Probe library for high-throughput detection of mouse and hamster related species-specific viruses in CHO cell library and application of probe library

Abstract

The invention provides a probe library for high-throughput detection of mouse and hamster related species-specific viruses in a CHO cell library and application of the probe library. The probe library is used for detecting a mouse pox virus, a hantavirus, a K virus, a lactic dehydrogenase virus, a lymphocytic choriomeningitis virus, a mouse microvirus, a mouse adenovirus, a mouse cytomegalovirus, a mouse encephalomyelitis virus, a mouse hepatitis virus, a mouse rotavirus, a mouse pneumovirus, a multiple oma virus, a reovirus type 3 and a sendai virus; sV5 and thymoviruses and variant viruses thereof. The high-throughput sequencing method based on the probe library can be used for distinguishing viral nucleic acid with infection risk and quickly checking whether a sample is polluted by specific live viruses or not. The detection limit reaches the level of a traditional PCR method, the capability of detecting all known variants of to-be-detected species-specific viruses is kept, 17 viruses can be detected only by implementing one detection item, and the method has the advantages of high efficiency, rapidness and high sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus diagnosis, and in particular relates to a probe library for high-throughput detection of species-specific viruses related to mice and hamsters in a CHO cell library and an application thereof. Background Art

[0002] The virus safety of biological products is one of the focuses of the product system of the biopharmaceutical industry. For cells used to produce biological products, specific virus testing of the species in which the cells are located is required. Detection of species-specific viruses in rodent cell lines, including Ectromelia Virus, Hantaan Virus, K Virus, Lactic Dehydrogenase Virus, Lymphocytic Choriomeningitis Virus, Minute Virus of Mice, Mouse Adenovirus, Mouse Cytomegalovirus, Mouse Encephalomyelitis Virus, Mouse Hepatitis Virus, Mouse Rotavirus, Pneumonia Virus of Mice, Polyoma Virus, Reovirus Type 3, Sendai Virus, SV5, and Thymic Virus. Virus), which can be detected using immunology-based mouse and hamster antibody production experiments (MAP and HAP); the samples are injected into highly susceptible natural hosts (i.e., mice or hamsters) and raised in an isolated environment for 4 weeks. Serum samples are collected, and the serum is tested for antibodies to these 17 viruses using enzyme-linked immunosorbent assay (ELISA) or immunofluorescence assay (IFA) to confirm whether these viruses are in the sample. This experiment requires the use of experimental animals, and the experimental cycle takes 8 weeks. From a GMP perspective, animal experiments are difficult to verify in terms of accuracy, specificity, and detection limit. PCR is also a commonly used detection method. Since the PCR method can only detect known specific viruses, potential variants may be missed, and method development and testing for each virus must be performed separately, which is a lot of work.

[0003] In recent years, the next-generation sequencing (NGS) technology has been gradually applied to the viral safety testing of biological products. The NGS method has the advantages of not requiring known sequence information, being able to conduct large-scale parallel testing, and in principle being able to identify all possible contaminant sequences in the sample. At the same time, the NGS experiment takes only one week, which is much shorter than animal experiments. In the updated version of the relevant regulations on cells used to produce biological products, it is explicitly mentioned that technologies such as NGS should be used to replace in vivo methods to obtain more sensitive test results and reduce the use of experimental animals.

[0004] The nucleic acid extraction products of the cell bank contain a large amount of host genome. Compared with the genome content of the host cells, the genome content of the virus is generally very small, which often leads to the fact that most of the measured sequences are useless host genome sequences, increasing the difficulty of subsequent information analysis and reducing the detection sensitivity. In order to obtain sufficient viral genome sequence coverage to determine positive detection, it is very important to reduce the impact of the host genome. The method of non-specific NGS to improve detection sensitivity is mainly to increase the amount of sequencing data, but because most of the data in the sequencing results are interference data of the host genome, this method is costly, inefficient, and wasteful. Therefore, it is urgent to provide a new, highly sensitive high-throughput sequencing method to detect 17 specified MAP / HAP-related viruses in cell banks. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a probe library for high-throughput detection of species-specific viruses related to mice and hamsters in CHO cell banks and its application. The present invention has developed a high-throughput sequencing method to detect 17 specified MAP / HAP-related viruses in cell banks. In order to improve the detection sensitivity of the NGS method, the present invention adds a probe hybridization magnetic bead capture step on the basis of the non-specific NGS method to effectively enrich the potential viral genomes to be detected, reduce the waste of sequencing data caused by the host background of the cell bank, and improve the detection sensitivity of NGS while maintaining the same sequencing depth, so that its detection limit reaches the level of the traditional PCR method, while maintaining the ability to detect all known variants of the species-specific viruses to be detected, and has the advantage of detecting 17 viruses with one detection item.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a probe library for high-throughput detection of species-specific viruses associated with mice and hamsters in a CHO cell library, wherein the probe library is used to detect mousepox virus, Hantavirus, K virus, lactate dehydrogenase virus, lymphocytic choriomeningitis virus, microvirus of mice, mouse adenovirus, mouse cytomegalovirus, mouse encephalomyelitis virus, mouse hepatitis virus, mouse rotavirus, mouse pneumonia virus, polyoma virus, reovirus type 3, Sendai virus, SV5 and thymitis virus and variant viruses thereof; the probe library comprises the following probe groups:

[0008] Probe set 1: used to detect Ectromelia Virus and its variants in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 1-42;

[0009] Probe Group 2: used to detect Hantaan Virus and its variants in a sample, comprising any one or a combination of at least two of the probes having nucleotide sequences as shown in SEQ ID NOs: 43-106;

[0010] Probe Group 3: used to detect K virus and its variants in a sample, comprising any one or a combination of at least two of the probes having nucleotide sequences as shown in SEQ ID NOs: 107-145;

[0011] Probe set 4: for detecting Lactic Dehydrogenase Virus and its variants in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 146-166;

[0012] Probe Group 5: for detecting Lymphocytic Choriomeningitis Virus and its variants in a sample, comprising any one or a combination of at least two of the probes having nucleotide sequences as shown in SEQ ID NOs: 167-262;

[0013] Probe Group 6: for detecting Minute Virus of Mice and its variants in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 263-287;

[0014] Probe Group 7: for detecting mouse adenovirus and its variant viruses in a sample, comprising any one or a combination of at least two of the probes having nucleotide sequences as shown in SEQ ID NOs: 288-332;

[0015] Probe Group 8: for detecting mouse cytomegalovirus and its variants in a sample, comprising any one or a combination of at least two of the probes having nucleotide sequences as shown in SEQ ID NOs: 333-387;

[0016] Probe group 9: for detecting mouse encephalomyelitis virus (Mouse Encephalomyelitis Virus) and its variant viruses in a sample, comprising any one or a combination of at least two of the probes having nucleotide sequences as shown in SEQ ID NOs: 388-410;

[0017] Probe set 10: for detecting mouse hepatitis virus (Mouse Hepatitis Virus) and its variant viruses in a sample, comprising any one or a combination of at least two of the probes having nucleotide sequences as shown in SEQ ID NOs: 411-431;

[0018] Probe group 11: used to detect mouse rotavirus and its variant viruses in a sample, comprising any one or a combination of at least two of the probes having nucleotide sequences as shown in SEQ ID NOs: 432-601;

[0019] Probe group 12: for detecting Pneumonia Virus of Mice and its variants in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 602-620;

[0020] Probe set 13: used for detecting polyoma virus and its variants in a sample, comprising any one or a combination of at least two of the probes having nucleotide sequences as shown in SEQ ID NOs: 621-658;

[0021] Probe set 14: for detecting Reovirus Type 3 and its variants in a sample, comprising any one or a combination of at least two of the probes having nucleotide sequences as shown in SEQ ID NOs: 659-1051;

[0022] Probe set 15: for detecting Sendai virus and its variants in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 1052-1070;

[0023] Probe set 16: for detecting SV5 and its variant viruses in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 1071-1089;

[0024] Probe group 17: used to detect thymic virus and its variants in a sample, comprising any one or a combination of at least two of the probes having nucleotide sequences as shown in SEQ ID NOs: 1090-1127.

[0025] In the present invention, the probe library is used to detect species-specific viruses related to mice and hamsters in a sample, and the probe library comprises at least one of the above 17 probe groups; wherein the species-specific viruses comprise: Ectromelia Virus, Hantaan Virus, K Virus, Lactic Dehydrogenase Virus, Lymphocytic Choriomeningitis Virus, Minute Virus of Mice, Mouse Adenovirus, Mouse Cytomegalovirus, Mouse Encephalomyelitis Virus, Mouse Hepatitis Virus, Mouse Rotavirus, Pneumonia Virus of Mice, Polyoma Virus, Reovirus Type 3, Sendai Virus Virus), SV5, thymic virus and at least one of the variants of the above viruses.

[0026] The probes involved in the present invention are designed to ensure the ability to detect different variants of the virus, and are designed according to the genome length, proportion of conserved regions, and number of mutants of different viruses. Therefore, the above probe combination can ensure its ability to detect different variants of the virus.

[0027] In some embodiments, the probe combination comprises any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or seventeen of the above 17 probe groups.

[0028] In a specific embodiment, the probe library may include probe sets for detecting pneumonia virus of mice, minute virus of mice, Sendai virus, reovirus type 3, SV5 and variant viruses thereof in a sample.

[0029] Preferably, the 5' end of the probe has a modification.

[0030] Preferably, the modification is biotin labeling.

[0031] In the present invention, the sample may be a cell bank or total RNA extracted from a cell bank.

[0032] In a second aspect, the present invention provides a kit, comprising the probe library for high-throughput detection of species-specific viruses associated with mice and hamsters in a CHO cell library as described in the first aspect.

[0033] Preferably, the kit further comprises an internal control viral RNA standard.

[0034] Preferably, the internal control viral RNA standard comprises RNA of a probe targeting a genomic sequence of MS2 phage and / or T4 phage.

[0035] In a third aspect, the present invention provides a method for enriching nucleic acid fragments of species-specific viruses associated with mice and hamsters in a sample, the method comprising the following steps:

[0036] (1) Obtaining total RNA from a sample;

[0037] (2) fragmenting the total RNA and synthesizing cDNA;

[0038] (3) Using the probe library for high-throughput detection of species-specific viruses associated with mice and hamsters in a CHO cell library described in the first aspect or the kit described in the second aspect for hybridization capture.

[0039] Preferably, the sample is also spiked with an internal control viral RNA standard;

[0040] Preferably, the internal control viral RNA standard comprises RNA of a probe targeting a genomic sequence of MS2 phage and / or T4 phage;

[0041] Preferably, the sample is a cell bank, and wherein the concentration of mouse and hamster-related species-specific viruses in the cell bank is at least 0.0001 copies / cell.

[0042] Preferably, the sample is total RNA extracted from a cell bank, wherein the concentration of mouse and hamster related species-specific viruses in the total RNA is at least 10 copies / μg RNA.

[0043] Preferably, step (1) further comprises rRNA removal.

[0044] In a fourth aspect, the present invention provides a method for detecting species-specific viruses associated with mice and hamsters in a sample, the method comprising the following steps:

[0045] (A) Obtaining total RNA from samples;

[0046] (B) constructing a DNA library;

[0047] (C) using the probe library for high-throughput detection of species-specific viruses associated with mice and hamsters in a CHO cell library described in the first aspect or the kit described in the second aspect for hybridization capture;

[0048] (D) performing PCR amplification on the product of step (C);

[0049] (E) performing high-throughput sequencing on the product of step (D);

[0050] (F) Using bioinformatics methods to determine whether the product of step (E) is positive for virus detection.

[0051] Preferably, the sample is also spiked with an internal control viral RNA standard;

[0052] Preferably, the internal control viral RNA standard comprises RNA of a probe targeting a genomic sequence of MS2 phage and / or T4 phage;

[0053] Preferably, the sample is a cell bank, and wherein the concentration of mouse and hamster-related species-specific viruses in the cell bank is at least 0.0001 copies / cell.

[0054] Preferably, the sample is total RNA extracted from a cell bank, wherein the concentration of mouse and hamster related species-specific viruses in the total RNA is at least 10 copies / μg RNA.

[0055] Preferably, step (A) further comprises rRNA removal.

[0056] Preferably, the bioinformatics method comprises:

[0057] (i) Data were split using the bcl2fastq program;

[0058] (ii) The reads were aligned with the MAP / HAP virus database using the BWA-MEM program;

[0059] (iii) The number of covered bases was calculated using Samtools.

[0060] The sequencing result data analysis process of the present invention is designed based on the above-mentioned method for detecting species-specific viruses related to mice and hamsters in specific samples. It is completely different from the bioinformatics analysis method of human genome capture, which is usually used in the scenario of conventional hybrid capture technology. The database and analysis code used are innovative. For example, BWA-MEM is used to compare with MAP / HAP virus database, snakemake workflow is used for multi-threaded parallel task processing, and Samtools is used to calculate the number of covered bases.

[0061] Preferably, step (i) comprises: using the bcl2fastq program to split the data according to the labels (index) corresponding to the samples.

[0062] Preferably, step (ii) comprises:

[0063] (a) processing the data obtained in step (i) using fastp software;

[0064] (b) The reads were aligned with the MAP / HAP virus database using the BWA-MEM program;

[0065] (c) Screening reads and their aligned viral genomes using read length ≥75 bp and alignment rate ≥90% as criteria;

[0066] (d) The filtered reads were aligned with each viral genome using the snakemake workflow.

[0067] Preferably, the number of covered bases is calculated based on the standard that the length of reads is ≥75bp and the alignment rate is ≥90%. When the number of covered bases is ≥200bp, the corresponding virus is judged to be detected positive.

[0068] As a preferred embodiment of the present invention, the present invention provides a method for detecting species-specific viruses associated with mice and hamsters in a sample, which comprises the steps of:

[0069] (1) RNAs targeting genomic sequences of 17 MAP / HAP-related species-specific viruses and two bacteriophage probes, MS2 and T4, were synthesized as RNA standards for each virus.

[0070] (2) 1×10 3The copy number of each MAP / HAP-related species-specific virus and MS2 phage RNA standard, and 1×10 10 The copy number of T4 phage RNA standard was spiked to 1×10 7 CHO cells were used as samples.

[0071] (3) Isolate and extract total RNA from samples.

[0072] (4) removing rRNA from the RNA / sample to obtain total RNA after removing rRNA.

[0073] (5) Use Illumina Stranded mRNA Prep kit to construct RNA library.

[0074] (6) Using a probe combination for hybridization capture, and performing PCR amplification and purification on the captured nucleic acid.

[0075] (7) Use the Illumina NextSeq550 system to complete high-throughput sequencing experiments.

[0076] (8) After sequencing, the bcl2fastq program was used to split the data according to the index corresponding to the sample, and the bioinformatics analysis process was started.

[0077] In the present invention, the probe design program CATCH was used to design all 17 MAP / HAP-related species-specific viruses included in the NCBI database, including: Ectromelia Virus, Hantaan Virus, K Virus, Lactic Dehydrogenase Virus, Lymphocytic Choriomeningitis Virus, Minute Virus of Mice, Mouse Adenovirus, Mouse Cytomegalovirus, Mouse Encephalomyelitis Virus, Mouse Hepatitis Virus, Mouse Rotavirus, Pneumonia Virus of Mice, Polyoma Virus, Reovirus Type 3, Sendai Virus, SV5 and Thymic Virus. Virus), as well as the known complete sequences of two bacteriophages, MS2 and T4, were used to design specific capture probes. The probes were single-stranded DNA, each 120 bp in length, allowing for 9 mismatches and a 50 bp coverage extension.

[0078] In the present invention, probes targeting 3500-4000bp of conserved genomic sequences within each virus are designed, thereby maintaining a certain capture efficiency for unknown virus variants while ensuring the capture efficiency for known virus variants, and minimizing the number of probes to reduce costs. The 5' end of the probe is labeled with biotin so that the hybrid complex is captured by magnetic beads coated with streptavidin.

[0079] In the present invention, the total RNA of the sample is extracted and an RNA library is constructed to distinguish viral nucleic acids with infectious risks and identify potential live virus contamination.

[0080] In the present invention, all samples are spiked with two internal control viral RNA standards, MS2 phage and T4 phage. The role of MS2 phage is to control the detection limit to ensure that the detection can reach the preset detection limit; the role of T4 phage is to control the process to ensure that the detected sample can obtain a certain amount of nucleic acid for sequencing after hybridization capture enrichment.

[0081] At present, a single library of a commercial RNA library construction kit can use up to 1 μg of total RNA for library construction. In order to increase the quality of the initial RNA in the library construction stage to improve the efficiency of sample processing, the present invention uses a RiboMinus rRNA removal Kit that can process 10 μg RNA / sample in a single reaction to remove rRNA accounting for 70%-80% of the total RNA, so as to achieve the purpose of enriching potential viral nucleic acids. Since the quality of the total RNA after rRNA removal by the kit still exceeds the upper limit of the loading of a single library of the RNA library construction kit, the subsequent library construction experiment divides the total RNA after rRNA removal into two parallel libraries using the same index (label) for library construction, and re-merges the two libraries in the hybridization stage, thereby increasing the quality of the total RNA of the cell library that can be processed by one experimental process by 10 times, and improving the detection sensitivity.

[0082] The present invention is a qualitative test, which only needs to obtain sequence information that can identify the presence of the virus, and does not need to obtain high-depth full-length virus information. Therefore, under the premise of high initial RNA, the number of PCR cycles for library construction and amplification is still a high 13 cycles, in order to obtain sufficient cDNA library to increase the subsequent hybridization probe capture on-target rate. Similarly, due to the nature of the qualitative detection of the present invention, the tolerance for PCR repetition is very high, and a high 17 cycles are used in the post-hybridization PCR amplification to increase the copy number of the cDNA library containing the virus sequence to be tested as much as possible, and to ensure that the cDNA library obtained after hybridization capture has a concentration suitable for sequencing on the machine.

[0083] In the present invention, when constructing a hybrid capture system, in order to increase the sample loading amount of hybrid capture and reduce the volume of the hybrid system to improve the hybridization efficiency, after merging the two cDNA libraries constructed in parallel for each sample, the library DNA is recovered using the AMPure XP magnetic bead purification system, and the pre-configured hybrid capture system is used instead of water for elution, thereby controlling the total volume of the hybrid system to within 20 μL. The EzyNGS universal blocking primer and salmon sperm DNA in the hybrid system act as blocking adapter primers and renatured double-stranded DNA libraries, which can significantly reduce nonspecific hybrid capture and improve the on-target rate.

[0084] In the present invention, a DNA probe labeled with biotin at the 5' end is hybridized with a constructed cDNA library, and a streptavidin-coated magnetic bead is used to capture the hybridization complex of the probe and the cDNA library through a coupling reaction between streptavidin and biotin, and the magnetic bead-probe-capture library complex carrying the target library is separated by a magnetic frame, the magnetic beads of the capture probe are washed, and the captured library is amplified by PCR and purified and recovered.

[0085] In the present invention, the cDNA library after hybridization capture was determined by Agilent 2100 bioanalyzer, and then the high-throughput sequencing experiment was completed using Illumina NextSeq550 system, and the data volume allocated to each sample was 10G.

[0086] In the present invention, the bioinformatics analysis process after sequencing includes:

[0087] (1) Quality control;

[0088] (2) Removal of process internal controls;

[0089] (3) Removal of host sequences;

[0090] (4) Comparison with MAP / HAP virus database;

[0091] (5) Screening out all the aligned viral reference genomes and reads;

[0092] (6) aligning the selected reads to each selected viral genome;

[0093] (7) Calculate the number of covered bases;

[0094] (8) Output the result with the highest number of bases covered by the same virus for positive judgment;

[0095] (9) If the number of bases covered is ≥200 bp, the virus is considered to be detected positive.

[0096] Among them, the host genome database includes the genomes of humans, mice, hamsters, and green monkeys to cover all cell line source species that may require MAP / HAP testing. The MAP / HAP virus database includes all 17 known complete sequences of MAP / HAP-related species-specific viruses included in the NCBI database and the RVDB database to ensure that all known related virus variants can be detected. When comparing with the MAP / HAP virus database, the screening criteria are read length ≥ 75bp and alignment rate ≥ 90%, which can not only ensure the specificity of detecting known variants of related viruses, but also maintain a certain detection capability for unknown variants of related viruses.

[0097] In a fifth aspect, the present invention provides the use of the probe combination for detecting species-specific viruses associated with mice and hamsters described in the first aspect or the kit described in the second aspect in high-throughput sequencing.

[0098] Compared with the prior art, the present invention has the following beneficial effects:

[0099] The method of the present invention can distinguish viral nucleic acids with infectious risks and has the advantage of quickly checking whether the sample is contaminated by specific live viruses. The traditional MAP / HAP method takes 8 weeks to detect, while the present method only takes 1 week. At the same time, its detection limit reaches the level of the traditional PCR method and maintains the ability to detect all known variants of the species-specific virus to be detected, with the advantage of detecting 17 viruses with one test item.

[0100] The detection limit of the method of the present invention is: after the total cellular RNA is mixed with viral RNA, a positive result of virus incorporation can be detected, and the detectable limit of incorporation is at least 10 viral genome copies / μg RNA or 0.0001 viral genome copies / cell.

[0101] Specificity of the method of the present invention: The examples also demonstrate that the 17 species-specific virus contaminations at the detection limit level required by regulations can be effectively detected without missed detections, and the result classification is displayed by the classification name of the species; no positive virus should be detected in any cell-negative sample. DETAILED DESCRIPTION

[0102] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0103] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0104] Example 1

[0105] This example uses targeted NGS technology to detect 17 species-specific viruses associated with mice / hamsters in a CHO cell library.

[0106] 1. Based on the specific capture probe sequence, 17 MAP / HAP-related species-specific viruses and two phage probes, MS2 and T4, targeting the genome sequence RNA were synthesized as RNA standards for each virus. The length of each standard was 3500-4000bp.

[0107] (1) The NCBI reference sequences and genomic regions corresponding to the sequences of the RNA standards of Ectromelia Virus include: JQ410350.1 Ectromelia virus ERPV culture-collection ATCC: VR-1431 complete genome: 1455-1676, 2980-3222, 6405-6624, 14857-15076, 19606-19831, 36649-36868, 41002-41221, 56047-56266, 124587-124806, 140897-141000, 144533-144650, 171703-171930, 177795-178026, 185851-186070, 194236-194476, 199896-200115, 204902-205122.

[0108] (2) The NCBI reference sequences and genomic regions corresponding to the sequences of Hantaan Virus RNA standards include:

[0109] MK548699.1Orthohantavirus hantanense strain Aa17-53-R segment L complete genome: 20-239, 1701-1920, 2051-2270, 2801-3020, 4321-4540, 4651-4870, 6351-6530.

[0110] MK548690.1Orthohantavirus hantanense strain Aa17-53-R segment M complete genome: 1-220, 801-1020, 1301-1520, 2101-2370, 2701-2920, 3151-3370, 3401-3581.

[0111] MK548681.1Orthohantavirus hantanense strain Aa17-53-R segment S complete genome: 1-220, 251-670, 1511-1696.

[0112] (3) The NCBI reference sequence and genomic regions corresponding to the sequence of the RNA standard of K virus include: NC_074971.1 Betapolyomavirus secumuris isolate #6018 complete genome: 1-240, 251-1720, 1751-2970, 3001-4994.

[0113] (4) The NCBI reference sequences and genomic regions corresponding to the sequences of the RNA standards of Lactic Dehydrogenase Virus include: NC_001639.1 Lactate dehydrogenase-elevating virus complete genome: 140-359, 3101-3529, 3751-4023, 5701-5920, 8498-8717, 10448-10667, 10798-11017, 11051-11270, 11658-11813, 12004-12223, 12304-12523, 12601-12820, 12848-13067, 13101-13320, 13401-13620, 13651-14067.

[0114] (5) The NCBI reference sequences and genomic regions corresponding to the sequences of the RNA standards of Lymphocytic Choriomeningitis Virus include:

[0115] NC_077806.1Lymphocytic choriomeningitis virus Armstrong 53b segmentRNAL complete genome: 2359-2578, 2850-3128, 3159-3378, 3509-3928, 3959-4178, 4959-5178, 6706-6968, 7051-7228.

[0116] NC_077807.1Lymphocytic choriomeningitis virus Armstrong 53b segmentRNA S complete genome: 1501-1715, 2669-2888, 2950-3319.

[0117] (6) The NCBI reference sequence and genomic regions corresponding to the sequence of the Minute Virus of Mice RNA standard include: NC_001510.1 Minute virus of mice complete genome: 52-570, 601-820, 851-1070, 1101-1320, 1351-1570, 1601-1820, 1851-2070, 2101-2320, 2351-2570, 2573-3009, 3040-3459, 3490-3923, 3954-4173, 4240-4820.

[0118] (7) The NCBI reference sequences and genomic regions corresponding to the sequences of the RNA standards of Mouse Adenovirus include: NC_014899.1 Murine adenovirus 2 complete genome: 1-220, 251-470, 501-720, 751-970, 1001-1220, 1251-1470, 1501-1720, 1751-1970, 2001-2220, 2251-2470, 2501-2720, 2751-2970, 3001-3220, 3251-3470, 3501-3720, 3751-3970, 4001-4220, 4251-4520.

[0119] (8) The NCBI reference sequences and genomic regions corresponding to the sequences of the RNA standards of Mouse Cytomegalovirus include: OK149191.1 Murid betaherpesvirus 1strain HaNa1 complete genome: 10301-10518, 42736-42955, 43510-43729, 43933-44152, 87054-87273, 87553-87772, 87876-88095, 88163-88382, 88589-88808, 89003-89222, 94651-94870, 114289-114508, 123452-123671, 124530-124749, 132885-133104, 152718-152937, 184436-184655.

[0120] (9) The NCBI reference sequence and genomic regions corresponding to the sequence of the RNA standard of Mouse Encephalomyelitis Virus include: M20562.1 Theiler murine encephalomyelitis complete genome: 151-369, 400-619, 651-1271, 1407-1626, 3796-4015, 4153-4372, 4411-4630, 4810-5029, 5151-5308, 5653-5872, 5960-6179, 6261-6480, 6704-7070, 7256-7475, 7504-7723, 7752-7971.

[0121] (10) The NCBI reference sequences and genomic regions corresponding to the RNA standards of Mouse Hepatitis Virus include: NC_001846.1 Murine hepatitis virus strain MHV-A59 C12mutant complete genome: 1-370, 401-620, 1751-1970, 2001-2220, 2401-2620, 2751-2970, 2992-3214, 3501-3720, 5001-5220, 5501-5720, 6801-7020, 7551-7770, 8601-8820, 9001-9220, 9551-9770, 9901-10120, 10151-10370, 22571-22689, 23879-23979, 27810-27924, 28046-28151, 31109-31334.

[0122] (11) The NCBI reference sequences and genomic regions corresponding to the sequences of the RNA standards of Mouse Rotavirus include:

[0123] MT276820.1Murine rotavirus A strain EDIM nonstructural protein 1(NSP1)gene complete coding sequence: 50-269.

[0124] MT276821.1 Murine rotavirus Astrain EDIM nonstructural protein 2 (NSP2) gene complete coding sequence: 42-261, 292-511.

[0125] MT276822.1Murine rotavirus A strain EDIM nonstructural protein 3 (NSP3) gene complete coding sequence: 98-517.

[0126] MT276823.1Murine rotavirus A strain EDIM nonstructural protein 4(NSP4)gene complete coding sequence: 1-181, 362-581.

[0127] MT276824.1Murine rotavirus A strain EDIM nonstructural protein 5 (NSP5) gene complete coding sequence: 43-262.

[0128] MT276817.1Murine rotavirus A strain EDIM VP1(VP1)gene complete coding sequence: 193-612.

[0129] MT276818.1Murine rotavirus A strain EDIM VP2(VP2)gene complete coding sequence: 1-214, 1666-1814.

[0130] MT276819.1Murine rotavirus A strain EDIM VP3 (VP3) gene complete coding sequence: 43-462.

[0131] MT276815.1Murine rotavirus A strain EDIM VP4(VP4)gene complete coding sequence: 1-208, 689-908, 1801-2020.

[0132] MT276816.1Murine rotavirus A strain EDIM VP6(VP6)gene complete coding sequence: 1-220, 351-570.

[0133] MT276814.1Murine rotavirus A strain EDIM VP7(VP7)gene complete coding sequence: 151-370, 751-970.

[0134] (12) The NCBI reference sequence and genomic regions corresponding to the sequence of the RNA standard of MS2 bacteriophage (MS2) include: MK213795.1 Escherichia phage MS2 complete genome: 1-3526.

[0135] (13) The NCBI reference sequences and genomic regions corresponding to the sequences of the RNA standards of Pneumonia Virus of Mice include: AY743910.1 Pneumonia virus of mice 15 complete genome: 1-320, 751-970, 1001-1220, 1401-1620, 1651-1870, 2151-2370, 2651-2870, 2901-3120, 3151-3370, 3401-3620, 3651-3870, 4952-5171, 5202-5421, 5852-6071, 6102-6321, 6352-6571, 6602-6821, 14227-14446.

[0136] (14) The NCBI reference sequences and genomic regions corresponding to the sequences of the RNA standards of polyoma virus include: NC_001515.2 Murine polyomavirus strain BG complete genome: 1-220, 251-470, 501-720, 751-970, 1001-1220, 1251-1520, 1654-1873, 2007-2226, 2257-2476, 2507-2726, 2858-3077, 3158-3377, 3408-3627, 3811-4030, 4061-4280, 4311-4530, 4561-4780, 5011-5230.

[0137] (15) The NCBI reference sequences and genomic regions corresponding to the sequences of the RNA standards of Reovirus Type 3 include:

[0138] NC_077839.1Mammalian orthoreovirus 3Dearing strain T3D segment L1 complete genome: 201-420, 3575-3794.

[0139] NC_077840.1Mammalian orthoreovirus 3Dearing strain T3D segment L2 complete genome: 1-320, 1651-1870.

[0140] NC_077842.1Mammalian orthoreovirus 3Dearing strain T3D segment L3 complete genome: 814-1033.

[0141] NC_077841.1Mammalian orthoreovirus 3Dearing strain T3D segment M1 complete genome: 951-1170, 1614-1833.

[0142] NC_077843.1Mammalian orthoreovirus 3Dearing strain T3D segment M2 complete genome: 1-170, 1555-1774, 2005-2207.

[0143] NC_077845.1Mammalian orthoreovirus 3Dearing strain T3D segment M3 complete genome: 1-170, 419-638, 1651-1870.

[0144] NC_077844.1Mammalian orthoreovirus 3Dearing strain T3D segment S1 complete genome: 201-420, 1211-1416.

[0145] NC_077846.1Mammalian orthoreovirus 3Dearing strain T3D segment S2 complete genome: 1-187, 401-620, 1101-1320.

[0146] NC_077837.1Mammalian orthoreovirus 3Dearing strain T3D segment S3 complete genome: 51-270, 301-520.

[0147] NC_077838.1Mammalian orthoreovirus 3Dearing strain T3D segment S4 complete genome: 1-320, 1001-1196.

[0148] (16) The NCBI reference sequences and genomic regions corresponding to the RNA standard of Sendai Virus include: AB065189.1 SENDAI VIRUS GENOMIC RNA, COMPLETE GENOME, CLONE:E30M15CL5 virus complementary sequence: 1-370, 1801-2020, 2201-2420, 2751-2970, 3901-4120, 5251-5470, 5651-5870, 9801-10020, 10551-10770, 11151-11370, 11801-12020, 12351-12570, 12851-13070, 13251-13470, 13701-13920, 13951-14170, 14201-14420, 14501-14720.

[0149] (17) The NCBI reference sequences and genomic regions corresponding to the sequences of the SV5 RNA standards include: MK423242.1 Parainfluenza virus 5 isolate N99 complete genome: 1-170, 851-1070, 2351-2570, 2601-2820, 3701-3920, 4551-4770, 4851-5070, 5151-5370, 7251-7470, 8351-8570, 8901-9120, 9151-9370, 9401-9620, 10101-10320, 10751-10970, 11851-12070, 12801-13020, 13851-14070, 14151-14370.

[0150] (18) The NCBI reference sequence and genomic regions corresponding to the sequence of the T4 bacteriophage (T4) RNA standard include: NC_000866.4Enterobacteria phage T4 complete genome: 85-4084.

[0151] (19) The NCBI reference sequences and genomic regions corresponding to the sequences of the thymic virus RNA standards include: KY355735.1 Murid betaherpesvirus 3 isolate YOK1 sequences: 6351-6570, 13646-13865, 14996-15215, 138245-138447, 145311-145570, 145911-146130, 148285-148520, 148885-149120, 150297-150516, 165452-165871, 165902-166121, 166152-166371, 167386-167605, 167636-167855, 167886-168255.

[0152] 2. According to Table 1 below, 1×10 3 The copy number of each MAP / HAP-related species-specific virus and MS2 phage RNA standard, and 1×10 10 The copy number of T4 phage RNA standard was spiked to 1×10 7 in CHO cells.

[0153] Table 1

[0154]

[0155] 3. For 1×10 spiked virus standards 7 The total RNA of the samples was isolated and extracted using Qiagen RNeasy Plus Mini Kit (Cat. No. 74134) according to the instructions of the kit.

[0156] 4. Use Thermo Fisher Scientific RiboMinus rRNA removal Kit (Cat. No.: K1550-02 and K1550-05) to remove rRNA from 10 μg RNA / sample, obtain total RNA after rRNA removal, perform corresponding operations according to the instructions of the kit, and finally use 22 μL of water to elute and recover RNA from the concentration column. After recovery, use Qubit fluorometer to quantify RNA, and the total amount of recovered RNA is about 1 μg RNA / sample.

[0157] 5. Use Illumina Stranded mRNA Prep kit (Cat. No.: 20040534) to construct RNA library, skip the polyA capture step, and start with RNA fragmentation and cDNA synthesis. Each sample is divided into two tubes for library construction. Add 8.5 μL of total RNA after rRNA removal in the previous step and 8.5 μL of EPH3 reagent to each tube, and start the RNA fragmentation procedure according to the kit instructions. Use Shanghai Biotech EzyNGS UDI adapter primer (Cat. No.: N608310-0048) to add index to the library, use standard PCR library amplification conditions and 13 PCR cycles to amplify the library, and finally obtain two libraries with the same index for each sample. Use Qubit fluorometer to quantify DNA library, and the total amount of DNA in each library is about 2-3 μg.

[0158] 6. Mix the two libraries with the same index for each sample in the above step and purify them using AMPure XPBead-Based Reagent (Cat. No.: 20327600), and prepare the following hybridization system (Table 2).

[0159] Table 2

[0160] Components Addition volume (μL) 2× Hybridization Buffer 9.5 Hybridization enhancer 3 Probe (0.5nM / probe) 2 Salmon sperm DNA (10 μg / μL) 0.5 EzyNGS Universal Blocking Primer 2 water 2 Total volume 19

[0161] The probes include probes for hybridization capture of 17 MAP / HAP-related species-specific viral RNA standards (SEQ ID NOs: 1-1127), probes for hybridization capture of MS2 bacteriophage RNA standards (SEQ ID NOs: 1128-1155), and probes for hybridization capture of T4 bacteriophage RNA standards (SEQ ID NOs: 1156-1187). The concentration of each probe in the system is 0.5 nM.

[0162] The 2× hybridization buffer and hybridization enhancer were from Shanghai Bio-Tech EzyNGS hybridization capture and elution buffer kit (Cat. No.: N608370-0016), EzyNGS universal blocking primer (Cat. No.: N608360-0016) was purchased from Shanghai Bio-Tech, and salmon sperm DNA (Cat. No.: D7656-1ML) was purchased from Merck. DNA library purification was performed according to the AMPure XP Bead-Based Reagent instructions, and the above hybridization system was used instead of water for elution in the elution step, and all supernatants were transferred to new corresponding PCR tubes to start the following hybridization procedure.

[0163] Hybridization program (heated lid 105°C, reaction volume 19 μL); 95°C 30 seconds; 65°C 16 hours; 65°C hold.

[0164] 7. After hybridization, perform streptavidin magnetic bead capture and washing according to the instructions of Shanghai Biotech EzyNGS Hybridization Capture and Elution Buffer Kit, and use KAPA HiFi HotStart Ready Mix (Cat. No.: KK2601) and Illumina P5 / P7 universal primers for post-hybridization PCR amplification. The system is prepared as shown in the following (Table 3).

[0165] Table 3

[0166]

[0167]

[0168] Post-hybridization PCR program (hot cover 105°C, reaction volume 50 μL); 98°C 45 seconds; 98°C 15 seconds-cycle start; 60°C 30 seconds; 72°C 30 seconds-cycle end, a total of 17 cycles; 72°C 5 minutes; 4°C hold.

[0169] 8. After PCR, the DNA library was purified according to the instructions of AMPure XP Bead-Based Reagent, the DNA library was quantified using Qubit fluorometer, the average fragment length of the library was determined using Agilent 2100 Bioanalyzer, and the molar concentration of the corresponding library was calculated. After the library was diluted to 1pM, the high-throughput sequencing experiment was completed using the Illumina NextSeq550 system, and the data volume allocated for each sample was 10G.

[0170] 9. After sequencing, use the bcl2fastq program to split the data according to the labels (index) corresponding to the samples and start the bioinformatics analysis process.

[0171] (1) The data were first processed by fastp to remove reads with too low sequencing scores (sequence data of small fragments generated by high-throughput sequencing platforms) and remove adapter sequences.

[0172] (2) The trimmed reads were aligned with the T4 phage standard sequence using the BWA-MEM program with a parameter of K-mer=31, and all reads aligned with the T4 phage sequence were removed.

[0173] (3) The BWA-MEM program was used to align the reads that were not aligned to the T4 phage standard sequence in the previous step with the host gene library, with the parameter K-mer=31, and all reads that were aligned to the host gene library were removed.

[0174] (4) The BWA-MEM program was used to align the unaligned reads in the previous step with the MAP / HAP virus database, which contains the known complete sequences of all 17 MAP / HAP-related species-specific viruses included in the NCBI database and the RVDB database. All matching reads and the viral genomes that these reads could align to were screened out using the read length ≥75 bp and the alignment rate ≥90% as the criteria.

[0175] (5) Use the snakemake workflow to align the reads selected in the previous step to each viral genome selected in the previous step. Use Samtools to calculate the number of bases covered in the alignment with a read length of ≥75 bp and a matching rate of ≥90% as the standard, and output the result with the highest number of bases covered by the same virus for positive judgment. If the number of bases covered is ≥200 bp, the virus is considered to be positive, and the virus species name is output to generate a sequencing report.

[0176] 10. In order to verify the effect of increasing the initial sample amount and performing hybridization capture enrichment on improving the detection sensitivity, this experimental example also conducted a standard library construction sequencing control experiment without hybridization. 5 or 1×10 6 The copy number of each MAP / HAP-related species-specific virus and MS2 phage RNA standard was spiked to 1×10 7 in CHO cells.

[0177] Table 4

[0178]

[0179] From 1×10 spiked virus standards 7 Total RNA of the sample was isolated and extracted from CHO cells using Qiagen RNeasy Plus Mini Kit (Cat. No.: 74134) according to the instructions of the kit. 1 μg of total RNA was removed from rRNA and RNA library was constructed using Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus (Cat. No.: 20040525) according to the instructions. The average fragment length of the library was determined using the Agilent 2100 Bioanalyzer, and the molar concentration of the corresponding library was calculated. After the library was diluted to 1 pM, the high-throughput sequencing experiment was completed using the Illumina NextSeq550 system, and the data volume allocated to each sample was 10G. The bioinformatics analysis process was the same as the hybridization experiment group, refer to step 9.

[0180] 11. The results are shown in Table 5.

[0181] Table 5

[0182]

[0183] As can be seen from the table above, all the virus standards spiked in the targeted sequencing experimental group were detected as positive, proving that the method can detect 17 species-specific viruses related to mice and hamsters at a concentration of at least 0.0001 copies / cell in the cell library to be tested. When the virus spiked in the non-hybridization control group was 0.1 copies / cell and 0.01 copies / cell, all the spiked virus standards could not be detected, proving the advantage of the present invention over non-targeted NGS in detection sensitivity when the sequencing depth is the same.

[0184] The probes designed in the method of the present invention are specifically designed for the purpose of detecting species-specific viruses related to mice and hamsters, which is different from the conventional human genome hybridization capture method; the method of the present invention needs to detect extremely low possible viral contamination under extremely high cell background. When the viral contamination concentration is very low, the detectable nucleic acid library concentration cannot be obtained after hybridization capture, and subsequent sequencing experiments cannot be performed; therefore, the present invention adds T4 phage as a process control in the detection method to ensure that the hybridization experiment can continue when the concentration of the virus to be tested is extremely low, while the conventional human genome capture experiment does not require such a design, because the captured region must exist in the sample and the abundance is determined by the sequencing depth. By the same principle, the present invention needs to reduce the host cell background as much as possible through a special rRNA removal step, and improve the detection sensitivity by increasing the initial nucleic acid amount and merging the two libraries, while human genome capture does not require such a design.

[0185] Example 2

[0186] This example uses targeted NGS technology to detect 17 mouse / hamster-related species-specific viruses in total RNA extracts from CHO cells.

[0187] 1. In this example, viral standard RNA is incorporated into total cell RNA and the sample is tested using the method of the present invention.

[0188] 2. The difference between the targeted NGS part of this embodiment and that of embodiment 1 is that 1×10 7 Total RNA of CHO cell samples was then divided into 1×10 2 The copy number of each MAP / HAP-related species-specific virus and MS2 phage RNA standard, and 1×10 8The copies of T4 phage were spiked into 10 μg of total RNA from CHO cells.

[0189] Table 6

[0190]

[0191] 3. Use Thermo Fisher Scientific RiboMinus rRNA removal Kit (Cat. No.: K1550-02 and K1550-05) to remove the rRNA in the 10 μg RNA / sample spiked with the virus standard to obtain the total RNA after rRNA removal. The subsequent steps are the same as in Example 1.

[0192] 4. Similar to Example 1, in order to verify the effect of increasing the initial sample load and performing hybridization capture enrichment on the detection sensitivity, this experimental example also conducted a standard library construction sequencing control experiment without hybridization. 4 or 1×10 3 The copy number of each MAP / HAP-related species-specific virus and MS2 phage RNA standard was spiked into 1 μg of CHO cell total RNA.

[0193] Table 7

[0194]

[0195] 1 μg of total RNA was subjected to rRNA removal and RNA library construction using Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus (Cat. No.: 20040525) according to the instructions, and the subsequent steps were the same as in Example 1.

[0196] 6. The results are shown in Table 8.

[0197] Table 8

[0198]

[0199]

[0200] As can be seen from the table above, all the virus standards spiked into the targeted sequencing experimental group were detected as positive, proving that this method can detect 17 species-specific viruses related to mice / hamsters with a concentration of at least 10 copies / μg RNA after total RNA was extracted from the cell library to be tested. 4 The internal reference virus bacteriophage MS2 was not detected at 10 copies / μg RNA, and the virus spiked in was 10 3When the number of copies / μg RNA was 1.54, all viruses were not detected, which proved the advantage of the present invention over non-targeted NGS in detection sensitivity at the same sequencing depth.

[0201] Example 3

[0202] This example detects live viruses that infect cells and viral nucleic acids that are incorporated into cellular nucleic acids.

[0203] Since both Examples 1 and 2 used viral RNA standards designed based on viral sequences to simulate viral samples, this example extracted total RNA from cells infected with the virus, quantified the number of viral RNA copies using RT-qPCR, and spiked it into 1×10 7 The detection ability of the present invention for real virus-infected cells was evaluated by using 10 μg of CHO cells or 10 μg of CHO cell total RNA.

[0204] 1. After the five hamster or mouse specific viruses, pneumonia virus of mice, minute virus of mice, Sendai virus, reovirus type 3, and SV5, were amplified by cell culture, total RNA was extracted using Qiagen RNeasy Plus Mini Kit (Cat. No.: 74134), and the viral RNA copy number was quantified by RT-qPCR.

[0205] 2. According to Table 9 below, 1×10 7 The copy number of Sendai virus and reovirus type 3 RNA was spiked to 1×10 7 in CHO cells.

[0206] Table 9

[0207] Sample name Viral RNA (copy number) Host cells Sendai virus pre-injection <![CDATA[1×10 7 ]]> <![CDATA[1×10 7 CHO cells]]> Reovirus type 3 pre-injection <![CDATA[1×10 7 ]]> <![CDATA[1×10 7 CHO cells]]>

[0208] 3. From 1×10 7 The total RNA of the sample was isolated and extracted from CHO cells using Qiagen RNeasy Plus Mini Kit (Cat. No.: 74134) according to the kit instructions, and the subsequent experimental procedures were referred to Example 1.

[0209] 4. According to the following table 10, 1×10 2 The number of copies of five hamster or mouse specific viral RNAs, namely, pneumonia virus of mice, minute virus of mice, Sendai virus, reovirus type 3, and SV5, were spiked into 10 μg of total RNA of CHO cells.

[0210] Table 10

[0211]

[0212]

[0213] 5. Use Thermo Fisher Scientific RiboMinus rRNA removal Kit (Cat. No.: K1550-02 and K1550-05) to remove the rRNA in the 10 μg RNA / sample spiked with viral RNA to obtain total RNA after rRNA removal. The subsequent steps are the same as in Example 1.

[0214] The probes used include a probe group for detecting mouse pneumonia virus (SEQ ID NO: 602-620), a probe group for detecting mouse minute virus 263-287), a probe group for detecting Sendai virus (SEQ ID NO: 1052-1070), a probe group for detecting reovirus type 3 (SEQ ID NO: 659-1051), and a probe group for detecting SV5 (SEQ ID NO: 1071-1089).

[0215] 6. The results are shown in Table 11.

[0216] Table 11

[0217] Viral RNA Read segment Coverage base number (bp) Sendai virus pre-injection 268820 15360 Reovirus type 3 pre-injection 390635 23452 Mouse pneumonia virus 12 353 Microvirus of mice 185 2624 Sendai virus post-introduction 56 604 Reovirus type 3 post-introduction 186 1084 SV5 post-doping 50 523

[0218] It can be seen from the above table that, for RNA of live virus-infected cell samples simulating real conditions, the present invention can detect it normally and reach the corresponding detection limit.

[0219] In summary, the present invention provides a probe combination for detecting species-specific viruses related to mice and hamsters in samples and its application in high-throughput sequencing. The high-throughput sequencing method in the present invention can distinguish viral nucleic acids with infectious risks and has the advantage of quickly checking whether the sample is contaminated by specific live viruses. The detection limit of the present invention reaches the level of the traditional PCR method, and maintains the ability to detect all known variants of the species-specific virus to be tested, and can detect 17 viruses with only one detection item, with the advantages of high efficiency and high sensitivity.

[0220] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A probe library for high-throughput detection of species-specific viruses associated with mice and hamsters in CHO cell libraries, characterized in that: The probe library is used to detect mousepox virus, Hanta virus, K virus, lactate dehydrogenase virus, lymphocytic choriomeningitis virus, mouse microvirus, mouse adenovirus, mouse cytomegalovirus, mouse encephalomyelitis virus, mouse hepatitis virus, mouse rotavirus, mouse pneumonia virus, polyoma virus, reovirus type 3, Sendai virus, SV5 and thymocyte virus and its variant virus; the probe library includes the following probe groups: Probe set 1: used to detect mousepox virus and its variants in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 1-42; Probe Group 2: used to detect Hantavirus and its variants in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 43-106; Probe Group 3: used to detect K virus and its variant viruses in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 107-145; Probe group 4: used to detect lactate dehydrogenase virus and its variant viruses in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 146-166; Probe set 5: for detecting lymphocytic choriomeningitis virus and its variants in a sample, comprising any one or a combination of at least two of the probes having nucleotide sequences as shown in SEQ ID NOs: 167-262; Probe Group 6: for detecting minute virus of mice and its variant viruses in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 263-287; Probe Group 7: for detecting mouse adenovirus and its variant viruses in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 288-332; Probe Group 8: for detecting mouse cytomegalovirus and its variants in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 333-387; Probe set 9: for detecting mouse encephalomyelitis virus and its variant viruses in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 388-410; Probe set 10: for detecting mouse hepatitis virus and its variant viruses in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 411-431; Probe group 11: used for detecting mouse rotavirus and its variant viruses in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 432-601; Probe group 12: for detecting mouse pneumonia virus and its variant viruses in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 602-620; Probe group 13: for detecting multiple tumor viruses and their variants in a sample, comprising any one or a combination of at least two of the probes having nucleotide sequences as shown in SEQ ID NOs: 621-658; Probe set 14: for detecting reovirus type 3 and its variants in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 659-1051; Probe set 15: for detecting Sendai virus and its variants in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 1052-1070; Probe set 16: for detecting SV5 and its variant viruses in a sample, comprising any one or a combination of at least two of the probes with nucleotide sequences as shown in SEQ ID NOs: 1071-1089; Probe group 17: used to detect thymic virus and its variant viruses in a sample, comprising any one or a combination of at least two of the probes having nucleotide sequences as shown in SEQ ID NOs: 1090-1127.

2. The probe library according to claim 1, characterized in that The 5' end of the probe is modified; Preferably, the modification comprises a biotin label.

3. A kit, characterized in that: The kit comprises the probe library for high-throughput detection of mouse and hamster-related species-specific viruses in a CHO cell library as described in claim 1 or 2; Preferably, the kit further comprises an internal control viral RNA standard; Preferably, the internal control viral RNA standard comprises RNA of a probe targeting a genomic sequence of MS2 phage and / or T4 phage.

4. A method for enriching nucleic acid fragments of species-specific viruses associated with mice and hamsters in a sample, characterized in that: The method comprises the following steps: (1) Obtaining total RNA from a sample; (2) fragmenting the total RNA and synthesizing cDNA; (3) Using the probe library for high-throughput detection of species-specific viruses associated with mice and hamsters in a CHO cell library according to claim 1 or 2, or the kit according to claim 3 for hybridization capture.

5. The method according to claim 4, characterized in that The sample is also spiked with an internal control viral RNA standard; Preferably, the internal control viral RNA standard comprises RNA of a probe targeting a genomic sequence of MS2 phage and / or T4 phage; Preferably, the sample is a cell bank, and the concentration of mouse and hamster-related species-specific viruses in the cell bank is at least 0.0001 copies / cell; Preferably, the sample is total RNA extracted from a cell bank, wherein the concentration of mouse and hamster-related species-specific viruses in the total RNA is at least 10 copies / μg RNA; Preferably, step (1) further comprises rRNA removal.

6. A method for detecting species-specific viruses associated with mice and hamsters in a sample, characterized in that: The method comprises the following steps: (A) Obtaining total RNA from samples; (B) constructing a DNA library; (C) using the probe library for high-throughput detection of species-specific viruses associated with mice and hamsters in a CHO cell library according to claim 1 or 2, or the kit according to claim 3 for hybridization capture; (D) performing PCR amplification on the product of step (C); (E) performing high-throughput sequencing on the product of step (D); (F) Using bioinformatics methods to determine whether the product of step (E) is positive for virus detection.

7. The method for detecting species-specific viruses associated with mice and hamsters in a sample according to claim 6, characterized in that: The sample is also spiked with an internal control viral RNA standard; Preferably, the internal control viral RNA standard comprises RNA of a probe targeting a genomic sequence of MS2 phage and / or T4 phage; Preferably, the sample is a cell bank, and the concentration of mouse and hamster-related species-specific viruses in the cell bank is at least 0.0001 copies / cell; Preferably, the sample is total RNA extracted from a cell bank, wherein the concentration of mouse and hamster-related species-specific viruses in the total RNA is at least 10 copies / μg RNA; Preferably, step (A) further comprises rRNA removal.

8. The method for detecting species-specific viruses associated with mice and hamsters in a sample according to claim 6 or 7, characterized in that: The bioinformatics method comprises: (i) Data were split using the bcl2fastq program; (ii) the reads were aligned to the MAP / HAP virus database using the BWA-MEM program; (iii) The number of covered bases was calculated using Samtools.

9. The method for detecting species-specific viruses associated with mice and hamsters in a sample according to claim 8, characterized in that: Step (i) comprises: using the bcl2fastq program to split the data according to the labels corresponding to the samples; Preferably, step (ii) comprises: (a) processing the data obtained in step (i) using fastp software; (b) The reads were aligned to the MAP / HAP virus database using the BWA-MEM program; (c) screening reads and their aligned viral genomes using read length ≥75 bp and alignment rate ≥90% as criteria; (d) Use the snakemake workflow to align the filtered reads with each viral genome; Preferably, the number of covered bases is calculated based on the standard that the read length is ≥75bp and the alignment rate is ≥90%. When the number of covered bases is ≥200bp, the corresponding virus is judged to be detected positive.

10. Use of the probe library for high-throughput detection of species-specific viruses associated with mice and hamsters in a CHO cell library according to claim 1 or 2 or the kit according to claim 3 in high-throughput sequencing.

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