total microorganisms in the initial pile of wine compensation and the traceability analysis results (Disposed_quantity) based on the weighted total amount of microorganisms. In the figure, D, S, P, A, T, and Unknown are different traceability sources of microorganisms, among which D is Daqu, S is the drying hall, P is the aisle, A is air, T is the operating tool, and Unknown is the traceability source of unknown origin. DETAILED DESCRIPTION

[0078] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0079] Unless otherwise specified, the materials, equipment, software, etc. used in the following examples can be obtained from commercial or public sources.

[0080] Example 1

[0081] A microbial traceability method based on the weighting of the total amount of microorganisms, the specific steps are as follows:

[0082] (1) Determine the sampling locations of the traceability source and traceability target: According to the production process characteristics and actual operation of a certain liquor company, the microbial traceability source is divided into five parts, namely Daqu, drying hall, aisle, air and operating tools. Sampling is carried out from these five locations and the initial pile of mash at the traceability target.

[0083] (2) Collect samples at the determined sampling sites: Different sampling methods were used to obtain samples from the six different locations. The grouping information of the microbial traceability source samples was D, S, P, A, and T, respectively, and the grouping of the traceability target samples was named sink. The specific sampling arrangement is shown in Table 2.

[0084] Table 2. Sample sampling locations and their information

[0085]

[0086] (3) Obtaining microbial sequencing information at the sampling site:

[0087] ① DNA extraction of samples: After pretreatment, the collected samples were used to extract total DNA from the samples using a genomic DNA extraction kit, and 3 μL of the obtained total DNA solution was taken for 1% agarose gel electrophoresis to detect the DNA quality.

[0088] Among them, sample pretreatment and total DNA extraction were entrusted to a professional sequencing company.

[0089] The qualified standards for DNA quality testing are: the total amount of DNA extracted from the sample is >1 μg, 1.5 <OD 260 / OD 280 <2.2, OD 260 / OD 230 ≥1, no obvious degradation.

[0090] ② Amplicon sequencing: After the DNA test quality is qualified, it is sent for amplicon sequencing, and the sequencing fragment is the full length of 16S rRNA.

[0091] ③ Species classification of microorganisms in each sample based on sequencing results: The amplicon sequencing results of the sequencing fragments were divided into operational taxonomic units (OTUs) according to 99% similarity.

[0092] ④Complete species annotation based on the species database: Compare the result sequence of the amplicon sequencing obtained in step ② with the SILVA v138 reference database to complete species annotation and obtain microbial data based on amplicon sequencing.

[0093] (4) Run the microbial traceability model to obtain the original traceability results. The microbial data based on amplicon sequencing obtained in step (3)④ and the sample grouping information in step (2) are imported into the microbial traceability model FEAST using the "Load_CountMatrix()" and "Load_metadata()" statements, respectively. The FEAST() function is run for traceability calculation. The model setting parameters are: the samples are flattened to the same value according to the OTU results in step (3)③, and different_sources_flag = 1. According to the model calculation, the output results are shown in the "Traceability Analysis Results (%)" column in Table 3. The number corresponding to each position point is the contribution value of the traceability source position point to the total microorganisms of the traceability target, that is, the original percentage of the traceability source is obtained.

[0094] (5) Detection of the number of microorganisms in the sample: The dilution coating method is used to detect the number of microorganisms in the sample, and the unit microbial content of each sample is calculated. The unit microbial content results of the samples obtained from the same source are averaged as the unit microbial content detection value of the source. The specific calculation process is as follows:

[0095] Formula 1:

[0096] In formula 1, B i is the unit microbial content of the ith traceable source; B ij is the unit microbial content of the jth sample from the i-th traceability source; k is the total number of samples collected from the i-th traceability source.

[0097] (6) Determine the total amount of microorganisms at each source location: According to the sampling method of each sample and the production process of the product, determine the total amount of microorganisms at each traceable source, where the total amount of microorganisms of D is the product of the mass of D and the number of microorganisms per unit mass of the source; the total amount of microorganisms of S, P and T is the product of the total surface area and the number of microorganisms per unit area; the total amount of microorganisms of A is the product of the volume of air above and the number of microorganisms per unit volume of air. The specific calculation process is as follows:

[0098] Formula 2: Y i =A i ×B i

[0099] In formula 2, Y i is the total amount of microorganisms at the ith traceable source, in CFU; A i is the value of the ith traceable source; B i is the unit microbial content of the i-th traceable source.

[0100] Taking the calculation process of the total amount of microorganisms at the source D as an example, A i 160000g, B i =38650000 CFU / g. The total amount of microorganisms Y at the traceable source D is calculated according to formula 2. D =160000g×38650000CFU / g=6.18E+12CFU.

[0101] (7) Calculate the weighted coefficient: Take the minimum value of the total amount of microorganisms in each source, which is the value of source A, 1368000 CFU. The total amount of microorganisms in each source divided by the minimum value is the weighted coefficient of the source. The weighted coefficients calculated according to formulas 1 and 2 are shown in Table 3. The specific calculation process is as follows:

[0102] Formula 3:

[0103] In formula 3, C i is the weighting coefficient of the ith tracing source; Y i is the total amount of microorganisms at the ith traceable source, in CFU; Y min is the minimum value of the total amount of microorganisms in all traceable sources, that is, Y A , 1,368,000 CFU.

[0104] Taking the weighted coefficient calculation process of tracing source D as an example, Y i Y D =6.18E+12CFU,Y min Y A = 1368000 CFU, calculate the weighting coefficient of the traceable source D according to formula 3

[0105] (8) Perform a weighted calculation on the original traceability results to obtain a weighted traceability analysis result: keep the proportion of unknown sources (Unknown) unchanged, and perform a weighted calculation on the existing traceability sources. The specific calculation results are shown in Table 3. The specific calculation process is as follows:

[0106] Formula 4:

[0107] In formula 4, X' i is the weighted percentage of the ith traceable source; C i is the weighting coefficient of the ith tracing source; X i is the original percentage of the ith traceable source; X un is the original percentage of unknown sources; m is the total number of traceable sources other than unknown sources, It is the sum of the original percentages of the remaining traceable sources except the unknown sources multiplied by the corresponding source weighting coefficients.

[0108] Taking the weighted percentage calculation of the traceable source D as an example, C i C D =4520467.8,X i For X D =2.80%,X un is 4.29E-07%, It is the sum of the original percentages of the traceable sources D, S, P, A, and T except the unknown sources and the product of the corresponding source weight coefficients, that is, C D ×X D +C S ×X S +C P ×X P +C A ×X A +CT ×X T , calculate the weighted percentage of the traceable source D according to formula 4

[0109] Table 3. Comparison of traceability analysis results of Maotai-flavor liquor fermentation microorganisms

[0110]

[0111] The numerical values ​​in this article are expressed in integers, decimals, scientific notation, etc. Among them, scientific notation "e -x " is equivalent to the exponential counting method "1×10 -x For example, "6.18E+12" in scientific notation is equivalent to "6.18×10 12 ”.

[0112] (9) Comparative analysis of calculation results

[0113] Figure 1 The figure shows a comparison of the original result (Original_quantity) of the traceability model analysis and the traceability analysis result (Disposed_quantity) based on the weighted total microbial quantity using the method of the present invention. Figure 1 The analysis shows that the traceability results before and after weighting are quite different. The original traceability model analysis results indicate that the microorganisms at the traceability source T (operating tools) are the main contributors to the microorganisms in the initial piled wine compensation. However, in actual production, the factor with a greater impact on the piled fermentation microorganisms should be D (Daqu), followed by S (drying hall).

[0114] The weighted traceability analysis results obtained according to the method of the present invention indicate that Daqu and Liangtang are the main sources of microorganisms in the initial piled wine compensation, which is basically consistent with the actual situation and closer to the actual production, and can reveal the importance of Daqu and Liangtang microorganisms to piled fermentation.

[0115] The experimental results show that the correction of microbial traceability results using the weighted method of total microbial volume is better than simple traceability analysis, is more consistent with actual conditions, the results obtained are more reasonable and reliable, and can more accurately reflect the contribution of potential sources of traceability targets.

Claims

1. A method for tracing the origin of liquor pile fermentation microorganisms, characterized in that: The following steps are involved: S1. Determine the sampling sites of the traceability source and traceability target; S2. Collect samples at the sampling sites of the traceability source and the traceability target respectively to obtain multiple samples; S3. Obtaining microbial sequencing information at the sampling site; S4. Record the sampling information of the sampling site, import the sampling information of the sampling site and the microbial sequencing information into the traceability model to obtain the original traceability result, wherein the original traceability result includes the original percentage of each traceability source; S5. Perform weighted calculation on the original traceability results based on the total amount of microorganisms at the traceability source to obtain a weighted traceability analysis result; the specific steps of performing weighted calculation on the original traceability results based on the total amount of microorganisms at the traceability source described in S5 include: S51. Counting the microorganisms in the multiple samples described in S2, and calculating the unit microbial content of the multiple samples; S52. Calculate the unit microbial content of each traceable source according to the unit microbial content of the multiple samples described in S51, and calculate the total amount of microorganisms of each traceable source; S53. Calculate a weighting coefficient based on the result obtained in S51 and the result obtained in S52; S54. Based on the weighting coefficient described in S53, a weighted operation is performed on the original traceability result described in S4 to obtain a weighted traceability analysis result; The weighting coefficient is calculated by the following formula 3: Formula 3: In formula 3, C i is the weighting coefficient of the ith tracing source; Y i is the total amount of microorganisms at the ith traceable source, in CFU; Y min It is the minimum value of the total amount of microorganisms in all traceable sources; The weighted traceability analysis result described in S54 is calculated by the following formula 4: Formula 4: In formula 4, X i ' is the weighted percentage of the ith traceable source; C i is the weighting coefficient of the ith tracing source; X i is the original percentage of the ith traceable source; X un is the original percentage of unknown sources; m is the total number of traceable sources excluding unknown sources; It is the sum of the original percentages of the remaining traceable sources except the unknown sources multiplied by the corresponding source weighting coefficients.

2. The method according to claim 1, characterized in that The counting method in S51 adopts a dilution coating method and / or a fluorescence quantitative counting method.

3. The method according to claim 2, characterized in that The counting requirement of the dilution coating method is that the total number of colonies on the plate is 30 to 300.

4. The method according to claim 2, characterized in that In the fluorescent quantitative counting method, the amplified gene fragment is selected from one or more combinations of 16S rRNA gene fragment, 18S rRNA gene fragment, and ITS gene fragment.

5. The method according to claim 2, characterized in that In the fluorescent quantitative counting method, the amplified gene fragment is within the range of the 16S rRNA gene fragment.

6. The method according to claim 1, characterized in that In S51, the number of counts for each sample is ≥3, and the counting result is calculated as an average value.

7. The method according to claim 1, characterized in that The unit microbial content of each traceable source described in S52 is calculated by the following formula 1: Formula 1: In formula 1, B i is the unit microbial content of the ith traceable source; B ij is the unit microbial content of the jth sample from the i-th traceability source; k is the total number of samples collected from the i-th traceability source.

8. The method according to claim 7, characterized in that The unit microbial content of the sample includes the number of microorganisms per unit mass, the number of microorganisms per unit area, and the number of microorganisms per unit volume. The units of the unit microbial content of the sample include CFU / g, CFU / m 2 、CFU / m 3 .

9. The method according to claim 1, characterized in that The total amount of microorganisms at each traceable source described in S52 is calculated by the following formula 2: Formula 2: Y i = A i ×B i In formula 2, Y i is the total amount of microorganisms at the ith traceable source, in CFU; A i is the sampling volume of the ith traceable source; B i is the unit microbial content of the i-th traceable source.

10. The method according to claim 9, characterized in that The unit microbial content of the traceable source includes the number of microorganisms per unit mass, the number of microorganisms per unit area, and the number of microorganisms per unit volume. The units of the unit microbial content of the traceable source include CFU / g, CFU / m 2 、CFU / m 3 .

11. The method according to claim 9, characterized in that The measurement methods of the sampling amount for tracing the source include mass, area, and volume. The units of the sampling amount for tracing the source include g, m 2 、m 3 .

12. The method according to claim 1, characterized in that The traceability source also includes unknown sources. When performing weighted calculation on the original traceability result described in S54, the original percentage of unknown sources in the original traceability result is kept unchanged, and weighted calculation is performed on the remaining known sources.

13. The method according to claim 1, characterized in that The method of obtaining the microbial sequencing information of the sampling site described in S3 includes extracting the DNA of the sample, performing PCR amplification and amplicon sequencing on specific fragments, classifying the microorganisms in each sample into species according to the sequencing results, and completing species annotation based on the species database.

14. The method according to claim 13, characterized in that The specific fragment is selected from one or more combinations of 16S rRNA gene fragments, 18S rRNA gene fragments, and ITS gene fragments.

15. The method according to claim 13, characterized in that The specific fragment is a 16S rRNA gene fragment.

16. The method according to claim 13, characterized in that The species classification was completed with 99% similarity.

17. The method according to claim 13, characterized in that Samples with the same traceability source or the same traceability target use a unified high-throughput sequencing platform and species database.

18. The method of claim 1, wherein: The microbial sequencing information described in S3 is obtained from all samples obtained from S2 in the same batch.

19. The method according to claim 1, wherein: The sampling information described in S4 includes sample grouping information, sample quantity, sampling method, and sample unit.

20. The method of claim 1, wherein: The microbial sequencing information is flattened and then imported into the traceability model.

21. The method of claim 1, wherein: The number of traceability sources described in S1 is greater than or equal to 3.

22. The method of claim 1, wherein: The samples described in S2 are packaged in sterile sampling bags, and after collection, steps S3 and S5 are immediately performed.

23. The method of claim 1, wherein: The sample described in S2 is solid, and / or liquid, and / or gaseous.

24. The method of claim 1, wherein: When the sample described in S2 is in liquid form, the collection further includes membrane filtration.

25. The method of claim 1, wherein: When the sample described in S2 is in gaseous state, the collection also includes using an air sampler to perform air extraction and filtration.

26. The method of claim 1, wherein: The liquor is a sauce-flavor liquor.

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