Composite standard substance aiming at animal pathogenic microorganisms as well as preparation method and application of composite standard substance

Abstract

The invention provides a composite standard substance aiming at animal pathogenic microorganisms and a preparation method and application thereof, the composite standard substance comprises microorganisms, and the microorganisms specifically comprise more than one bacteria, more than one fungi, more than one prokaryote, more than one protist and more than one virus. The pathogen classification family, genus and species of each microorganism in the composite standard substance are different; the nucleic acid concentration of any virus in the composite standard substance is greater than or equal to the nucleic acid concentration of any pathogenic microorganism in the non-virus microorganisms. The microorganisms in the composite standard substance cover five boundary classes of bacteria, fungi, prokaryotes, protists and viruses, and can meet quantitative / qualitative analysis or metagenome sequencing requirements of animal clinical pathogenic microorganism diversity; moreover, the composite standard substance disclosed by the invention is beneficial to ensuring that virus and non-virus microorganisms can be detected in one-time detection, and the possibility of false negative phenomenon when the total nucleic acid concentration of a sample is too low is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogen detection or high-throughput sequencing, and in particular to a composite standard substance for animal pathogenic microorganisms, and a preparation method and application thereof. Background Art

[0002] Metagenomic high-throughput sequencing technology does not rely on traditional microbial culture and does not require specific amplification. It directly performs high-throughput sequencing on nucleic acids in clinical samples. It can be used to detect all microorganisms in animals and animal products, including viruses, bacteria, fungi, protozoa, prokaryotes, etc.

[0003] During the clinical detection and application of metagenomic sequencing technology for animal pathogenic microorganisms, positive nucleic acid standard substances or quality control products and reference substances should be used to confirm the metagenomic sequencing methods, instruments and equipment (including fully automated library construction equipment and sequencing equipment), key reagents and consumables (nucleic acid extraction reagents, library construction reagents, sequencing reagents), etc. and verify their performance to ensure that the sequencing methods, instruments and equipment and key reagents and consumables meet the detection requirements.

[0004] Since metagenomic sequencing can detect the characteristics of all microorganisms indiscriminately, the performance verification of this method requires high requirements for nucleic acid standard materials, quality control materials, and positive control samples, and it is necessary to consider the diversity of animal clinical pathogenic microorganisms, such as common viruses and bacteria. However, there are no dedicated animal pathogenic microorganism metagenomic sequencing-related standard materials, quality control materials, positive control samples, and application solutions in the existing relevant technical fields. Most laboratories use a single or small amount of virus or bacterial culture, or use clinical positive samples for method confirmation and performance verification, which has certain defects in species richness, reagent uniformity, and stability. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a composite standard substance for animal pathogenic microorganisms, a preparation method and an application thereof. The pathogenic microorganisms in the composite standard substance of the present invention cover five kingdoms of bacteria, fungi, prokaryotes, protozoa and viruses, and can meet the quantitative / qualitative analysis or metagenomic sequencing requirements of the diversity of clinical pathogenic microorganisms in animals; and in the composite standard substance of the present invention, the nucleic acid concentration of any virus is greater than or equal to the nucleic acid concentration of any non-viral microorganism, which is beneficial to ensure that both viruses and non-viral microorganisms can be detected in one test, reducing the possibility of false negatives when the total nucleic acid concentration of the sample is too low.

[0006] The purpose of the present invention is achieved through the following technical solutions: In the first aspect, the present invention provides a composite standard substance for animal pathogenic microorganisms, wherein the composite standard substance contains pathogenic microorganisms derived from animals, wherein the pathogenic microorganisms include viruses and non-viral microorganisms, and the pathogenic microorganisms specifically include more than one bacteria, more than one fungi, more than one prokaryotes, more than one protists, and more than one viruses, and the family, genus, and species of microbial classification to which each pathogenic microorganism in the composite standard substance belongs are different; based on the nucleic acid concentration corresponding to the pathogenic microorganism, the concentration of any virus in the composite standard substance is greater than or equal to the concentration of any pathogenic microorganism in the non-viral microorganisms.

[0007] In some embodiments of the present invention, the concentration of any one virus in the composite standard substance is greater than the concentration of any one pathogenic microorganism in the non-viral microorganism.

[0008] In some embodiments of the present invention, the non-viral microorganisms in the composite standard substance refer to bacteria, fungi, prokaryotes, and protists in the composite standard substance.

[0009] In some embodiments of the present invention, based on the nucleic acid concentration corresponding to the pathogenic microorganism, the ratio of the concentration of any virus in the composite standard substance to the concentration of any pathogenic microorganism in the non-viral microorganism is between (1 and 100):1, preferably between (2 and 80):1, more preferably between (5 and 50):1, further preferably between (6 and 20):1, further preferably between (8 and 16):1, and most preferably 10:1.

[0010] In some embodiments of the present invention, the ratio of the concentration of any virus in the composite standard substance to the concentration of any pathogenic microorganism in the non-viral microorganism, measured by the nucleic acid concentration corresponding to the pathogenic microorganism, can be any of the following ratios or between any two of the following ratios: 1:1, 1.1:1, 2:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1.

[0011] In some embodiments of the present invention, based on the nucleic acid concentration corresponding to the pathogenic microorganisms, the concentration of each pathogenic microorganism belonging to bacteria, fungi, prokaryotes or protozoa in the composite standard substance is 10 to 10,000 copies / μL, and the concentration of each virus in the composite standard substance is 100 to 100,000 copies / μL.

[0012] In some embodiments of the present invention, based on the nucleic acid concentration corresponding to the pathogenic microorganisms, the concentration of each pathogenic microorganism belonging to bacteria, fungi, prokaryotes or protozoa in the composite standard substance is 10 copies / μL, and the concentration of each virus in the composite standard substance is 100 copies / μL.

[0013] In some embodiments of the present invention, based on the nucleic acid concentration corresponding to the pathogenic microorganisms, the concentration of each pathogenic microorganism belonging to bacteria, fungi, prokaryotes or protozoa in the composite standard substance is 100 copies / μL, and the concentration of each virus in the composite standard substance is 1000 copies / μL.

[0014] In some embodiments of the present invention, based on the nucleic acid concentration corresponding to the pathogenic microorganisms, the concentration of each pathogenic microorganism belonging to bacteria, fungi, prokaryotes or protists in the composite standard substance is 1000 copies / μL, and the concentration of each virus in the composite standard substance is 10000 copies / μL.

[0015] In some embodiments of the present invention, based on the nucleic acid concentration corresponding to the pathogenic microorganisms, the concentration of each pathogenic microorganism belonging to bacteria, fungi, prokaryotes or protozoa in the composite standard substance is 10,000 copies / μL, and the concentration of each virus in the composite standard substance is 100,000 copies / μL.

[0016] In some embodiments of the present invention, the pathogenic microorganisms in the composite standard substance are inactivated pathogenic microorganisms.

[0017] In some embodiments of the present invention, the pathogenic microorganisms in the composite standard substance include more than one bacteria, more than one fungus, more than one prokaryotic organism, more than one protist, more than two viruses, and the more than two viruses include RNA viruses and DNA viruses.

[0018] In some embodiments of the present invention, the pathogenic microorganisms in the composite standard substance include 1 to 50 bacteria, 1 to 10 fungi, 1 to 10 prokaryotes, 1 to 10 protozoa, and 2 to 20 viruses.

[0019] In some embodiments of the present invention, the genome length of each pathogenic microorganism in the composite standard material is between 0.002M bp and 70M bp.

[0020] In some embodiments of the present invention, the composite standard material contains pathogenic microorganisms with a genome length between 0.002M bp and 40M bp, and contains pathogenic microorganisms with a genome length between 40M bp and 70M bp.

[0021] In some embodiments of the present invention, the bacteria are selected from pathogenic microorganisms including Brucellaceae, Clostridiaceae, Erysipelothrixaceae, Enterobacteriaceae, Pseudomonadaceae, Enterobacteriaceae, Staphylococcaceae, and the like.

[0022] In some embodiments of the present invention, the bacteria are selected from pathogenic microorganisms including Brucella, Clostridium, Erysipelothrix, Escherichia, Pseudomonas, Salmonella, Staphylococcus, etc.

[0023] In some embodiments of the present invention, the fungus is selected from pathogenic microorganisms including Saccharomyces family and the like.

[0024] In some embodiments of the present invention, the fungus is selected from pathogenic microorganisms including Candida and the like.

[0025] In some embodiments of the present invention, the prokaryotic organism is selected from pathogenic microorganisms including Mycoplasma family and the like.

[0026] In some embodiments of the present invention, the prokaryotic organism is selected from pathogenic microorganisms including Mycoplasma and the like.

[0027] In some embodiments of the present invention, the protists are selected from pathogenic microorganisms including Sarcocystis family and the like.

[0028] In some embodiments of the present invention, the protists are selected from pathogenic microorganisms including Toxoplasma and the like.

[0029] In some embodiments of the present invention, the virus is selected from pathogenic microorganisms including Coronaviridae, Caliciviridae, etc.

[0030] In some embodiments of the present invention, the virus is selected from pathogenic microorganisms including alphacoronavirus, circovirus, etc.

[0031] In some embodiments of the present invention, the composite standard substance includes Brucella, Candida albicans, Clostridium perfringens, Erysipelothrix rhusiopathiae, Escherichia coli, Mycoplasma hyopneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis, Staphylococcus aureus, Toxoplasma gondii, porcine epidemic diarrhea virus, porcine circovirus type 2, etc.

[0032] In some embodiments of the present invention, the microorganisms in the composite standard substance are Brucella, Candida albicans, Clostridium perfringens, Erysipelothrix rhusiopathiae, Escherichia coli, Mycoplasma hyopneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis, Staphylococcus aureus, Toxoplasma gondii, porcine epidemic diarrhea virus, and porcine circovirus type 2.

[0033] In some embodiments of the present invention, the microbial preservation solution of the composite standard substance is a commercially available nucleic acid protective agent; for example, the nucleic acid (DNA / RNA) protective agent (Cat. No.: SAE0402) of Luoyang Saiao Bioengineering Technology Co., Ltd.

[0034] In a second aspect, the present invention provides a method for preparing the composite standard substance according to the first aspect, the preparation method comprising the following steps: (1) Conduct quality inspection on raw materials of bacteria, fungi, prokaryotes, protozoa, and viruses to ensure the accuracy of nucleic acid concentration in the raw materials and that the nucleic acid concentration meets the requirements; (2) The raw materials of each component are mixed according to a certain nucleic acid ratio, and then diluted with a nucleic acid protective agent to the target nucleic acid concentration to obtain a finished composite standard substance.

[0035] In some embodiments of the present invention, the step (1) can be quality-checked by real-time fluorescence quantitative PCR, digital PCR and other technologies.

[0036] In some embodiments of the present invention, the mixing ratio of step (2) satisfies: based on the nucleic acid concentration corresponding to the pathogenic microorganism, the ratio of the concentration of any virus in the composite standard substance to the concentration of any pathogenic microorganism in the non-viral microorganism is between (1-100):1, preferably between (2-80):1, more preferably between (5-50):1, further preferably between (6-20):1, further preferably between (8-16):1, and most preferably 10:1.

[0037] In some embodiments of the present invention, the target nucleic acid concentration of step (2) satisfies: based on the nucleic acid concentration corresponding to the pathogenic microorganism, the concentration of each pathogenic microorganism of the non-viral microorganism in the composite standard substance is 10 to 10,000 copies / μL, and the concentration of each virus in the composite standard substance is 100 to 100,000 copies / μL.

[0038] In some embodiments of the present invention, the nucleic acid protective agent in step (2) is a commercially available nucleic acid protective agent; for example, the nucleic acid (DNA / RNA) protective agent (Cat. No.: SAE0402) of Luoyang Saio Bioengineering Technology Co., Ltd.

[0039] In some embodiments of the present invention, the preparation method further comprises step (3): performing a quality inspection on the finished product by a metagenomic sequencing method, and after confirming that the finished product meets the expected requirements, placing the finished product in a -80°C long-term cryopreservation. Preferably, the finished product can be packaged into 1.5 mL screw-capped spiral tubes and then placed in a -80°C long-term cryopreservation.

[0040] In a third aspect, the present invention provides a composite standard substance as described in the first aspect or a composite standard substance prepared by the preparation method as described in the second aspect for use in quantitative / qualitative analysis or metagenomic sequencing of animal pathogenic microorganisms.

[0041] In some embodiments of the present invention, the metagenomic sequencing can be performed on a first-generation sequencing platform, a second-generation sequencing platform, or a nanopore sequencing platform.

[0042] In some embodiments of the present invention, the application includes using the composite standard substance described in the first aspect or the composite standard substance prepared by the preparation method described in the second aspect to prepare a quality control product or a reference product.

[0043] The beneficial effects of the present invention are as follows: The microorganisms in the composite standard substance of the present invention cover five kingdoms: bacteria, fungi, prokaryotes, protozoa, and viruses, and can meet the quantitative / qualitative analysis or metagenomic sequencing requirements of the diversity of animal clinical pathogenic microorganisms; and in the composite standard substance of the present invention, the nucleic acid concentration of any virus is greater than or equal to the nucleic acid concentration of any viral microorganism in the non-viral microorganisms, which is beneficial to ensure that both viruses and non-viral microorganisms can be detected in one test, reducing the possibility of false negatives when the total nucleic acid concentration of the sample is too low.

[0044] The composite standard substance of the present invention has good application prospects in quantitative / qualitative analysis or metagenomic sequencing of animal pathogenic microorganisms. DETAILED DESCRIPTION

[0045] The following examples further illustrate the technology of the present invention. These examples are for illustration and example of the present invention and are not intended to limit the scope of the present invention in any form.

[0046] Some of the experimental reagents used in the examples of the present invention are shown in Table 1: Table 1 Experimental reagents

[0047] Some of the experimental instruments and equipment used in the embodiments of the present invention are shown in Table 2: Table 2 Experimental instruments and equipment

[0048] The composition of pathogenic microorganisms in the composite standard substance used in the embodiment of the present invention is shown in Table 3: Table 3 Composition of pathogenic microorganisms in composite standard substances

[0049] Example 1 A composite standard substance, wherein the pathogenic microorganisms in the composite standard substance are Brucella, Candida albicans, Clostridium perfringens, Erysipelothrix rhusiopathiae, Escherichia coli, Mycoplasma hyopneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis, Staphylococcus aureus, Toxoplasma gondii, porcine epidemic diarrhea virus, and porcine circovirus type 2. The specific conditions of each microorganism are shown in Table 3.

[0050] The preparation method of the composite standard substance comprises the following steps: (1) Use digital PCR technology to conduct quality inspection on Brucella, Candida albicans, Clostridium perfringens, Erysipelothrix rhusiopathiae, Escherichia coli, Mycoplasma hyopneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis, Staphylococcus aureus, Toxoplasma gondii, porcine epidemic diarrhea virus, and porcine circovirus type 2 to ensure the accuracy of the nucleic acid concentration in each microorganism and that the nucleic acid concentration meets the requirements; (2) The microorganisms were mixed in a ratio of 1:0.1 according to the nucleic acid concentration of each virus and each non-viral microorganism (i.e., Brucella, Candida albicans, Clostridium perfringens, Erysipelothrix rhusiopathiae, Escherichia coli, Mycoplasma hyopneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis, Staphylococcus aureus, Toxoplasma gondii), and then diluted to the target nucleic acid concentration (see Table 4 for details) using the nucleic acid (DNA / RNA) protective agent (Cat. No.: SAE0402) of Luoyang Saiao Bioengineering Technology Co., Ltd., to obtain the finished composite standard substances (a total of 3 composite standard substances with different concentrations).

[0051] (3) The finished composite standard substance obtained in step (2) is quality inspected by the metagenomic sequencing method. After confirming that it meets the expected requirements, the finished product is packaged into 1.5 mL screw-capped spiral tubes and then stored in a -80°C freezer for long-term storage. The specific operation process of quality inspection of the finished composite standard substance obtained in step (2) by the metagenomic sequencing method is as follows: 1. Nucleic acid extraction: Take 3 portions of the 3 different concentrations of composite standard substances obtained in step (2), 100 μL each; perform nucleic acid extraction using the automatic nucleic acid extractor KINGFISHER FLEX in the biosafety cabinet BSC-1304IIA2 according to the instructions of the DNA & RNA nucleic acid extraction kit; 2. Reverse transcription and library construction: According to the instructions of the DNA & RNA co-library construction kit, reverse transcribe and build the library for the nucleic acid extracted in step 1; 3. DNB preparation and sequencing: Calculate the required ssDNA library volume according to the requirements of the DNBSEQ-G99RS high-throughput sequencing reagent set (SE100), and prepare DNBs from the ssDNA library obtained in step 2 according to the required ssDNA library volume. DNB concentration ≥ 8 ng / μL can be sequenced on the machine, and sequencing is performed using the high-throughput sequencer DNBSEQ-G99RS, with the number of reads for each sample ≥ 20 M; 4. Bioinformatics analysis: Fastp software was used to perform quality control on the data, BWA software was used to align the data to the reference genome, and samtools software was used to convert the file format and count the number of reads. The average RPTM value of each concentration sample was calculated based on the number of reads per ten million (RPTM) detected for each replicate at each concentration, and the coefficient of variation CV value of each replicate sample was calculated based on the average RPTM value. The results are shown in Table 4. According to the data in Table 4, on the high-throughput sequencer DNBSEQ-G99RS, metagenomic sequencing was performed using the DNA & RNA co-construction library kit and the DNBSEQ-G99RS high-throughput sequencing reagent set (G99FCL SE100) sequencing kit. The nucleic acid concentration of the virus was 10 copies / μL, and the nucleic acid concentration of bacteria, fungi, prokaryotes, and protists was 1 copy / μL. Almost all of them can be detected, indicating that under the metagenomic sequencing method, the detection limit of viruses in animal clinical samples is about 10 copies / μL, and the detection limit of bacteria, fungi, prokaryotes, and protists is about 1 copy / μL; the present invention stipulates that the concentration of each pathogenic microorganism of non-viral microorganisms in the composite standard substance is 10 to 10000 copies / μL, and the concentration of each virus in the composite standard substance is 100 to 100000 copies / μL, which meets the detection limit requirements.

[0052] Table 4 Sequencing results of composite standard substances at different concentrations

[0053] The two groups of data with large differences in RPTM detection between different dilution multiples in Table 4 were removed, and the statistical RPTM mean values ​​of viruses and non-viral microorganisms were shown in Table 5. The RPTM ratios of viruses and non-viral microorganisms with different nucleic acid concentrations were shown in Table 6. According to Tables 5 and 6, it can be concluded that when the nucleic acid concentration of the virus is 10 or 100 times that of the non-viral microorganism nucleic acid, RPTM 病毒 ≈RPTM非病毒微生物 , viruses and non-viral microorganisms can be detected simultaneously; when the nucleic acid concentration of the virus is 10 times lower than that of the non-viral microorganism, RPTM 病毒 <RPTM 非病毒微生物 If the total nucleic acid concentration of the sample is too low, the virus may be missed; when the viral nucleic acid concentration is 100 times higher than the non-viral microbial nucleic acid concentration, RPTM 病毒 >RPTM 非病毒微生物 If the total nucleic acid concentration of the sample is too low, non-viral microorganisms may be missed.

[0054] Table 5 Average number of RPTM detected at different nucleic acid concentrations for viruses and non-viral microorganisms

[0055] Table 6 Analysis of RPTM ratios of viral and non-viral microorganisms detected at different nucleic acid concentrations

[0056] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to the embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A composite standard substance for animal pathogenic microorganisms, characterized in that: The composite standard substance contains pathogenic microorganisms derived from animals, the pathogenic microorganisms include viruses and non-viral microorganisms, and the pathogenic microorganisms specifically include more than one bacteria, more than one fungi, more than one prokaryotes, more than one protists, and more than one viruses, and the family, genus, and species of biological classification to which each pathogenic microorganism in the composite standard substance belongs are all different; based on the nucleic acid concentration corresponding to the pathogenic microorganisms, the ratio of the concentration of any virus in the composite standard substance to the concentration of any pathogenic microorganism in the non-viral microorganisms is between (1 and 100):

1.

2. The composite standard substance according to claim 1, characterized in that: The pathogenic microorganisms in the composite standard substance include more than one bacteria, more than one fungus, more than one prokaryotic organism, more than one protist, and more than two viruses, and the more than two viruses include RNA viruses and DNA viruses.

3. The composite standard substance according to claim 2, characterized in that: The pathogenic microorganisms in the composite standard substance include 1 to 50 kinds of bacteria, 1 to 10 kinds of fungi, 1 to 10 kinds of prokaryotes, 1 to 10 kinds of protozoa, and 2 to 20 kinds of viruses.

4. The composite standard substance according to any one of claims 1 to 3, characterized in that: The genome length of each pathogenic microorganism in the composite standard material is between 0.002M bp and 70M bp.

5. The composite standard substance according to claim 4, characterized in that: The composite standard material contains pathogenic microorganisms with genome lengths between 0.002M bp and 40M bp, and contains pathogenic microorganisms with genome lengths between 40M bp and 70M bp.

6. The composite standard substance according to any one of claims 1 to 3 and 5, characterized in that: The bacteria are selected from pathogenic microorganisms including Brucellaceae, Clostridiaceae, Erysipelothrixaceae, Enterobacteriaceae, Pseudomonadaceae, Enterobacteriaceae, and Staphylococcaceae; and / or, the fungus is selected from pathogenic microorganisms including the family Saccharomyces; and / or, the prokaryotes are selected from pathogenic microorganisms including the family Mycoplasma; and / or, the protists are selected from pathogenic microorganisms including the family Sarcocystis; And / or, the virus is selected from pathogenic microorganisms including Coronaviridae and Caliciviridae.

7. The composite standard substance according to claim 6, characterized in that: The bacteria are selected from pathogenic microorganisms including Brucella, Clostridium, Erysipelothrix, Escherichia, Pseudomonas, Salmonella, and Staphylococcus; and / or, the fungus is selected from pathogenic microorganisms including Candida spp.; and / or, the prokaryotes are selected from pathogenic microorganisms including Mycoplasma; and / or, the protists are selected from pathogenic microorganisms including Toxoplasma; And / or, the virus is selected from pathogenic microorganisms including alphacoronavirus and circovirus.

8. The composite standard substance according to claim 1, characterized in that: The composite standard substance includes Brucella, Candida albicans, Clostridium perfringens, Erysipelothrix rhusiopathiae, Escherichia coli, Mycoplasma hyopneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis, Staphylococcus aureus, Toxoplasma gondii, porcine epidemic diarrhea virus, and porcine circovirus type 2.

9. The composite standard substance according to any one of claims 1 to 3, 5, 7 to 8, characterized in that: Based on the nucleic acid concentration corresponding to the pathogenic microorganism, the ratio of the concentration of any virus in the composite standard substance to the concentration of any microorganism in the non-viral microorganism is between (2 and 80):

1.

10. The composite standard substance according to claim 9, characterized in that: Measured in terms of nucleic acid concentration corresponding to pathogenic microorganisms, the concentration of each pathogenic microorganism belonging to bacteria, fungi, prokaryotes or protozoa in the composite standard substance is 10 to 10,000 copies / μL, and the concentration of each virus in the composite standard substance is 100 to 100,000 copies / μL.

11. The method for preparing a composite standard substance according to any one of claims 1 to 10, characterized in that: The preparation method comprises the following steps: (1) Conduct quality inspection on raw materials of bacteria, fungi, prokaryotes, protozoa, and viruses to ensure the accuracy of nucleic acid concentration in the raw materials and that the nucleic acid concentration meets the requirements; (2) The raw materials of each component are mixed according to a certain nucleic acid ratio, and then diluted with a nucleic acid protective agent to the target nucleic acid concentration to obtain a finished composite standard substance.

12. Use of the composite standard substance according to any one of claims 1 to 10 or the composite standard substance prepared by the preparation method according to claim 11 in quantitative / qualitative analysis or metagenomic sequencing of animal pathogenic microorganisms.

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