Specific primer group for simultaneously detecting 54 key pathogens as well as related method and application of specific primer group

Abstract

The invention discloses a specific primer group for simultaneously detecting 54 key pathogens as well as a related method and application thereof, the specific primer group is designed aiming at the 54 key pathogens, a target sequence is amplified by combining multiple PCR (Polymerase Chain Reaction) and a nanopore sequencing technology, and then purification, enrichment, library building and sequencing are carried out, so that the specific primer group can be used for simultaneously detecting the 54 key pathogens. A detection technology system capable of detecting 54 key pathogens at the same time is established, the method is rapid and accurate in detection and high in sensitivity, required reagents and instruments are portable, pathogen detection work can be carried out in a field environment, beneficial data support can be provided for source tracing of the pathogens, analysis of mutation sites can be carried out, and the detection efficiency is improved. The method has very prominent significance on early prevention and control of sudden new infectious diseases caused by high-pathogenicity pathogenic microorganisms.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a specific primer set for the simultaneous detection of 54 key pathogens, as well as related methods and applications. Background Technology

[0002] Highly pathogenic pathogens (HPPathogens) are microorganisms that can cause serious illness or high mortality rates. These include viruses, bacteria, fungi, and parasites, and are typically characterized by high pathogenicity, high transmissibility, and a high risk of causing large-scale infectious disease outbreaks, posing significant public health risks and biosafety threats. According to the "List of Human Infectious Pathogenic Microorganisms" (2023 edition) published by the National Health Commission of China, pathogens are classified into four levels, from Level I to Level II. Level I and Level II pathogens are listed as highly pathogenic pathogens and are the focus of research and monitoring in the field of pathogen detection and identification.

[0003] Highly pathogenic pathogens pose a serious threat to global public health security. These pathogens are mainly transmitted through mosquitoes, ticks, and animal hosts, causing infectious disease outbreaks and patient casualties. According to the World Health Organization, Ebola virus infection is usually accompanied by a high mortality rate. In the 2014-2016 West African outbreak, the Zaire strain of Ebola virus resulted in 28,616 confirmed cases and 11,310 deaths. Dengue virus is a major carrier of mosquito-borne diseases globally, infecting approximately 390 million people annually, of whom 96 million show obvious clinical symptoms. Japanese encephalitis virus and West Nile virus can easily cause encephalitis, paralysis, and even death after infection. Therefore, the detection of highly pathogenic pathogens is of irreplaceable importance in global infectious disease control.

[0004] Currently, most pathogenic pathogens are detected using viral culture and serological testing, which are technically demanding and time-consuming, making them unsuitable for rapid on-site pathogen detection and screening. While real-time quantitative PCR (qPCR) offers high sensitivity, it has a limited range of detectable pathogens on-site and carries a risk of false positives. Therefore, there is an urgent need for a technique that can accurately, rapidly, and sensitively detect highly pathogenic pathogens on-site. Multiplex PCR, with its reaction conditions and principles similar to singlex PCR, involves adding two or more primer pairs to the same reaction system to amplify different templates, yielding different target fragments. This allows for the simultaneous detection of multiple genes. Nanopore sequencing, a newly developed detection technology in recent years, offers advantages such as high throughput, small size, portability, and ease of operation. It can acquire nucleic acid fragment sequence information in samples in real time, enabling source tracing analysis of pathogens and simultaneous detection of a wide variety of pathogenic microorganisms. Compared to traditional detection techniques, it significantly reduces detection time and costs, making it suitable for on-site testing environments. Summary of the Invention

[0005] In order to overcome the above-mentioned technical problems existing in the field, the purpose of this invention is to provide a specific primer set for the simultaneous detection of 54 key pathogens and related methods and applications.

[0006] This study aims to establish a key pathogen detection system combining multiplex PCR and nanopore sequencing technologies. Fifty-four key pathogens were screened, specific amplification primers were designed, multiplex PCR amplification conditions were optimized, and the pathogen detection sensitivity was investigated. Evaluation showed that the detection system and method provided by this invention achieve rapid, accurate, and highly sensitive detection of target pathogens.

[0007] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0008] A first aspect of the present invention provides a specific primer set for the simultaneous detection of 54 key pathogens.

[0009] Furthermore, the primer set includes:

[0010] The first primer set for detecting Argentine arena virus consists of primers with nucleotide sequences as shown in SEQ ID NO: 1-2;

[0011] The second primer set is used to detect avian influenza virus type 1. The second primer set consists of primers with nucleotide sequences as shown in SEQ ID NO: 3-6.

[0012] A third primer set for detecting Chikungunya virus, the third primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO: 7-10;

[0013] A fourth primer set for detecting Dori virus, the fourth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:11-14;

[0014] The fifth primer set for detecting Evogliez virus consists of primers with nucleotide sequences as shown in SEQ ID NO:15-18;

[0015] The sixth primer set for detecting Guanareto sand virus consists of primers with nucleotide sequences as shown in SEQ ID NO:19-22;

[0016] The seventh primer set for detecting Hantavirus, the seventh primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:23-26;

[0017] The eighth primer set for detecting hepatitis A virus, the eighth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:27-28;

[0018] The ninth primer set for detecting human gamma herpesvirus type 8, the ninth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:29-32;

[0019] The tenth primer set for detecting Japanese encephalitis virus, the tenth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:33-36;

[0020] The eleventh primer set for detecting Khosanur forest disease virus, the eleventh primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:37-38;

[0021] The twelfth primer set for detecting Lhasa fever virus consists of primers with nucleotide sequences as shown in SEQ ID NO:39-42;

[0022] The thirteenth primer set for detecting Marburg virus consists of primers with nucleotide sequences as shown in SEQ ID NO:43-46;

[0023] The fourteenth primer set for detecting Mayaro virus, the fourteenth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:47-50;

[0024] The fifteenth primer set for detecting Middlesbrough virus, the fifteenth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:51-54;

[0025] The sixteenth primer set for detecting the Mojiang virus consists of primers with nucleotide sequences as shown in SEQ ID NO: 55-58;

[0026] The seventeenth primer set for detecting Mukamb virus consists of primers with nucleotide sequences as shown in SEQ ID NO:59-62;

[0027] The eighteenth primer set for detecting Ndum virus, the eighteenth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO: 63-66;

[0028] The nineteenth primer set for detecting Nipah virus, the nineteenth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO: 67-70;

[0029] The twentieth primer set is used to detect Omsk hemorrhagic fever virus. The twentieth primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:71-72.

[0030] The 21st primer set for detecting Bovasen virus consists of primers with nucleotide sequences as shown in SEQ ID NO:73-74;

[0031] The 22nd primer set for detecting Rochenberg virus consists of primers with nucleotide sequences as shown in SEQ ID NO:75-78;

[0032] The 23rd primer set for detecting Sindbis virus consists of primers with nucleotide sequences as shown in SEQ ID NO:79-81;

[0033] The 24th primer set is used to detect the forest-type Ebola virus. The 24th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:82-85.

[0034] The 25th primer set is used to detect tick-borne encephalitis virus. The 25th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:86-87.

[0035] The 26th primer set for detecting West Nile virus consists of primers with nucleotide sequences as shown in SEQ ID NO:88-89;

[0036] The 27th primer set is used to detect the Zaire Ebola virus. The 27th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:90-93.

[0037] The 28th primer set for detecting Bear Canyon Sandworm consists of primers with nucleotide sequences as shown in SEQ ID NO:94-97;

[0038] The 29th primer set for detecting Brazilian arena virus consists of primers with nucleotide sequences as shown in SEQ ID NO:98-101;

[0039] The thirtieth primer set is used to detect the Bendibugyo type of Ebola virus, and the thirtieth primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:102-105;

[0040] The 31st primer set for detecting Bunianvila virus, the 31st primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:106-109;

[0041] The 32nd primer set for detecting California encephalitis virus, the 32nd primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:110-113;

[0042] The 33rd primer set for detecting Xinjiang hemorrhagic fever virus consists of primers with nucleotide sequences as shown in SEQ ID NO:114-117;

[0043] The 34th primer set for detecting dengue virus, the 34th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO: 118-119;

[0044] The 35th primer set for detecting Eastern equine encephalitis virus, wherein the 35th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:120-123;

[0045] The 36th primer set for detecting flaxseed virus, the 36th primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:124-125;

[0046] The 37th primer set for detecting foot-and-mouth disease virus consists of primers with nucleotide sequences as shown in SEQ ID NO:126-127;

[0047] The 38th primer set for detecting Gehrig's virus, the 38th primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:128-131;

[0048] The 39th primer set for detecting Hendra virus, the 39th primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:132-133;

[0049] The 40th primer set for detecting Iliovirus, the 40th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:134-135;

[0050] The 41st primer set for detecting Machupo isovirus consists of primers with nucleotide sequences as shown in SEQ ID NO:136-139;

[0051] The 42nd primer set for detecting monkeypox virus consists of primers with nucleotide sequences as shown in SEQ ID NO:140-141;

[0052] Primer set 43 for detecting Nairobi sheep disease virus, the primer set 43 consists of primers with nucleotide sequences as shown in SEQ ID NO:142-145;

[0053] The forty-fourth primer set for detecting oral thrush virus, the forty-fourth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:146-147;

[0054] Primer set 45 for detecting oropche virus, the primer set 45 consists of primers with nucleotide sequences as shown in SEQ ID NO:148-151;

[0055] The 46th primer set for detecting Reston-type Ebola virus, the 46th primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:152-155;

[0056] The 47th primer set for detecting Lift Valley sandfly virus consists of primers with nucleotide sequences as shown in SEQ ID NO:156-159;

[0057] The 48th primer set for detecting Sepik virus, the 48th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:160-161;

[0058] Primer set 49 for detecting St. Louis encephalitis virus, wherein the primer set 49 consists of primers with nucleotide sequences as shown in SEQ ID NO:162-163;

[0059] The fiftieth primer set for detecting Venezuelan equine encephalitis virus, said fiftieth primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:164-167;

[0060] Primer set 51 for detecting Western equine encephalitis virus, wherein the primer set 51 consists of primers with nucleotide sequences as shown in SEQ ID NO:168-171;

[0061] Primer set 52 for detecting yellow fever virus, wherein the primer set 52 consists of primers with nucleotide sequences as shown in SEQ ID NO:172-173;

[0062] Primer set 53 for detecting Takarib Sassavirus, wherein the primer set 53 consists of primers with nucleotide sequences as shown in SEQ ID NO:174-175;

[0063] The 54th primer set for detecting smallpox virus consists of primers with nucleotide sequences as shown in SEQ ID NO:140-141.

[0064] Furthermore, the concentration of each primer is 0.2 μmol / L.

[0065] A second aspect of the present invention provides a primer pool combination for the simultaneous detection of 54 key pathogens.

[0066] Furthermore, the primer pool includes: Mix1 and Mix2;

[0067] The Mix1 consists of the first primer set to the twenty-seventh primer set as described in the first aspect of the present invention;

[0068] The Mix2 consists of primer sets 28 to 54 as described in the first aspect of this invention;

[0069] The 54 key pathogens are: Bear Canyon Arena Virus, Machupo Arena Virus, Lassa Fever Virus, Flexo Arena Virus, Guanareto Arena Virus, Tacarib Arena Virus, Argentine Arena Virus, Brazilian Arena Virus, Xinjiang Hemorrhagic Fever Virus, Zaire Ebola Virus, Reston Ebola Virus, Great Forest Ebola Virus, Bendibugyo Ebola Virus, Marburg Virus, Nipah Virus, Hendra Virus, Merjan Virus, Venezuelan Equine Encephalitis Virus, Western Equine Encephalitis Virus, Eastern Equine Encephalitis Virus, Chikungunya Virus, Yellow Fever Virus, Khosanur Forest Disease Virus, and Omsk Hemorrhagic Fever Virus. St. Louis encephalitis virus, tick-borne encephalitis virus, dengue virus, monkeypox virus, smallpox virus, Bunianvila virus, California encephalitis virus, Dori virus, Everglades virus, foot-and-mouth disease virus, Gata virus, Hantavirus, hepatitis A virus, human gamma herpesvirus 8, West Nile virus, Japanese encephalitis virus, Sindbis virus, Mayaro virus, Middleburg virus, Mukamb virus, oral thrush virus, Sepik virus, Ilius virus, Lift Valley sandfly virus, Rochenberg virus, Powassen virus, Oropuch virus, Nairobi sheep disease virus, Ndum virus, and / or avian influenza virus type 1.

[0070] Furthermore, the concentration of each primer is 0.2 μmol / L.

[0071] A third aspect of the present invention provides a reagent for the simultaneous detection of 54 key pathogens using multiplex PCR and nanopore sequencing technologies;

[0072] Furthermore, the reagent comprises the primer pool combination described in the second aspect of the present invention;

[0073] The 54 key pathogens mentioned are the 54 key pathogens described in the second aspect of this invention.

[0074] In some implementation schemes, multiplex PCR, compared to singlex PCR, can amplify within the same reaction system, offering advantages such as time savings and high detection efficiency. However, in previous studies, multiplex PCR products were mostly identified and analyzed using traditional methods such as electrophoresis gels. This method places high demands on target sequence length and primer design, requiring consideration of differences in amplified product length and the compatibility and specificity of primer pairs. It suffers from problems such as low detection sensitivity, limited detection of pathogenic microorganisms, significant primer dimerization, and inability to obtain amplified fragment sequences. Furthermore, the bulky nature of the detection instruments prevents operation in field settings outside the laboratory, limiting its widespread application.

[0075] In some implementation schemes, nanopore sequencing is a rapidly developing third-generation sequencing technology in recent years. Its advantages include portable instruments, making it suitable for field operation, and the ability to acquire gene fragment sequence information in samples in real time, facilitating data analysis. However, directly sequencing samples can easily affect detection sensitivity due to high background sequence levels.

[0076] In a specific embodiment of the present invention, the present invention combines multiplex PCR and nanopore sequencing technologies, and uses the primer pool combination described in the second aspect of the present invention to achieve simultaneous detection of 54 key pathogens. This overcomes the shortcomings of the two technologies (multiplex PCR and nanopore sequencing), and the detection is rapid, accurate, and highly sensitive. The required reagents and instruments are portable, which is beneficial for pathogen detection in the field.

[0077] A fourth aspect of the present invention provides a kit for simultaneously detecting 54 key pathogens using multiplex PCR and nanopore sequencing technologies.

[0078] Furthermore, the kit comprises the reagents described in the third aspect of the present invention;

[0079] The 54 key pathogens mentioned are the 54 key pathogens described in the second aspect of this invention.

[0080] In some embodiments, the kit further includes instructions for use or a label, a positive control, a negative control, a buffer, an adjuvant, or a solvent, and one or more containers for containing the compositions contained in the kit. The compositions may be in liquid form or lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from a variety of materials, including glass or plastic. The instructions for use or label details how to use the kit to test samples.

[0081] In some embodiments, the kit may also include a variety of different reagents suitable for practical use (e.g., for different detection methods), and is not limited to the reagents listed in this invention. Any reagent that is based on the primer pool described above to identify 54 key pathogens or to detect whether a sample contains 54 key pathogens falls within the protection scope of this invention.

[0082] The fifth aspect of the present invention provides a method for identifying 54 key pathogens for non-disease diagnosis purposes or a method for detecting whether a sample to be tested contains any one or more of the 54 key pathogens for non-disease diagnosis purposes.

[0083] Furthermore, the method includes using multiplex PCR and nanopore sequencing technology in combination with the primer pool combination described in the second aspect of the present invention to detect the bacteria to be tested or the sample to be tested;

[0084] The 54 key pathogens mentioned are the 54 key pathogens described in the second aspect of this invention.

[0085] In some implementations, the method includes the following steps:

[0086] (1) Extract DNA from the bacteria to be tested or the sample to be tested;

[0087] (2) Use the primers in the above primer pool combination to perform multiple PCR reaction to obtain PCR products;

[0088] (3) Construct a library from the PCR products obtained in step (2) above;

[0089] (4) Sequencing was performed using nanopore sequencing technology.

[0090] In a specific embodiment of the present invention, the multiplex PCR reaction system comprises: 5 μL of 2×Phusion U MultiplexPCR Master Mix, 2.5 μL of mixed primers, and 2.5 μL of mixed template. PCR reaction conditions are: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, for 35 cycles; and 72℃ extension for 10 min. The PCR products are used to construct a library according to the protocol of the rapid library construction kit RBK096.

[0091] In a specific embodiment of the present invention, the sequencing platform used in the process of sequencing the purified product using nanopore sequencing technology is a MinION MK1C sequencer paired with an R9.4.1 Flowcell chip. The steps are as follows: Prepare 9 μL of PCR product, add 1 μL of the corresponding barcode, incubate at 30°C for 2 min, incubate at 80°C for 2 min, and then cool on ice. Mix all barcode-labeled libraries, purify with AMPure XP beads, add sequencing adapters to the purified product, and then perform sequencing over 4 hours.

[0092] The sixth aspect of the present invention provides the application of the primer set of the first aspect of the present invention, the primer pool combination of the second aspect of the present invention, the reagent of the third aspect of the present invention, or the kit of the fourth aspect of the present invention in identifying 54 key pathogens in non-disease diagnosis destinations or detecting whether 54 key pathogens are present in a sample to be tested in non-disease diagnosis destinations.

[0093] The 54 key pathogens mentioned are the 54 key pathogens described in the second aspect of this invention.

[0094] In some embodiments, the test sample refers to a composition obtained from or derived from the target subject, the test sample containing cellular entities and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical and / or physiological characteristics.

[0095] In some implementations, the test sample may be obtained from a subject's tissue sample, blood sample, or other fluid sample of biological origin, such as a biopsy tissue sample or tissue culture or cells derived therefrom. The source of the tissue sample may be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy tissue or aspirates; blood or any blood component; body fluids; cells from any stage of an individual's pregnancy or development; or plasma. Furthermore, the test sample includes biological samples that have been processed in any way after acquisition, such as being treated with reagents, stabilized, enriched for certain components (such as proteins or polynucleotides), or embedded in a semi-solid or solid matrix for sectioning purposes.

[0096] In some implementations, the sample to be tested includes, but is not limited to, bodily fluids, blood, serum, plasma, cerebrospinal fluid, sputum, tears, urine, saliva, sweat, semen, mucus, lymph, amniotic fluid, interstitial fluid, pulmonary lavage fluid, feces, tissue samples, peritoneal fluid, bladder irrigation fluid, secretions (e.g., breast secretions), oral irrigation fluid, swabs (e.g., oral swabs), touch preparations, fine needle aspiration materials, cell extracts, or any combination thereof from the subject.

[0097] In some embodiments, the subject of this invention refers to any animal, including both human and non-human animals. Non-human animals include all vertebrates, such as mammals like non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; as well as non-mammals such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human.

[0098] The seventh aspect of the present invention provides for any of the following applications:

[0099] (1) The application of the primer set described in the first aspect of the present invention, the primer pool combination described in the second aspect of the present invention, the reagent described in the third aspect of the present invention, or the kit described in the fourth aspect of the present invention in constructing a high-throughput amplicon sequencing library for simultaneous detection of 54 key pathogens;

[0100] (2) The application of the primer set described in the first aspect of the present invention, the primer pool combination described in the second aspect of the present invention, the reagent described in the third aspect of the present invention, or the kit described in the fourth aspect of the present invention in constructing a detection technology system for simultaneous detection of 54 key pathogens;

[0101] (3) The application of the primer set described in the first aspect of the present invention, the primer pool combination described in the second aspect of the present invention, the reagent described in the third aspect of the present invention, or the kit described in the fourth aspect of the present invention in the simultaneous detection of 54 key pathogen genes or mutation sites in 54 key pathogen genes at a non-disease diagnosis destination.

[0102] The 54 key pathogens mentioned are the 54 key pathogens described in the second aspect of this invention.

[0103] Compared with the prior art, the present invention has the following beneficial effects:

[0104] (1) This invention selects 54 pathogens with high transmissibility and high pathogenicity from the latest version of the National Catalogue of Pathogenic Microorganisms, including Ebola virus, and designs primers for these pathogens as target pathogens. For some pathogens, this invention designs multiple pairs of primers, and amplifies, purifies, enriches, constructs libraries, and sequences the target sequences using a combination of multiplex PCR and nanopore sequencing technologies. This establishes a detection technology system capable of simultaneously detecting 54 key pathogens. This method is time-efficient, highly sensitive, and can obtain amplified gene fragment sequence information for data statistical analysis.

[0105] (2) After verifying primer specificity, optimizing multiplex PCR reaction conditions, and statistically analyzing detection sensitivity data, it was determined that this method achieves optimal amplification effect and detection rate at a primer concentration of 0.2 μmol / L, stably detecting 54 pathogens. Further investigation of detection sensitivity revealed that within a single operation time of 4 hours, at least 51 key pathogens could be detected simultaneously, achieving a detection sensitivity of 10. 3 With a detection rate of copies / mL, the sequence information of pathogens can be accurately detected. Therefore, this research method combines the advantages of multiplex PCR and third-generation sequencing technologies, overcoming the shortcomings of both. It offers rapid, accurate, and highly sensitive detection, and the required reagents and instruments are portable, making it suitable for pathogen detection in field environments.

[0106] (3) The detection system established by this invention for 54 highly pathogenic key pathogens not only provides valuable data support for tracing the source of pathogens, but also enables the analysis of mutation sites. This is of great significance for the early prevention and control of emerging infectious disease outbreaks caused by highly pathogenic microorganisms. Attached Figure Description

[0107] Figure 1 : Single PCR gel image;

[0108] Figure 2 Results of full-template and full-primer amplification and sequencing of 54 pathogens;

[0109] Figure 3 Results of full-template amplification and sequencing of 54 pathogens;

[0110] Figure 4 : Primer concentration optimization results (image);

[0111] Figure 5 Images of multiplex PCR gels using full templates and primer pools for 54 pathogens. Image A: Thermo Fisher Scientific multiplex amplification enzyme-primer pool 1; Image B: Thermo Fisher Scientific multiplex amplification enzyme-primer pool 2; Image C: Kangwei Century multiplex amplification enzyme-primer pool 1; Image D: Kangwei Century multiplex amplification enzyme-primer pool 2.

[0112] Figure 6 Sensitivity studies of 54 pathogens were conducted. Figure A shows the sequencing results when the amplification enzyme was Thermo Fisher's multiplex amplification enzyme, and Figure B shows the sequencing results when the amplification enzyme was Kangwei Century's multiplex amplification enzyme.

[0113] Figure 7 Sensitivity exploration for 4 pathogens not detected: Figure A: Sequencing results when the amplification enzyme is Thermo Fisher multiplex amplification enzyme; Figure B: Sequencing results when the amplification enzyme is Combibloc multiplex amplification enzyme. Detailed Implementation

[0114] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents.

[0115] The products, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are typically performed under conventional conditions or as recommended by the manufacturer. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0116] Example: Establishment and validation of a key pathogen detection system combining multiplex PCR and nanopore sequencing technologies.

[0117] 1. Materials and Methods

[0118] 1.1 Materials and Reagents

[0119] Phusion U multiplex PCR premix, DNA loading buffer (Thermo Fisher Scientific (China) Co., Ltd.); 2000bp DNA marker (Sangon Biotech (Shanghai) Co., Ltd.); nuclease-free water (Ingen Biotech (China) Co., Ltd.); agarose and TAE electrophoresis buffer (Yisheng Biotech (Shanghai) Co., Ltd.); sequencing-related kits (SQK-RBK096, EXP-FLP002, EXP-WSH004), R9.4.1 Flowcell chip, MinION MK1C sequencer (Oxford Nanopore Technologies (Shanghai) Co., Ltd.).

[0120] 1.2 Instruments and Equipment

[0121] Sorvall Legend Micro 21R microcentrifuge (Thermo Fisher Scientific (China) Co., Ltd.); Bio-Rad Mini-Sub Cell GT System electrophoresis apparatus, BIO-RAD Gel Doc XR+System gel imaging system, Bio-Rad T100 Thermal Cycler (Borvad Life Sciences (Shanghai) Co., Ltd.); Siemens KG49EBI40M refrigerator (Siemens (China) Co., Ltd.); Corning LSE Vortex Mixer (Sailo Czech (Shanghai) Biotechnology Co., Ltd.); XPE series electronic balance (Changsha Xiangping Technology Development Co., Ltd.); BSC-ⅡA2 biosafety cabinet (Beijing Donglian Haer Instrument Manufacturing Co., Ltd.).

[0122] 1.3 Experimental Methods

[0123] 1.3.1 Complete the survey and data collection

[0124] 1.3.2 Determine the scope of screening for key pathogens

[0125] Based on the pathogen classifications in the "List of Human Infectious Pathogens" published by the Ministry of Health of the People's Republic of China in 2006 and the "List of Human Infectious Pathogens" (2023 edition) published by the National Health Commission of the People's Republic of China, and integrating common key pathogens internationally, we selected 54 pathogens with a hazard level of I or II or higher in the latest classification (see Table 1) as key pathogens to be tested after standardizing and correcting the pathogen species names. For pathogens with high infectivity and high concealment, the screening scope can be appropriately expanded to increase the identification of different subtypes of pathogens. Subsequent PCR primer design procedures were then carried out.

[0126] Table 1 List of 54 key pathogens

[0127]

[0128]

[0129] 1.3.3 Selection of target gene and construction of plasmid

[0130] The sequence information of the genomes (or gene coding regions, CDS) of the aforementioned 54 pathogens was downloaded and organized from GenBank at the National Center for Biotechnology Information (NCBI). Using multiple sequence alignment (MSA) and pairwise sequence alignment (PSA) methods, pathogen-specific fragments of suitable length and relatively stable GC content were selected from the genomes (or gene coding regions, CDS) of various pathogens as target fragments for amplification. The nucleic acid sequences of the target fragments were then compared with the NCBI database using BLAST to verify their specificity. The target gene fragment plasmids were then synthesized by Qingke Biotechnology Co., Ltd. (using pUC57 plasmid as a vector), with an initial plasmid concentration of 1×10⁻⁶. 12 copies / mL, serially diluted with nuclease-free water to 1×10⁻⁶. 1 Copy / mL.

[0131] 1.3.4 Design of multiplex PCR primer pool

[0132] Download the coding sequence files of all pathogen reference genomes, screen all coding sequences of all viruses, construct a similarity matrix, and identify the specific regions of each virus. After obtaining the target fragment for amplification, use Primer3 software to generate a candidate primer set. By constraining parameters such as primer length, GC content, and free energy, a PCR primer candidate set with relatively consistent annealing temperatures is obtained. Subsequently, use MFEprimer software to screen primer specificity, avoiding primers from different pathogens that cannot pair with each other and amplify the target fragment, as well as primer mismatches that would lead to amplification products that do not match expectations. Dimer check is used to screen for possible dimerization of the primers themselves, avoiding primer dimerization that would prevent binding to the target fragment. Finally, the BLAST method is used to compare the primers with genomic fragments of all existing species to analyze primer specificity and avoid false positive results due to off-target amplification during the amplification process. Primers were synthesized by General Biotechnology (Anhui) Co., Ltd., and purified using iPAGE. After synthesis, primers were diluted to a concentration of 10 μmol / L with ddH2O and stored at -20℃ for later use.

[0133] 1.3.5 Single PCR to verify primer specificity

[0134] We designed 90 target DNA sequences for 54 key pathogens, and constructed DNA template plasmids (Beijing Qingke Biotechnology Co., Ltd.) by extending the target DNA fragments 200 bp upstream and downstream. Using this plasmid DNA fragment as a template, we verified the specificity of the 90 primer pairs using singleton PCR. The PCR reaction system consisted of: 25 μL of 2×PCR mix (2×San Taq PCR premix), 1 μL each of forward and reverse primers (10 μmol / L), 10 μL of DNA template, and 13 μL of H2O, for a total of 50 μL. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 1 min, repeated 35 times; and 72℃ extension for 5 min. 25 μL of the PCR amplification product was analyzed by 1% agarose gel electrophoresis (see [link to analysis]). Figure 1 (The pathogen names corresponding to the numbers are shown in Table 2). Another 25 μL was taken for Sanger sequencing to verify the accuracy of the amplification.

[0135] 1.3.6 Nanopore sequencing for preliminary detection of multiple pathogens

[0136] Eighty-six synthesized target sequences for key pathogens were mixed in equal proportions, and corresponding 86 primer pairs were mixed in equal proportions to form a primer pool for multiplex PCR amplification. The multiplex PCR amplification system consisted of 5 μL of 2×Phusion U multiplex PCR premix, 2.5 μL of mixed primers, and 2.5 μL of mixed template. PCR reaction conditions were: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, for 35 cycles; and 72℃ extension for 10 min. The PCR products were then sequenced using nanopore sequencing.

[0137] 1.3.7 Primer Pool Combination Optimization

[0138] Because the number of primer pools in this study is relatively large, according to Figure 2 For the detection of pathogens, primers for 54 pathogens (as shown in Table 2) were divided into two primer pools (Mix1 and Mix2, with each primer having a final concentration of 10 μmol / L). The primer sequences in the two pools are shown in Tables 3 and 4 below. The optimal detection results under different primer pool combinations were explored. The mixed full template was amplified using the full primer pool, Primer Pool 1, and Primer Pool 2, respectively, under the same amplification conditions as in 1.3.6.

[0139] Table 2 Primers for 54 key pathogens after singlet PCR validation.

[0140]

[0141]

[0142]

[0143]

[0144] Table 3 lists the primers included in Mix1 (targeting 27 key pathogens).

[0145]

[0146]

[0147] Table 4 lists the primers included in Mix2 (targeting 27 key pathogens).

[0148]

[0149]

[0150]

[0151] 1.3.8 Primer Concentration Optimization

[0152] The optimal primer amplification concentrations were determined through multiplex amplification experiments with different primer concentration gradients. Primer pools 1 and 2 were diluted to the following concentrations: 6.4 μmol / L, 3.2 μmol / L, 1.6 μmol / L, 1.2 μmol / L, 0.8 μmol / L, 0.4 μmol / L, 0.2 μmol / L, 0.1 μmol / L, 0.05 μmol / L, 0.025 μmol / L, and 0.0125 μmol / L, respectively. The multiplex PCR reaction system is shown in 1.3.6. The amplification products from primer pools 1 and 2 with the same primer concentration were mixed, and the mixed product was used for library construction and sequencing.

[0153] 1.3.9 Sensitivity Validation of Multiplex PCR System

[0154] according to Figure 4 The optimal primer concentration was used to explore the DNA template detection sensitivity, and the possibility of different multiplex amplification enzymes improving the detection sensitivity was also investigated. Two multiplex amplification enzymes, one from Thermo Fisher Scientific and the other from Kangwei Century, were used, and the mixed template was serially diluted to 10⁻⁶. 6 copies / mL, 10 5 copies / mL, 10 4 copies / mL and 10 3 copies / mL, 10 2Copy / mL. Multiplex PCR amplification was performed using primer pool 1 and primer pool 2, respectively. The multiplex PCR reaction system is shown in step 1.3.6. The amplification products from primer pool 1 and primer pool 2 with the same template concentration were mixed. Half of the mixture was used for gel electrophoresis, and the other half was used for library construction and sequencing.

[0155] according to Figure 6 The results showed that the detection of four pathogens, reston_ebola, orf, getah and everglade, was unstable or even not detected. We mixed templates of these four pathogens, amplified them using primer pools 1 and 2 respectively, and then constructed libraries for sequencing.

[0156] 1.3.10 SQK-RBK110.96 Kit Nanopore Sequencing PCR

[0157] The products were used to construct libraries according to the protocol of the rapid library construction kit SQK-RBK110.9. The sequencing platform was a MinION MK1C sequencer with an R9.4.1 Flowcell chip. The simplified procedure was as follows: 9 μL of PCR product was prepared, and 1 μL of the corresponding barcode was added. The mixture was incubated at 30°C for 2 min, then at 80°C for 2 min, and finally cooled on ice. All barcode-labeled libraries were mixed and purified using AMPure XP beads. After adding sequencing adapters, the purified product was sequenced over 4 hours.

[0158] 1.3.11 Data and Statistical Analysis

[0159] The sequencing data was processed by the Guppy software built into the sequencer to split the barcodes, and the Blast analysis software was used to identify the species of the next-level data and count the number of target gene sequence fragments and the number of virus types.

[0160] 2. Experimental Results

[0161] 2.1 Primer Specificity

[0162] Using 90 pairs of specific primers, singleton PCR was performed on 54 key pathogens listed in Table 1. The results showed that primer pairs 69, 72, and 4 others amplified non-specific target bands, while primer pairs 77 and 78 failed to amplify the target bands. Apart from these, the remaining 86 primer pairs all amplified the target bands without any non-specific amplification. Figure 1 ).

[0163] After gel electrophoresis verification of the PCR products, the 86 pairs of products amplifying specific target bands were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequence alignment with the target gene showed a similarity rate of over 99.6%. The amplified products were highly consistent with the target gene sequence, indicating that the 86 pairs (172 primers) selected through singleton PCR specificity verification in this study possess high specificity.

[0164] 2.2 Nanopore sequencing for preliminary detection of multiple pathogens

[0165] Using 54 pathogens as complete templates for amplification, 86 pairs of primers were selected as complete primers for amplification, with a concentration of 10. 6 Multiplex PCR amplification was performed using a template of [copy / mL]. The amplified products were then used for library construction and sequencing. Data from 2.5 hours after sequencing were analyzed. Results are shown in [Table / Reference]. Figure 2 The number of sequence fragments and virus types detected in the amplification products were inconsistent and relatively low. Only 500-600 sequence fragments were detected, and at least 18 pathogens were detected. Amplification products using water as a template detected more than 6 pathogens, but the number of sequences was low, only 17-23. Considering the possibility of barcode misreading during nanopore sequencing, these sequences were likely due to barcode misreading. The results show that using a complete primer pool as amplification primers resulted in poor amplification performance, far exceeding expectations.

[0166] 2.3 Primer pool combination optimization

[0167] Given that the preliminary experimental results did not meet expectations, we considered that the excessive number of primers might be the cause. Therefore, we split the entire primer pool, merging the primers that detected the pathogen in the preliminary experiment into primer pool Mix1, and merging the undetected primers into Mix2. Amplification and sequencing were then performed again based on these primer pools. Data from 2.5 hours after sequencing were analyzed, and the results are as follows: Figure 3 The number of sequence fragments and virus types detected by the amplification products of both the whole primer pool and the separate primer pools were relatively stable. However, the whole primer pool showed a significant difference in the number of sequence fragments detected and the number of virus types detected compared to the separate primer pool. When the amplification primers were from the whole primer pool, the number of sequence fragments detected was relatively small, around 500, and the number of virus types detected was close to 30 target pathogens. When the amplification primers were from the separate primer pools Mix1 and Mix2, the number of sequence fragments reached 10. 5 The efficiency is on the order of magnitude, 100× that of the full primer pool. Simultaneously, it detects 50 viral species, achieving a detection efficiency of 1.75× that of the full primer pool. Therefore, when performing multiplex PCR amplification using separate primer pools, the sequencing detection efficiency is significantly improved, making it a suitable primer design for this detection system.

[0168] 2.4 Primer Concentration Optimization

[0169] Since pathogen detection rate is a key evaluation indicator, this study used a wider range of primer concentration gradients for multiplex PCR amplification experiments, and combined this with third-generation sequencing to explore the optimal primer concentration. Sequencing statistics are shown below. Figure 4 When the primer concentration was between 3.2 μmol / L and 0.1 μmol / L, the number of pathogens detected remained stable at over 50. A primer concentration of 0.2 μmol / L resulted in a relatively high number of detected pathogen sequences. Therefore, 0.2 μmol / L was selected as the optimal primer concentration for the multiplex PCR system.

[0170] 2.5 Sensitivity Validation of Multiplex PCR System

[0171] 2.5.1 Multiplex PCR electrophoresis gel

[0172] Using primer pool Mix1 and primer pool Mix2 as amplification primers respectively, 10 6 copies / mL-10 2 The mixed template of copies / mL was subjected to multiplex PCR detection using Thermo Fisher Scientific and Combibloc's multiplex amplification enzymes, respectively. The results are as follows: Figure 5 As shown.

[0173] The results showed that at a template concentration of 10 4 At a template concentration of 10 copies / mL, primer pool 1 can amplify the target band. 6 At a template concentration of 10 copies / mL, both enzyme systems stably amplified the product in both primer pools; 5 At a template concentration of 10 copies / mL, the two enzyme systems could amplify the target product in both primer pools, but reproducibility was poor; at a template concentration of 10... 4 At a concentration of 10 copies / mL, both enzymes amplified the target band only in primer pool 1, with poor reproducibility; at a target concentration of 10... 3 copies / mL and 10 2 At a concentration of 10 copies / mL, neither enzyme system amplified the target band. Therefore, it was determined that 10 4 The copy number per mL (C / mL) was the sequencing detection limit for the multiplex PCR system of 54 key pathogens. Based on this, multiplex PCR amplification and library construction were performed on five mixed template concentrations, and the detection results were statistically analyzed.

[0174] 2.5.2 Sensitivity evaluation of multiplex PCR system

[0175] Primer pool 1 and primer pool 2 were used at a primer concentration of 0.2 μmol / L, along with two multiplex amplification enzymes from Thermo Fisher Scientific and Conversely, respectively, to achieve a 10 6 copies / mL-10 2The copy number / mL represents the template concentration for multiplex PCR amplification, followed by library construction and sequencing. Data from 1 hour after sequencing were statistically analyzed, and the results are as follows: Figure 6 .

[0176] When the multiplex PCR amplification enzyme was 2×Phusion U Multiplex PCR Master, the data after 1 hour of amplification, library preparation, and sequencing were statistically analyzed. The template concentration was 10... 6 and 10 5 At a copy / mL concentration, the number of detected sequence fragments in the amplified products is unstable. The template concentration is 10... 6 When the number of copies / mL is 10, the detection quantity is within 10. 5 Of the above fragments, at least 52 virus types were detected. 10 5 The number of amplified sequences detected at 10 copies / mL 4 -10 5 The number of virus types detected varied considerably, ranging from a minimum of 35 to a maximum of 48. 4 -10 2 At a rate of copies / mL, the number of detected sequence fragments and virus types drops sharply and is not stable; the number of detected sequences is around 10. 1 -10 2 Between the samples, a minimum of 17 virus types and a maximum of 30 virus types were detected.

[0177] When the multiplex PCR amplification enzyme was Kangwei Century multiplex amplification enzyme, the data after 1 hour of amplification, library preparation, and sequencing were statistically analyzed. The template concentration was 10... 6 At a concentration of 1 copy / mL, the amplified products showed a relatively high and stable number of detected sequence fragments, averaging 5 × 10⁶. 5 The number of virus types detected is consistently at least 52 pathogens. 10 5 At a copy / mL ratio, the results for the number of amplified sequences and the number of virus types were relatively unstable, with the number of pathogen fragments concentrated around 10. 5 More than 10 samples were collected, with a minimum of 41 virus types detected. 4 -10 2 At a copy / mL, the number of detected sequence fragments drops sharply but remains relatively stable, generally around 10. 3 -10 4 Between the lines. The number of virus types has decreased significantly, generally around 27 types can be detected.

[0178] according to Figure 6 The results showed that the detection of four pathogens—reston_ebola, orf, getah, and everglade—was unstable or even non-detectable. We mixed templates of these four pathogens, amplified them using primer pools 1 and 2 respectively, constructed libraries, and sequenced them. The results are as follows: Figure 7 As shown. When the multiplex PCR amplification enzyme is 2×Phusion U Multiplex PCR Master, the template concentration is 10. 6 At a concentration of 4 copies / mL, the amplified products showed a relatively large and stable number of detected sequence fragments, with an average detection rate of 4 × 10⁻⁶. 5 One fragment. Template concentration is 10. 5 At a copy / mL, the number of sequences detected was 10. 3 Strip, template concentration of 10 4 At a concentration of 10 copies / mL, stable detection was still possible, but the number of sequences was low. When the multiplex PCR amplification enzyme was Kangwei Century multiplex amplification enzyme, the template concentration was 10. 6 At a copy / mL, the number of sequences detected can reach 10. 6 Orders of magnitude, but when the template concentration is 10 5 -10 2 At a copy / mL, the detection performance is not much different from that of Thermo Fisher Scientific amplification enzyme.

Claims

1. A specific primer set for the simultaneous detection of 54 key pathogens, characterized in that, The primer set includes: The first primer set for detecting Argentine arena virus consists of primers with nucleotide sequences as shown in SEQ ID NO:1-2; The second primer set is used to detect avian influenza virus type 1. The second primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:3-6. A third primer set for detecting Chikungunya virus, the third primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:7-10; A fourth primer set for detecting Dori virus, the fourth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:11-14; The fifth primer set for detecting Evogliez virus consists of primers with nucleotide sequences as shown in SEQ ID NO:15-18; The sixth primer set for detecting Guanareto sand virus consists of primers with nucleotide sequences as shown in SEQ ID NO:19-22; The seventh primer set for detecting Hantavirus, the seventh primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:23-26; The eighth primer set for detecting hepatitis A virus, the eighth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:27-28; The ninth primer set for detecting human gamma herpesvirus type 8, the ninth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO: 29-32; The tenth primer set for detecting Japanese encephalitis virus, the tenth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:33-36; The eleventh primer set for detecting Khosanur forest disease virus, the eleventh primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:37-38; The twelfth primer set for detecting Lhasa fever virus consists of primers with nucleotide sequences as shown in SEQ ID NO:39-42; The thirteenth primer set for detecting Marburg virus consists of primers with nucleotide sequences as shown in SEQ ID NO:43-46; The fourteenth primer set for detecting Mayaro virus, the fourteenth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:47-50; The fifteenth primer set for detecting Middlesbrough virus, the fifteenth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO: 51-54; The sixteenth primer set for detecting the Mojiang virus consists of primers with nucleotide sequences as shown in SEQ ID NO:55-58; The seventeenth primer set for detecting Mukamb virus consists of primers with nucleotide sequences as shown in SEQ ID NO:59-62; The eighteenth primer set for detecting Ndum virus, the eighteenth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:63-66; The nineteenth primer set for detecting Nipah virus, the nineteenth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:67-70; The twentieth primer set is used to detect Omsk hemorrhagic fever virus. The twentieth primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:71-72. The 21st primer set for detecting Bovasen virus consists of primers with nucleotide sequences as shown in SEQ ID NO:73-74; The 22nd primer set for detecting Rochenberg virus consists of primers with nucleotide sequences as shown in SEQ ID NO:75-78; The 23rd primer set for detecting Sindbis virus consists of primers with nucleotide sequences as shown in SEQ ID NO:79-81; The 24th primer set is used to detect the forest-type Ebola virus. The 24th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:82-85. The 25th primer set is used to detect tick-borne encephalitis virus. The 25th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:86-87. The 26th primer set for detecting West Nile virus consists of primers with nucleotide sequences as shown in SEQ ID NO:88-89; The 27th primer set is used to detect the Zaire Ebola virus. The 27th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:90-93. The 28th primer set for detecting Bear Canyon Sandworm consists of primers with nucleotide sequences as shown in SEQ ID NO:94-97; The 29th primer set for detecting Brazilian arena virus consists of primers with nucleotide sequences as shown in SEQ ID NO:98-101; The thirtieth primer set is used to detect the Bendibugyo type of Ebola virus, and the thirtieth primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:102-105; The 31st primer set for detecting Bunianvila virus, the 31st primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:106-109; The 32nd primer set for detecting California encephalitis virus, the 32nd primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:110-113; The 33rd primer set for detecting Xinjiang hemorrhagic fever virus consists of primers with nucleotide sequences as shown in SEQ ID NO:114-117; The 34th primer set for detecting dengue virus, the 34th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO: 118-119; The 35th primer set for detecting Eastern equine encephalitis virus, wherein the 35th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:120-123; The 36th primer set for detecting flaxseed virus, the 36th primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:124-125; The 37th primer set for detecting foot-and-mouth disease virus, the 37th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO: 126-127; The 38th primer set for detecting Gehrig's virus, the 38th primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO: 128-131; The 39th primer set for detecting Hendra virus, the 39th primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO: 132-133; The 40th primer set for detecting Iliovirus, the 40th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO: 134-135; The 41st primer set for detecting Machupo isovirus consists of primers with nucleotide sequences as shown in SEQ ID NO:136-139; The 42nd primer set for detecting monkeypox virus consists of primers with nucleotide sequences as shown in SEQ ID NO: 140-141; Primer set 43 for detecting Nairobi sheep disease virus, the primer set 43 consists of primers with nucleotide sequences as shown in SEQ ID NO:142-145; The forty-fourth primer set for detecting oral thrush virus, the forty-fourth primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO: 146-147; Primer set 45 for detecting oropche virus, the primer set 45 consists of primers with nucleotide sequences as shown in SEQ ID NO:148-151; The 46th primer set for detecting Reston-type Ebola virus, the 46th primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO:152-155; The 47th primer set for detecting Lift Valley sandfly virus consists of primers with nucleotide sequences as shown in SEQ ID NO:156-159; The 48th primer set for detecting Sepik virus, the 48th primer set consists of primers with nucleotide sequences as shown in SEQ ID NO: 160-161; Primer set 49 for detecting St. Louis encephalitis virus, wherein the primer set 49 consists of primers with nucleotide sequences as shown in SEQ ID NO:162-163; The fiftieth primer set for detecting Venezuelan equine encephalitis virus, said fiftieth primer set consists of primers with nucleotide sequences as shown in SEQ ID NO:164-167; Primer set 51 for detecting Western equine encephalitis virus, wherein the primer set 51 consists of primers with nucleotide sequences as shown in SEQ ID NO:168-171; The 52nd primer set for detecting yellow fever virus consists of primers with nucleotide sequences as shown in SEQ ID NO: 172-173; Primer set 53 for detecting Takarib Sassavirus, wherein the primer set 53 consists of primers with nucleotide sequences as shown in SEQ ID NO:174-175; The 54th primer set for detecting smallpox virus consists of primers with nucleotide sequences as shown in SEQ ID NO: 140-141.

2. The primer set according to claim 1, characterized in that, The concentration of each primer is 0.2 μmol / L.

3. A primer pool combination for the simultaneous detection of 54 key pathogens, characterized in that, The primer pool includes: Mix1 and Mix2; The Mix1 consists of the first primer set to the twenty-seventh primer set as described in claim 1; The Mix2 consists of primer sets 28 to 54 as described in claim 1; The 54 key pathogens are: Bear Canyon Arena Virus, Machupo Arena Virus, Lassa Fever Virus, Flexo Arena Virus, Guanareto Arena Virus, Tacarib Arena Virus, Argentine Arena Virus, Brazilian Arena Virus, Xinjiang Hemorrhagic Fever Virus, Zaire Ebola Virus, Reston Ebola Virus, Great Forest Ebola Virus, Bendibugyo Ebola Virus, Marburg Virus, Nipah Virus, Hendra Virus, Merjan Virus, Venezuelan Equine Encephalitis Virus, Western Equine Encephalitis Virus, Eastern Equine Encephalitis Virus, Chikungunya Virus, Yellow Fever Virus, Khosanur Forest Disease Virus, and Omsk Hemorrhagic Fever Virus. St. Louis encephalitis virus, tick-borne encephalitis virus, dengue virus, monkeypox virus, smallpox virus, Bunianvila virus, California encephalitis virus, Dori virus, Everglades virus, foot-and-mouth disease virus, Gata virus, Hantavirus, hepatitis A virus, human gamma herpesvirus 8, West Nile virus, Japanese encephalitis virus, Sindbis virus, Mayaro virus, Middleburg virus, Mukamb virus, oral thrush virus, Sepik virus, Ilius virus, Lift Valley sandfly virus, Rochenberg virus, Powassen virus, Oropuch virus, Nairobi sheep disease virus, Ndum virus, and / or avian influenza virus type 1.

4. The primer pool combination according to claim 3, characterized in that, The concentration of each primer is 0.2 μmol / L.

5. A reagent for simultaneously detecting 54 key pathogens using multiplex PCR and nanopore sequencing technology, characterized in that, The reagent comprises the primer pool combination as described in claim 3 or 4; The 54 key pathogens are those described in claim 3.

6. A kit for simultaneously detecting 54 key pathogens using multiplex PCR and nanopore sequencing technologies, characterized in that, The kit comprises the reagent of claim 5; The 54 key pathogens are those described in claim 3.

7. A method for identifying 54 key pathogens for non-disease diagnosis purposes, or a method for detecting whether a sample contains one or more of the 54 key pathogens for non-disease diagnosis purposes, characterized in that... The method includes using multiplex PCR and nanopore sequencing technology in combination with the primer pool combination described in claim 3 or 4 to detect the bacteria to be tested or the sample to be tested. The 54 key pathogens are those described in claim 3.

8. The application of the primer set of claim 1 or 2, the primer pool combination of claim 3 or 4, the reagent of claim 5, or the kit of claim 6 in identifying 54 key pathogens in non-disease diagnosis destinations or detecting whether a sample to be tested contains 54 key pathogens in a non-disease diagnosis destination; The 54 key pathogens are those described in claim 3.

9. The application of the primer set of claim 1 or 2, the primer pool combination of claim 3 or 4, the reagent of claim 5, or the kit of claim 6 in the simultaneous detection of 54 key pathogen genes or mutation sites in 54 key pathogen genes at a non-disease diagnostic destination; The 54 key pathogens are those described in claim 3.

10. Applied to any of the following aspects: (1) The application of the primer set of claim 1 or 2, the primer pool combination of claim 3 or 4, the reagent of claim 5, or the kit of claim 6 in constructing a high-throughput amplicon sequencing library for simultaneous detection of 54 key pathogens; (2) The application of the primer set of claim 1 or 2, the primer pool combination of claim 3 or 4, the reagent of claim 5, or the kit of claim 6 in constructing a detection technology system for simultaneous detection of 54 key pathogens; The 54 key pathogens are those described in claim 3.

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