Method for detecting unknown pathogens and application thereof

Abstract

The application discloses a detection method for unknown pathogens and application thereof, and the method does not need to separate the unknown pathogens, only needs to perform metagenome sequencing on diseased animal lesion tissues, and then the primer and the probe for the unknown pathogens can be obtained through the method, efficient and convenient detection of the unknown pathogens is realized, and further, according to the detection result, the disease occurrence of a breeding farm and epidemiological investigation and prevention and control can be mastered, the spread of the unknown pathogens can be prevented and controlled in a short time, and the harm of the pathogens to the breeding farm is reduced.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method and application for detecting unknown pathogens. Background Technology

[0002] Poultry farming is a crucial pillar of my country's livestock industry, playing an irreplaceable role in ensuring the supply of livestock products, promoting agricultural economic development, and increasing farmers' income. With the rapid promotion of intensive and large-scale farming models, the scale of poultry farming has continued to expand. However, this has also led to the frequent outbreaks and prevalence of poultry diseases, becoming a core bottleneck restricting the high-quality development of the poultry farming industry. This not only causes huge economic losses but also poses a potential threat to public health and safety.

[0003] Currently, the prevalence of diseases in poultry farming is showing significant complexity and diversification, mainly falling into four categories: viral diseases, bacterial diseases, parasitic diseases, and nutritional and metabolic diseases. Among these, viral and bacterial diseases account for the highest proportion and cause the most serious damage. Of particular concern is the hidden spread and sudden outbreaks of unknown pathogens, which have become a major new safety hazard in the livestock farming sector, with their harm even exceeding that of known diseases, causing unpredictable losses to the poultry farming industry.

[0004] The harm caused by unknown pathogens to livestock farming is characterized by its insidious nature, sudden onset, high pathogenicity or depletion potential, and difficulties in prevention and control. Specifically: First, insidious transmission is difficult to detect. The pathogenicity of unknown pathogens is unknown, and many outbreaks initially present as asymptomatic infections, with poultry showing no obvious clinical symptoms. However, they can spread silently through contact, air, and feed. By the time obvious symptoms such as mass disease and death appear, the disease has already spread widely throughout the livestock population, missing the optimal control opportunity. Second, sudden outbreaks result in severe losses. The transmission routes of unknown pathogens are often unclear, making it impossible to grasp their epidemic patterns. Once they breach the host's immune barrier, they can easily trigger large-scale outbreaks, leading to mass mortality in poultry, a sharp drop in egg production, or even complete loss of egg production. Furthermore, the recovery period is extremely long, and some... Farms even face the risk of bankruptcy; third, the pathogenic mechanism is unclear. The gene sequence, pathogenic targets, and transmission routes of unknown viruses or pathogens are not clear, making it impossible to quickly analyze their damage mechanism to the reproductive and immune systems of poultry, and making it difficult to formulate targeted prevention and control measures; fourth, there are no effective means of prevention and control. Due to the lack of knowledge about unknown pathogens, there are currently no corresponding detection methods, vaccines, or specific drugs. Traditional prevention and control models are completely ineffective, and only extreme measures such as blockade and culling can be taken, further increasing breeding costs and economic losses. At the same time, unknown pathogens may also pose a risk of cross-species transmission, which poses a potential threat to public health and safety. For example, some unknown avian viruses may break through the species barrier and infect mammals or even humans, causing public health risks.

[0005] For the reasons mentioned above, there is an urgent need to develop an efficient detection method for unknown pathogens to meet the needs of prevention and control of unknown pathogens, which has become a technical problem that the poultry farming industry urgently needs to solve. Summary of the Invention

[0006] The purpose of this invention is to provide a detection method for unknown pathogens, so as to establish an efficient detection method for unknown pathogens to meet the needs of prevention and control of unknown pathogens, and to reduce the harm of unknown pathogens to livestock farming.

[0007] According to a first aspect of the present invention, a method for detecting an unknown pathogen is provided, the method not intended for disease diagnosis or treatment, the method comprising the following steps: S1: Collect diseased tissues from multiple diseased animals for metagenomic sequencing; S2: Compare the metagenomic sequencing data of multiple animals obtained in step S1 with the reference genome data of the animal, remove the genome data belonging to the animal, and use splicing software to splice the remaining sequencing data to obtain contigs; S3: Construct a local non-redundant nucleic acid database to annotate contigs, remove contigs that can be annotated and matched, and retain contigs that have no annotation results after annotation for subsequent steps. S4: Contigs without annotation results in non-redundant nucleic acid databases are predicted using DeepVirFinder to obtain potential unknown pathogen gene sequences; S5: Filter the predicted sequences in step S4, collect the prediction results of each sample with a length greater than 500bp and a pvalue less than 0.05, and output contigs; S6: Perform homology analysis on the contigs output in step S5. Based on the homology analysis results, select similar sequence fragments in each sample, and design and synthesize detection primers and probes using the similar sequence fragments. S7: Use the detection primers and probes synthesized in step S6 to perform PCR detection on the unknown pathogen.

[0008] Therefore, this method eliminates the need to isolate the unknown pathogen. Instead, it only requires metagenomic sequencing of the lesion tissue from diseased animals. This method can then obtain primers and probes targeting the unknown pathogen, enabling rapid, efficient, and convenient detection. Furthermore, based on the detection results, the disease situation in the farm can be understood, and epidemiological investigations and control measures can be implemented. The spread of the unknown pathogen can be controlled in a short period of time, reducing the harm it poses to the farm.

[0009] In some embodiments, the method further includes detecting the sample using the detection primers and probes designed and synthesized in step S6, and screening for detection primers and probes with high detection rates.

[0010] In some implementations, step S5 collects prediction results for each sample that are longer than 500 bp and have a pvalue less than 0.05. For the prediction results, the longer sequences are selected first, and then, under the same conditions, the contigs with higher scores are selected for output.

[0011] According to a second aspect of the present invention, an application is provided for the detection of the aforementioned unknown pathogen in the non-therapeutic purpose of detecting an unknown virus causing duck reproductive disorder syndrome. This allows for rapid, efficient, and convenient detection of the unknown virus causing duck reproductive disorder syndrome, and further enables its control, minimizing its harm to livestock farms.

[0012] According to a third aspect of the present invention, a method for screening primers and probes for detecting unknown pathogens is provided, the method comprising the following steps: S1: Collect diseased tissues from multiple diseased animals for metagenomic sequencing; S2: Compare the metagenomic sequencing data of multiple animals obtained in step S1 with the reference genome data of the animal, remove the genome data belonging to the animal, and use splicing software to splice the remaining sequencing data to obtain contigs; S3: Construct a local non-redundant nucleic acid database to annotate contigs, remove contigs that can be annotated and matched, and proceed with subsequent steps for contigs that have no annotation results after annotation. S4: Contigs without annotation results in non-redundant nucleic acid databases are used to predict potential unknown viral gene sequences using DeepVirFinder; S5: Filter the predicted sequences in step S4, collect the prediction results of each sample with a length greater than 500bp and a pvalue less than 0.05, and output contigs; S6: Perform homology analysis on the contigs output in step S5. Based on the homology analysis results, select similar sequence fragments from each sample. Use these similar sequence fragments to design and synthesize detection primers and probes, and screen for primers and probes with high detection rates. Therefore, this method eliminates the need to isolate the pathogen. It only requires metagenomic sequencing of diseased animal tissues to obtain primers and probes targeting the unknown pathogen. These primers and probes can then be used for rapid, efficient, and convenient detection or epidemiological investigation of the unknown pathogen, facilitating further control and minimizing its harm to livestock farms.

[0013] According to a fourth aspect of the present invention, a screening method for detection primers and probes for the aforementioned unknown pathogens is provided, resulting in detection primers and probes that have been screened. Thus, rapid, efficient, and convenient detection of the corresponding pathogens can be achieved using these primers and probes, with simple and efficient operation.

[0014] According to a fifth aspect of the present invention, an application is provided of the above-mentioned method for screening primers and probes for the detection of unknown pathogens in the screening of primers and probes for the detection of unknown pathogens. Thus, through this application, rapid, efficient, and convenient detection of the corresponding pathogens can be achieved, with simple and efficient operation.

[0015] According to a sixth aspect of the present invention, a method for detecting an unknown virus causing duck reproductive disorder syndrome (DRS) without diagnostic or therapeutic purposes is provided. The method involves detecting the unknown virus causing DRS using the aforementioned unknown pathogen detection method, or screening primers and probes suitable for detecting the unknown virus using an unknown pathogen detection primer and probe screening method. Therefore, this method allows for rapid and efficient detection of the unknown virus causing DRS without the need for virus isolation, significantly improving detection efficiency and reducing detection costs.

[0016] According to a seventh aspect of the present invention, primers and probes for detecting an unknown virus causing duck reproductive disorder syndrome are provided, wherein the primer sequences are shown in SEQ ID NO:1 and SEQ ID NO:2 or in SEQ ID NO:4 and SEQ ID NO:5, and the probe sequences are shown in SEQ ID NO:3 or in SEQ ID NO:6. Thus, the primers and probes allow for more efficient, simple, and convenient detection of the unknown virus causing duck reproductive disorder syndrome. The process does not require isolation of the unknown virus; detection can be achieved simply by using the primers and probes and performing PCR detection on the lesions. The detection method is efficient, convenient, and low-cost. Furthermore, since the virus is a novel and unknown virus, the detection primers and probes not only provide a detection method for the virus but also enable immediate control of the virus and lay the foundation for subsequent research on the virus.

[0017] According to an eighth aspect of the present invention, a detection kit containing the aforementioned detection primers and probes is provided. Thus, this kit allows for more efficient, simple, and convenient detection of an unknown virus causing duck reproductive disorder syndrome, without the need to isolate the unknown virus during use; detection can be achieved simply by using the kit and performing PCR detection on the lesions. The detection method is efficient, convenient, and low-cost.

[0018] According to a ninth aspect of the present invention, there is provided an application of the aforementioned detection primers and probes or the aforementioned kit for non-diagnostic or therapeutic purposes in the detection of an unknown virus causing duck reproductive disorder syndrome. Thus, this application allows for more efficient, simple, and convenient detection of an unknown virus causing duck reproductive disorder syndrome, and the detection method is efficient, convenient, and low-cost.

[0019] The beneficial effects of this invention are: 1. This invention discloses a method and application for detecting unknown pathogens. This method does not require the isolation of the unknown pathogen. It only requires metagenomic sequencing of the lesion tissue of diseased animals. Then, primers and probes for the unknown pathogen can be obtained through this method, realizing rapid, efficient and convenient detection of the unknown pathogen. Furthermore, based on the detection results, the disease situation in the farm can be understood and epidemiological investigations and prevention and control can be carried out. The spread of the unknown pathogen can be controlled in a short period of time, reducing the harm of the pathogen to the farm.

[0020] 2. This invention also discloses a method and application for screening primers and probes for detecting unknown pathogens. This method eliminates the need to isolate the pathogen; it only requires metagenomic sequencing of diseased animal tissues. Primers and probes targeting the unknown pathogen can then be obtained using this method. These primers and probes can then be used to efficiently and conveniently detect the unknown pathogen or conduct epidemiological investigations, facilitating further control of the pathogen and minimizing its harm to livestock farms.

[0021] 3. This invention also discloses a non-diagnostic or therapeutic detection method for an unknown virus causing duck reproductive disorder syndrome. This method can quickly and efficiently detect the unknown virus causing the duck reproductive disorder syndrome without the need to isolate the virus, which greatly improves detection efficiency, reduces detection costs, and allows for timely control of the virus causing the disease, thereby reducing breeding losses.

[0022] 4. This invention also discloses a detection primer and probe for an unknown virus causing duck reproductive disorder syndrome, as well as a kit containing the detection primer and probe and its application. Using this primer and probe or kit, the unknown virus causing duck reproductive disorder syndrome can be detected more efficiently, simply, and conveniently. The unknown virus does not need to be isolated during the process; detection can be achieved simply by PCR detection of the lesions. The detection method is efficient, convenient, and low-cost. Attached Figure Description

[0023] Figure 1 The prediction results and scores are for samples with a length greater than 500bp and a p-value less than 0.05. Figure 2 To detect the standard curve of primers and probes; Figure 3 The image shows the results of the primer and probe specificity verification. Detailed Implementation

[0024] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various changes and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention.

[0025] Unless otherwise specified, all chemical substances, proteins, enzymes, or reagent kits used in this invention are commercially available.

[0026] Unless otherwise stated, the methods used in this invention, such as PCR amplification, employ conventional experimental procedures in the field. Related operations can be performed with reference to commonly used experimental technical manuals or the operating instructions for commercial reagent kits and instruments.

[0027] Example 1: Detection method for unknown pathogens.

[0028] When livestock farms show symptoms of infection with unknown pathogens, the following methods can be used for rapid detection: S1: Collect diseased tissues from multiple diseased animals for metagenomic sequencing; S2: Compare the metagenomic sequencing data of multiple animals obtained in step S1 with the reference genome data of the animal, remove the genome data belonging to the animal, and use splicing software to splice the remaining sequencing data to obtain contigs; S3: Construct a local non-redundant nucleic acid database to annotate contigs, remove contigs that can be annotated and matched, and retain contigs that have no annotation results after annotation for subsequent steps. S4: Contigs without annotation results in non-redundant nucleic acid databases are predicted using DeepVirFinder to obtain potential unknown pathogen gene sequences; S5: Filter the predicted sequences in step S4, collect the prediction results of each sample with a length greater than 500bp and a pvalue less than 0.05, prioritize the longer sequences, and then select the contigs with higher scores for output under the same conditions. S6: Perform homology analysis on the contigs output in step S5. Based on the homology analysis results, select similar sequence fragments in each sample, and design and synthesize detection primers and probes using the similar sequence fragments. S7: Use the detection primers and probes synthesized in step S6 to perform PCR detection on the unknown pathogen.

[0029] When the test results are positive in sick animals and negative in healthy animals, it indicates that the selected primers and probes are suitable for detecting this unknown pathogen. This set of primers and probes can be used on a large scale to screen for this unknown pathogen and implement control measures to prevent the spread of the pathogen and reduce losses.

[0030] Example 2: Detection of an unknown virus causing duck reproductive disorder syndrome.

[0031] Taking a Muscovy duck farm experiencing a decline in egg production and discovering endometrial hemorrhage upon necropsy of affected ducks as an example, lesion tissue was collected from the farm and tested using existing methods for detecting known pathogens. The results showed no detection, leading to the preliminary determination that the cause of the disease was an unknown pathogen. Because the main clinical symptoms of this disease are decreased egg production and endometrial hemorrhage, and because the tissue tropism is towards the reproductive system, the disease was named "Duck Reproductive Disorder Syndrome" (DRS). Duck Reproductive Disorder Syndrome, DRDS However, through the isolation of bacteria and mycoplasma from the diseased tissue, no bacteria or mycoplasma could be isolated. Therefore, it was inferred that the cause of the disease was a virus, and the unknown pathogen causing the disease was named "Duck Reproductive Disorder Syndrome Virus" (DRS). Duck Reproductive Disorder Syndrome Virus,DRDSV )".

[0032] 2.1 Design and preliminary screening of primer and probe sets.

[0033] PCR primer and probe design method: Lesion tissues were collected from ducks with egg drop syndrome. Metagenomic sequencing was performed on the oviduct and uterus. Bowtie2 was used to align to the Muscovy duck reference genome (GCA_018104995.1). Muscovy duck genome data was removed to obtain sequencing data that did not belong to Muscovy ducks. The data was then assembled using various Denovo assembly software such as Megahit and MetaSPAdes to obtain contigs. A local non-redundant nucleic acid database (nt) was then constructed to annotate the contigs. Contigs that could be annotated and matched were removed, and contigs without annotation results (including those without matching results) were retained for subsequent research. Contigs without annotation (or matching results) in the nt database were used to predict potential unknown viral gene sequences using DeepVirFinder. A machine learning model in DeepVirFinder was used to train a model to predict contigs that could not be annotated (or matched) in the database, thereby finding potential viral gene sequences. Then, prediction results with a length greater than 500bp and a pvalue less than 0.05 are collected from each sample, and the prediction results are sorted from highest to lowest score. The closer the score is to 1, the more reliable the result. Figure 1(The higher the score, the greater the likelihood that it is a genome sequence of an unknown pathogen), and outputs the contigs with higher scores. Homology analysis is performed on the output contigs. Based on the homology analysis results, similar sequence fragments are selected from each sample. Two pairs of specific amplification primers and corresponding probes are designed and synthesized using these similar sequence fragments. The specific sequences of the primers and probes are shown in Table 1: including upstream primers DRDSV-F1 and DRDSV-F2, downstream primers DRDSV-R1 and DRDSV-R1, and probes DRDSV-P1 and DRDSV-P2, whose nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:6, respectively. The specific sequences are listed in Table 1 below (the two pairs of specific amplification primers and corresponding probes are represented by primer set 1 and primer set 2, respectively). Table 1 Primer and probe sequences for quantitative real-time PCR

[0034] As shown in Table 1, primer set 1 includes upstream primer DRDSV-F1, downstream primer DRDSV-R1, and probe DRDSV-P1, while primer set 2 includes upstream primer DRDSV-F2, downstream primer DRDSV-R2, and probe DRDSV-P2. These primers and probes were synthesized by a commercial company and used for subsequent research.

[0035] 2.2 Primer screening, sensitivity and specificity verification.

[0036] (1) Further screening of primers and probes for real-time PCR.

[0037] The two primer pairs and probes initially screened in Table 1 were used in different duck farms to detect the pathogen exhibiting the above-mentioned "clinical symptoms of duck reproductive disorder syndrome virus". The detection results are shown in Table 2 below. The results show that both primer pairs and probe combinations can detect positive results, indicating that both primer pairs are suitable for detecting duck reproductive disorder syndrome virus. After comparing the two primer pairs, it was found that the sensitivity of primer group 1 was slightly higher than that of primer group 2. Primer group 1 was selected for further research.

[0038] Table 2. Screening results of primers and probes for real-time PCR.

[0039] (2) Establishment of standard curve and sensitivity analysis.

[0040] The DRDSV target gene fragment was amplified using primer set 1 (using the reverse transcription of nucleic acid from extracted lesion tissue as a cDNA template; the lesion tissue contained viral RNA). The amplified product was separated by agarose gel electrophoresis, purified using a gel extraction kit, and the purified target fragment was cloned into the pMD-19T plasmid vector (TaKaRa, 6013) to construct a standard recombinant plasmid. This recombinant plasmid served as a DRDSV positive control template.

[0041] The standard recombinant plasmid was serially diluted 10-fold, and quantitative real-time PCR amplification was performed using the diluted template. A standard curve was plotted using Ct values ​​against the logarithm of the template copy number. The standard curve equation and correlation coefficient were obtained through linear regression analysis, and the PCR amplification efficiency was calculated using the formula: E = 10^(-1 / slope)-1.

[0042] The results showed that the standard curve equation was Y = 50.02 - 4.63X, where Y represents the Ct value and X represents the logarithm of the template copy number. The lowest detection limit was 2.16 copies (e.g., ...). Figure 2 As shown in the figure, this indicates that the detection method has high sensitivity.

[0043] (3) Specificity verification.

[0044] Nucleic acids from several common duck-derived pathogens were selected as templates for specific detection, including Muscovy duck parvovirus, avian reovirus, duck chlamydia, duck mycoplasma, avian influenza virus, duck egg drop syndrome virus, and duck plague virus. After total nucleic acid extraction, the nucleic acids of each pathogen were amplified by real-time quantitative PCR using the primers and probes listed in Table 1, with negative controls included.

[0045] The reaction system consisted of: 10 μL reaction buffer, 0.8 μL premix, 0.4 μL upstream primer, 0.4 μL downstream primer, 0.2 μL probe, 2 μL template nucleic acid, and nuclease-free water to a final volume of 20 μL. The amplification conditions were: 50℃ for 20 min; 95℃ for 5 min; 95℃ for 15 s; 60℃ for 20 s, for a total of 40 cycles, with fluorescence signals collected at the 60℃ stage.

[0046] The results are as follows Figure 3 As shown, only the duck reproductive disorder syndrome virus template produced a specific amplification curve, while no amplification signal was observed in the other duck-derived pathogens and the negative control, indicating that the primer and probe set described in this invention has good specificity.

[0047] Meanwhile, the primers and probes in Table 1 can be used to prepare a real-time PCR kit, which includes: real-time PCR premix, reaction buffer, nuclease-free water, upstream primers DRDSV-F1 / DRDSV-F2, downstream primers DRDSV-R1 / DRDSV-R2 and probes DRDSV-P1 / DRDSV-P2 shown in Table 1. The premix contains one or more of the following: Hifair® V Reverse Transcriptase, UNICON® HotStart Taq DNA Polymerase, RNase inhibitor, and UDG enzyme. The reaction buffer contains dNTPs, dUTPs, and stabilizers.

[0048] 2.3 Clinical sample testing and application validation.

[0049] (1) Sample processing Anal swab samples were collected from ducks of different ages in a breeding farm suspected of having duck reproductive disorder syndrome. The anal swabs were added to 5 volumes of sterile PBS buffer, thoroughly vortexed, and centrifuged at 10,000 r / min for 10 min at 4°C. The supernatant was collected and used. After dissection of affected laying ducks, tissues and organs were thoroughly ground, frozen and thawed, and then centrifuged at 10,000 r / min for 10 min at 4°C. The supernatant was collected and used.

[0050] (2) Nucleic acid extraction Follow the instructions for the nucleic acid extraction kit to extract total nucleic acid from the sample.

[0051] (3) qPCR amplification reaction Perform real-time PCR amplification according to the reaction system and amplification conditions described in section 2.2.

[0052] (4) Result determination and analysis When the negative control has no Ct value and the positive control has a Ct value less than 30, the detection system is deemed effective. When the Ct value of the sample to be tested is less than 30, it is determined to be positive for duck reproductive disorder syndrome virus; If the Ct value of the sample to be tested is greater than or equal to 30, it is judged as a suspected positive and needs to be resampled and retested. If the sample to be tested has no Ct value, it is considered negative.

[0053] The collected clinical anal swab samples were tested, and the results are shown in Table 3. Analysis of the results showed that in laying ducks, the DRDSV positivity rate was 67% in samples from diseased farms, while all samples from disease-free farms were negative. In male ducks, the DRDSV positivity rate was 34% in samples from diseased farms, while all samples from disease-free farms were negative. Comparison of the test results with the corresponding egg production index of the laying duck flock revealed that a higher DRDSV positivity rate was associated with a relatively lower egg production index, consistent with the clinical symptom of decreased egg production associated with reproductive system infections. This indicates that the primers and probes used for DRDSV screening have good specificity, and the detection method is effective.

[0054] Table 3. Detection results of DRDSV in diseased and healthy duck flocks.

[0055] The internal organ tissue samples collected from diseased female ducks were tested, and the results are shown in Table 4. Analysis of the test results showed that the reproductive system of diseased female ducks had the highest viral load, consistent with the clinical symptom of decreased egg production associated with reproductive system infection. This further demonstrates that the primers and probes used for DRDSV screening have good specificity, and the detection method is effective.

[0056] Table 4. Viral load detection in the visceral tissues of diseased female ducks. Throat swab 2.62 ileum 3.71 anal swab 3.90 rectum 3.75 Fallopian tube swab 4.21 pancreas 2.60 brain 2.94 esophagus 2.01 Heart 3.94 Gizzard 3.10 liver 2.71 proventriculus 3.87 spleen 2.94 Theca interna 4.14 lung 3.46 serum 0.00 kidney 3.09 blood clots 1.41 Trachea (at the fork in the road) 4.03 Fallopian tube endometrium 6.39 throat 2.54 Fallopian tubes and uterus 6.38 Duodenum 2.76 Fallopian tube uterine secretions 6.77 cecal tonsils 3.27 Infundibulum of the fallopian tube 4.70 terminal cecum 3.23 dilatation of the fallopian tube 5.04 jejunum 3.03 Isthmus of the fallopian tube 4.87 The above analysis results indicate that the current disease is diagnosed as duck reproductive disorder syndrome virus infection. The quantitative real-time PCR detection method for duck reproductive disorder syndrome virus described in this invention has good practicality and applicability. Its detection results have a good correlation with changes in clinical production performance and can meet the detection needs of this virus in Muscovy duck farming production practice.

[0057] The method described in this invention is effective not only for detecting duck reproductive disorder virus (DPRK virus, a novel and unknown virus), but also for detecting any other unknown pathogens. This enables rapid, efficient, and convenient detection of unknown pathogens, playing a crucial role in further pathogen control. Furthermore, this method does not require isolation of the unknown pathogen; it only requires metagenomic sequencing of diseased tissue from infected animals. Primers and probes targeting the unknown pathogen can then be obtained using this method, enabling rapid, efficient, and convenient detection. Based on the detection results, the disease situation in farms can be assessed, and epidemiological investigations and control measures can be implemented. This allows for the rapid control of the spread of the unknown pathogen, reducing its harm to farms.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Various modifications or equivalent substitutions made to the present invention by those skilled in the art without departing from the essence of the technical solutions of the present invention should fall within the scope of protection of the present invention.

Claims

1. A method for detecting an unknown pathogen, wherein, The method is not intended for disease diagnosis or treatment, and includes the following steps: S1: Collect diseased tissues from multiple diseased animals for metagenomic sequencing; S2: Compare the metagenomic sequencing data of multiple animals obtained in step S1 with the reference genome data of the animal, remove the genome data belonging to the animal, and use splicing software to splice the remaining sequencing data to obtain contigs; S3: Construct a local non-redundant nucleic acid database to annotate contigs, remove contigs that can be annotated and matched, and retain contigs that have no annotation results after annotation for subsequent steps. S4: Contigs without annotation results in non-redundant nucleic acid databases are predicted using DeepVirFinder to obtain potential unknown pathogen gene sequences; S5: Filter the predicted sequences in step S4, collect the prediction results of each sample with a length greater than 500bp and a pvalue less than 0.05, and output contigs; S6: Perform homology analysis on the contigs output in step S5. Based on the homology analysis results, select similar sequence fragments in each sample, and design and synthesize detection primers and probes using the similar sequence fragments. S7: Use the detection primers and probes synthesized in step S6 to perform PCR detection on the unknown pathogen.

2. The detection method according to claim 1, wherein, In step S5, prediction results with a length greater than 500bp and a pvalue less than 0.05 are collected for each sample. For the prediction results, the longer sequences are selected first, and then the contigs with higher scores are selected for output under the same conditions.

3. The application of the detection method of claim 1 or 2 for non-therapeutic purposes in the detection of an unknown virus causing duck reproductive disorder syndrome.

4. A method for screening primers and probes for detecting unknown pathogens, wherein, The method includes the following steps: S1: Collect diseased tissues from multiple diseased animals for metagenomic sequencing; S2: Compare the metagenomic sequencing data of multiple animals obtained in step S1 with the reference genome data of the animal, remove the genome data belonging to the animal, and use splicing software to splice the remaining sequencing data to obtain contigs; S3: Construct a local non-redundant nucleic acid database to annotate contigs, remove contigs that can be annotated and matched, and retain contigs that have no annotation results after annotation for subsequent steps. S4: Contigs without annotation results in non-redundant nucleic acid databases are predicted using DeepVirFinder to obtain potential unknown pathogen gene sequences; S5: Filter the predicted sequences in step S4, collect the prediction results of each sample with a length greater than 500bp and a pvalue less than 0.05, and output contigs; S6: Perform homology analysis on the contigs output in step S5. Based on the homology analysis results, select similar sequence fragments in each sample, and use the similar sequence fragments to design and synthesize detection primers and probes. Screen for detection primers and probes with high sensitivity.

5. Detection primers and probes screened using the method described in claim 4.

6. The application of the method described in claim 4 in the screening of primers and probes for the detection of unknown pathogens.

7. Detection methods for unknown viruses causing duck reproductive disorder syndrome for non-diagnostic or therapeutic purposes, including, The method involves detecting the unknown virus causing the duck reproductive disorder syndrome using the detection method described in claim 1, or screening primers and probes that can be used to detect the unknown virus causing the duck reproductive disorder syndrome using the screening method for detection primers and probes described in claim 4.

8. Primers and probes for detecting an unknown virus causing duck reproductive disorder syndrome, wherein, The primer sequences are shown in SEQ ID NO:1 and SEQ ID NO:2 or in SEQ ID NO:4 and SEQ ID NO:5, and the probe sequences are shown in SEQ ID NO:3 or in SEQ ID NO:

6.

9. A detection kit containing the detection primers and probes as described in claim 8.

10. The use of the detection primers and probes of claim 8 or the kit of claim 9 for non-diagnostic or therapeutic purposes in the detection of an unknown virus causing duck reproductive disorder syndrome.

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