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

Abstract

The invention provides a composite standard substance for animal viruses and a preparation method and application thereof, the composite standard substance comprises more than one NDA virus and more than one RNA virus, the genus and species of each virus in the composite standard substance are different, and viruses belonging to different families exist; the concentrations of various viruses in the composite standard substance are known; based on the nucleic acid concentration corresponding to the virus, the concentration of each virus in the composite standard substance is the same or different, and the concentration ratio is 1: (0.1-10). The composite standard substance disclosed by the invention can meet the characteristic requirements of animal clinical virus diversity, and the evaluation comprehensiveness of multi-type virus detection is improved; and the concentrations of various viruses in the composite standard substance are known, and the concentration difference is small, so that the inaccuracy of the detection result caused by the large concentration difference can be effectively overcome.

Description

Technical Field

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

[0002] In recent years, animal infectious diseases such as African swine fever, avian influenza, and foot-and-mouth disease have caused significant losses to the livestock industry. At the same time, infectious pathogens from animals pose a serious threat to food safety and public health. According to the World Health Organization, about 60% of emerging infectious diseases and 75% of emerging human pathogens in the world come from animals. Effective monitoring and control of the occurrence of animal pathogens is increasingly important for maintaining the "One Health" of humans, animals, and ecosystems.

[0003] Metagenome 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. By comparing and analyzing with the database, it determines the type of pathogenic microorganisms contained in the sample based on the compared sequence information. It can quickly and objectively detect all microorganisms in clinical samples and can be used for sequencing microorganisms (including viruses, bacteria, fungi, protozoa, prokaryotes, etc.) in animals and animal products (animal-derived foods, vaccines, feed, biological materials, cultures or isolates, etc.), and is suitable for second- and third-generation high-throughput sequencing platforms.

[0004] The volume and data volume of viruses in high-throughput sequencing results are smaller than those of other pathogenic microorganisms. Therefore, in the application of animal virus metagenomic microbial high-throughput sequencing technology, it is necessary to use viral nucleic acid standard substances or quality control substances and reference substances to confirm and verify the metagenomic sequencing method, related instruments and equipment (including fully automated library construction equipment and sequencing equipment) and reagents (nucleic acid extraction reagents, library construction reagents, sequencing reagents) to ensure that the detection method meets the detection requirements. The viral nucleic acid standard substances or quality control substances and reference substances selected for method confirmation should contain both DNA viruses and RNA viruses, and at the same time control the detection effects of deoxyribonucleic acid and ribonucleic acid.

[0005] Since metagenomic sequencing can indiscriminately detect the characteristics of all microorganisms, the method has high requirements for the viral nucleic acid standard substances or quality control products, positive samples, and reagents used for performance verification of the method. The diversity of animal clinical virus types needs to be considered. However, there are no dedicated viral metagenomic sequencing-related standard substances, quality control products, positive controls, and application solutions in the existing relevant technical fields. Most laboratories use a single virus or clinical positive sample for method confirmation and performance verification: a single virus cannot comprehensively evaluate the detection effects of multiple types of viruses in clinical practice; the types of viruses in clinical samples are limited, and there is also unevenness and instability in microbial concentration. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a composite standard substance for animal viruses and its preparation method and application. The composite standard substance of the present invention can meet the characteristic requirements of animal clinical virus diversity and improve the comprehensiveness of the evaluation of multiple types of virus detection; and the concentrations of various viruses in the composite standard substance of the present invention are known, and the concentration difference is small, which can effectively overcome the inaccuracy of the detection results caused by the large concentration difference.

[0007] The purpose of the present invention is achieved through the following technical solutions: In a first aspect, the present invention provides a composite standard substance for animal viruses, the composite standard substance comprising one or more NDA viruses and one or more RNA viruses, the genera and species to which the viruses in the composite standard substance belong are different, and there are viruses belonging to different families; the concentrations of various viruses in the composite standard substance are known; based on the nucleic acid concentration corresponding to the virus, the concentrations of the viruses in the composite standard substance are the same or different, and the concentration ratio is between 1:(0.1 and 10). In some embodiments of the present invention, the composite standard material includes more than two kinds of NDA viruses and more than two kinds of RNA viruses.

[0008] In some embodiments of the present invention, the composite standard material includes 2 to 10 NDA viruses and 2 to 20 RNA viruses.

[0009] In some embodiments of the present invention, the genome length of each virus in the composite standard material is between 0.002 kbp and 200 k bp.

[0010] In some embodiments of the present invention, the composite standard material contains viruses with a genome length between 0.002k bp and 100k bp, and contains viruses with a genome length between 100k bp and 200k bp. In some embodiments of the present invention, the composite standard substance includes viruses derived from different hosts; preferably, the viruses can come from animals such as pigs, chickens, ducks, dogs, cats, cattle, horses, sheep, and donkeys.

[0011] In some embodiments of the present invention, the composite standard material includes a virus having a single-stranded nucleic acid and a virus having a double-stranded nucleic acid.

[0012] In some embodiments of the present invention, the composite standard material includes a virus having a linear nucleic acid and a virus having a circular nucleic acid.

[0013] In some embodiments of the invention, the composite standard material includes viruses having segmented nucleic acids and viruses having non-segmented nucleic acids.

[0014] In some embodiments of the present invention, the composite standard material includes enveloped viruses and non-enveloped viruses.

[0015] In some embodiments of the present invention, the composite standard substance includes viruses of different shapes; preferably, it can include viruses having any shape of circular, elliptical, spherical, nearly spherical, icosahedral, hexagonal, polymorphic, etc.

[0016] In some embodiments of the present invention, the composite standard substance is selected from viruses including Herpesviridae, Paramyxoviridae, Arteriviridae, Coronaviridae, Caliciviridae, Flaviviridae, Parvoviridae, etc.

[0017] In some embodiments of the present invention, the composite standard substance is selected from viruses including the genus Varicellavirus, the genus Paramyxovirus, the genus Measlesvirus, the genus Porcine Arteritisvirus, the genus Alphacoronavirus, the genus Circovirus, the genus Flavivirus, the genus Pestivirus, the genus InfluenzaAvirus, the genus Parvovirus, and the like.

[0018] In some embodiments of the present invention, the composite standard substance includes pseudorabies virus, Newcastle disease virus, canine distemper virus, porcine reproductive and respiratory syndrome virus type 2, porcine epidemic diarrhea virus, porcine circovirus type 2, Japanese encephalitis virus, bovine viral diarrhea virus type 1, avian influenza H7N9, porcine parvovirus, etc.

[0019] In some embodiments of the present invention, the viruses in the composite standard substance are pseudorabies virus, Newcastle disease virus, canine distemper virus, porcine reproductive and respiratory syndrome virus type 2, porcine epidemic diarrhea virus, porcine circovirus type 2, Japanese encephalitis virus, bovine viral diarrhea virus type 1, avian influenza H7N9, and porcine parvovirus.

[0020] In some embodiments of the present invention, the concentration of each virus in the composite standard substance is 100 to 100,000 copies / μL, based on the nucleic acid concentration corresponding to the virus.

[0021] In some embodiments of the present invention, the virus preservation solution of the composite standard substance is selected from commercially available nucleic acid protective agents; for example, the nucleic acid (DNA / RNA) protective agent (Cat. No.: SAE0402) of Luoyang Saiao Bioengineering Technology Co., Ltd.

[0022] 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 each animal virus raw material to be used in the preparation of composite reference materials to ensure the accuracy of the nucleic acid concentration in each animal virus raw material and that the nucleic acid concentration meets the requirements; (2) The animal virus raw materials are mixed and diluted with a nucleic acid protective agent to the target nucleic acid concentration to obtain a finished composite standard substance.

[0023] In some embodiments of the present invention, step (1) can be quality-checked by real-time fluorescence quantitative PCR, digital PCR and other technologies.

[0024] 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 virus, the concentration ratio of each virus in the composite standard substance is between 1: (0.1 and 10) and the concentration of each virus is 100 to 100,000 copies / μL.

[0025] 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.

[0026] 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.

[0027] 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 viruses.

[0028] 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.

[0029] 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.

[0030] The beneficial effects of the present invention are as follows: The composite standard substance of the present invention can meet the characteristic requirements of animal clinical virus diversity and improve the comprehensiveness of the evaluation of multi-type virus detection; moreover, the concentrations of various viruses in the composite standard substance of the present invention are known and the concentration difference is small, which can effectively overcome the inaccuracy of the detection results caused by the large concentration difference.

[0031] The composite standard substance of the present invention has good application prospects in quantitative / qualitative analysis or metagenomic sequencing of animal viruses. DETAILED DESCRIPTION

[0032] 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.

[0033] Some of the experimental reagents used in the examples of the present invention are shown in Table 1: Table 1 Experimental reagents

[0034] 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

[0035] The virus composition in the composite standard substance used in the embodiment of the present invention is shown in Table 3: Table 3 Virus composition in composite standard substances

[0036] Example 1 A composite standard substance, wherein the viruses in the composite standard substance are pseudorabies virus, Newcastle disease virus, canine distemper virus, porcine reproductive and respiratory syndrome virus type 2, porcine epidemic diarrhea virus, porcine circovirus type 2, Japanese encephalitis virus, bovine viral diarrhea virus type 1, avian influenza H7N9, and porcine parvovirus. The specific conditions of each virus are shown in Table 3. The nucleic acid concentration of each virus is 1000 copies / μL.

[0037] The preparation method of the composite standard substance comprises the following steps: (1) Use digital PCR technology to conduct quality inspection on each animal virus raw material to be used for the preparation of composite standard materials to ensure the accuracy of the nucleic acid concentration in each animal virus raw material and that the nucleic acid concentration meets the requirements; (2) Mix the animal virus raw materials in equal proportions based on the number of nucleic acid copies, and then dilute them with the nucleic acid (DNA / RNA) protective agent (Cat. No.: SAE0402) produced by Luoyang Saio Bioengineering Technology Co., Ltd. until the nucleic acid concentration of each virus is 1000 copies / μL, thus obtaining the finished composite standard material.

[0038] (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 composite standard substance 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 an appropriate amount of the finished composite standard substance obtained in step (2) and dilute it 10 times and 100 times respectively to obtain 3 composite standard substance samples of different concentrations (wherein the nucleic acid concentration of each virus in sample 1 is 1000 copies / μL, the nucleic acid concentration of each virus in sample 2 is 100 copies / μL, and the nucleic acid concentration of each virus in sample 3 is 10 copies / μL), set 3 replicates for each concentration, and obtain 9 samples to be tested. Then take 100 μL of each sample to be tested and use the nucleic acid automatic extractor KINGFISHER FLEX in the biological safety cabinet BSC-1304IIA2 according to the instructions of the DNA & RNA nucleic acid extraction kit for nucleic acid extraction; 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 MGISEQ G99RS High-throughput Sequencing Reagent Set (PE150), and prepare DNB 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.

[0039] 4. Bioinformatics analysis: Fastp software was used to control the quality of the data, BWA software was used to align it 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 Tables 4, 5, and 6. According to the data in Tables 4, 5, and 6, on the high-throughput sequencer DNBSEQ-G99RS, using the DNA & RNA co-construction library kit and the DNBSEQ-E25RS high-throughput sequencing reagent set (FCL PE150) for metagenomic sequencing, viral nucleic acids can almost be detected at 100 copies / μL, with a coverage ratio of >5%, and most viruses have a repeated detection CV of <20%; when the viral nucleic acid concentration is 10 copies / μL, most viruses can be detected, but some viruses cannot be detected. This indicates that the detection limit of the animal virus metagenomic sequencing method is around 100 copies / μL, and the concentration of each virus in the composite standard substance is limited to 100 to 100,000 copies / μL in the present invention, which meets the requirements.

[0040] Table 4 Sequencing results of composite standard sample 1 (the concentration of each viral nucleic acid was 1000 copies / μL)

[0041] Table 5 Sequencing results of composite standard sample 2 (the concentration of each viral nucleic acid is 100 copies / μL)

[0042] Table 6 Sequencing results of composite standard sample 3 (the concentration of each viral nucleic acid is 10 copies / μL)

[0043] 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 viruses, characterized in that: The composite standard substance includes more than one NDA virus and more than one RNA virus. The genera and species of the viruses in the composite standard substance are different, and there are viruses belonging to different families. The concentrations of various viruses in the composite standard substance are known. Based on the nucleic acid concentration corresponding to the virus, the concentrations of the viruses in the composite standard substance are the same or different, and the concentration ratio is between 1: (0.1 and 10).

2. The composite standard substance according to claim 1, characterized in that: The composite standard substance includes more than two kinds of NDA viruses and more than two kinds of RNA viruses.

3. The composite standard substance according to claim 2, characterized in that: The composite standard substance comprises 2 to 10 kinds of DNA viruses and 2 to 20 kinds of RNA viruses.

4. The composite standard substance according to any one of claims 1 to 3, characterized in that: The genome length of each virus in the composite standard material is between 0.002k bp and 200k bp.

5. The composite standard substance according to claim 4, characterized in that: The composite standard material contains viruses with genome lengths between 0.002k bp and 100k bp, and contains viruses with genome lengths between 100k bp and 200k bp.

6. The composite standard substance according to any one of claims 1 to 3 and 5, characterized in that: The composite standard substance is selected from viruses including the genus Varicellavirus, the genus Paramyxovirus, the genus Measlesvirus, the genus Porcine Arteritisvirus, the genus Alphacoronavirus, the genus Circovirus, the genus Flavivirus, the genus Pestivirus, the genus InfluenzaAvirus, and the genus Parvovirus.

7. The composite standard substance according to claim 6, characterized in that: The composite standard substances include pseudorabies virus, Newcastle disease virus, canine distemper virus, porcine reproductive and respiratory syndrome virus type 2, porcine epidemic diarrhea virus, porcine circovirus type 2, Japanese encephalitis virus, bovine viral diarrhea virus type 1, avian influenza H7N9, and porcine parvovirus.

8. The composite standard substance according to any one of claims 1 to 3, 5 and 7, characterized in that: Calculated based on the nucleic acid concentration corresponding to the virus, the concentration of each virus in the composite standard substance is 100 to 100,000 copies / μL.

9. The method for preparing a composite standard substance according to any one of claims 1 to 8, characterized in that: The preparation method comprises the following steps: (1) Conduct quality inspection on each animal virus raw material to be used in the preparation of composite reference materials to ensure the accuracy of the nucleic acid concentration in each animal virus raw material and that the nucleic acid concentration meets the requirements; (2) The animal virus raw materials are mixed and diluted with a nucleic acid protective agent to the target nucleic acid concentration to obtain a finished composite standard substance.

10. Use of the composite standard substance according to any one of claims 1 to 8 or the composite standard substance prepared by the preparation method according to claim 9 in quantitative / qualitative analysis or metagenomic sequencing of animal viruses.

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