Primer group and kit for simultaneously detecting multiple pathogenic microorganisms and application of primer group and kit in detection of mouse-borne pathogenic microorganisms

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a primer group and a kit for simultaneously detecting multiple pathogenic microorganisms and application of the primer group and the kit in detection of mouse-borne pathogenic microorganisms. The nucleotide sequences of the primer group are as shown in SEQ ID NO: 1 to SEQ ID NO: 122. In each PCR detection system, when the template quantity reaches 20 copies or more concentration, 22 pathogenic microorganism mock plasmids can be completely detected, which indicates that the primer group can effectively detect pathogenic microorganisms in mice and parasites carried by the mice, and the detection sensitivity is equivalent to that of PCR. Meanwhile, the detection method has the advantages of rapidness, high efficiency, low cost, wide and accurate detection range and capability of meeting the requirements of high sensitivity and low sample size.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and in particular relates to a primer set and a kit for simultaneously detecting multiple pathogenic microorganisms, and an application of the primer set and a kit in detecting mouse-borne pathogenic microorganisms. Background Art

[0002] Rats are a prominent species of disease vector, acting as reservoirs or vectors for numerous pathogens. They are known to directly or indirectly transmit over 50 diseases to humans, including plague, hemorrhagic fever, leptospirosis, murine typhoid, and tick-borne relapsing fever, which are particularly prevalent. Rats transmit diseases in three ways: 1. External parasites in rats act as vectors, transmitting pathogens to humans through bites and bloodsucking; 2. Rats harboring pathogens contaminate food or water through their activities or feces, causing illness in humans after consumption; and 3. Rats bite humans directly or pathogens enter through wounds, causing infection. Therefore, accurate detection of rat-borne pathogens is crucial for the prevention and control of infectious diseases. It is a crucial tool for regional rat-borne pathogen risk assessment and a crucial component of risk control for vector-borne diseases at ports of entry.

[0003] PCR is currently the most commonly used method for detecting rodent-borne diseases. However, PCR has limited throughput and can only detect one or a few specific pathogens at a time, making it inadequate for detecting mixed infections or infections with unknown pathogens. Metagenomic sequencing (mNGS) is also expensive, requires complex bioinformatics analysis, is difficult to interpret, requires difficult operation, and requires long testing times, limiting its application in field laboratories and large-scale screening.

[0004] Therefore, there is an urgent need to provide a fast, efficient, low-cost, wide-range and accurate detection method that meets the requirements of high sensitivity and low sample volume for high-throughput detection of rodent-borne pathogens. Summary of the Invention

[0005] The purpose of the present invention is to provide a primer set and a kit for simultaneously detecting multiple pathogenic microorganisms and their application in detecting rodent-borne pathogenic microorganisms. The primer set and kit of the present invention can effectively detect rodent-borne pathogens, and the detection sensitivity is comparable to that of PCR.

[0006] The present invention provides a primer set for simultaneously detecting multiple pathogenic microorganisms, the nucleotide sequences of the primer set are shown in SEQ ID NO: 1 to SEQ ID NO: 122.

[0007] As a preferred embodiment, the pathogenic microorganisms include: one or more of: Hantavirus, Human Bocavirus, Lassa Fever Virus, Forest Encephalitis Virus, Crimean-Congo Hemorrhagic Fever Virus, New Bunyavirus, Langya Virus, Sand Fly Fever Virus, Yersinia pestis, Leptospira, Borrelia burgdorferi, Q Fever Rickettsia, Rickettsia ersi, Orientia tsutsugamushi, Francisella tularensis, Anaplasma phagocytophilum, Rickettsia, Brucella, Bartonella, Trypanosoma, Henipavirus and Leishmania.

[0008] The present invention also provides a kit for simultaneously detecting multiple pathogenic microorganisms, wherein the kit comprises the primer set and PCR-related reagents as described above.

[0009] As a preferred solution, the PCR-related reagents include 5×Multi-PCR Mix, 2×Multiplex PCRMix and PCR product purification magnetic beads.

[0010] The present invention also provides the use of the primer set for simultaneously detecting multiple pathogenic microorganisms or the kit for simultaneously detecting multiple pathogenic microorganisms in preparing a product for detecting pathogenic microorganisms of mice and parasites carried by mice.

[0011] As a preferred embodiment, the parasites include one or more of ticks, sand flies, fleas and chiggers.

[0012] The present invention also provides a method for detecting pathogens in mice and parasites carried by them for non-diagnostic purposes, comprising the following steps: using DNA and cDNA of a sample to be tested as a template, performing multiple PCR amplification using the primer set for simultaneously detecting multiple pathogenic microorganisms as described above or the kit for simultaneously detecting multiple pathogenic microorganisms as described above, purifying the multiple PCR products to obtain purified PCR products, adding sample-specific barcode primers to perform a second round of PCR amplification; constructing a sequencing library using the recovered and purified second-round PCR amplification products, performing high-throughput sequencing on the library of qualified quality, and identifying the types of pathogenic microorganisms through bioinformatics analysis.

[0013] As a preferred embodiment, the multiplex PCR amplification reaction system, measured in 20 μL, includes: 2-4 μL of template, 4 μL of 0.1 μM primer set mixture, 4 μL of 5×Multi-PCR Mix, and ddH2O to 20 μL; the multiplex PCR amplification program is: 95°C for 3 min; 95°C for 15 s, 58°C for 30 s, 72°C for 15 s, 36 cycles; 72°C for 3 min, and storage at 4°C.

[0014] As a preferred solution, the reaction system of the second round of PCR amplification is 50 μL, including: 3-5 μL of purified PCR product, 2 μL of sample-specific barcode primer, 25 μL of 2×Multiplex PCR Mix, and ddH2O to make up to 50 μL.

[0015] As a preferred solution, the program of the second round of PCR amplification is: 94°C for 2 min; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 8 cycles; 72°C for 5 min, and insulation at 10°C.

[0016] Beneficial Effects: The present invention provides a primer set for the simultaneous detection of multiple pathogenic microorganisms, the nucleotide sequences of which are shown in SEQ ID NO:1 to SEQ ID NO:122. In each PCR detection system of the present invention, when the template amount reached a concentration of 20 copies or above, all 22 pathogenic microorganism mock plasmids were detected, demonstrating that the primer set of the present invention can effectively detect pathogenic microorganisms in mice and the parasites they carry, with detection sensitivity comparable to that of PCR. The detection method of the present invention is rapid and efficient, low-cost, and has a wide and accurate detection range, meeting the requirements of high sensitivity and low sample volume.

[0017] The present invention uses tNGS technology to demonstrate significant advantages in the detection of pathogenic microorganisms in mice and their parasites: (1) Wide and accurate detection range: It can detect a variety of pathogenic microorganisms closely related to mice and their parasites at one time, such as Yersinia pestis, Hantavirus, Borrelia burgdorferi, etc., covering different types of pathogens such as bacteria, viruses, and fungi, and has effective detection capabilities for both common and rare pathogenic microorganisms. Through carefully designed primer sets, it can accurately identify and amplify specific regions of target pathogenic microorganisms, such as designing primers for conserved nucleic acid sequences of different pathogens, ensuring the accuracy and specificity of detection, avoiding nonspecific amplification and false positive results, and providing strong support for a comprehensive understanding of the spectrum of pathogens transmitted by mice and their parasites. (2) High sensitivity and low sample volume requirements: Efficient detection can still be achieved even with limited sample volume. Only a small amount of nucleic acid from mouse and parasite samples, such as a small amount of nucleic acid in blood, tissue fluid or tissue grinds, is required to enrich the target pathogenic microorganism nucleic acid through multiple PCR amplification technology, significantly improving the detection sensitivity. Compared with traditional detection methods, it can more sensitively capture low-load pathogens and effectively avoid the problem of missed detection due to low pathogen content in samples. It is of great significance in early screening and monitoring of diseases. (3) Efficient exclusion of human nucleic acids and cost control: During the library construction process, the nucleic acid of the target pathogen is targeted and amplified with the help of specific primers, which greatly reduces the amplification of a large amount of human nucleic acid in the sample and nucleic acid introduced by the library construction reagents, and effectively reduces the proportion of human nucleic acid in the sequencing data. Compared with metagenomic sequencing (mNGS), there is no need to excessively increase the amount of sample sequencing data to improve the sensitivity of pathogen detection. While ensuring the accuracy of detection, it significantly reduces the detection cost and improves the detection efficiency, making large-scale sample detection and clinical application more feasible. (4) Fast and efficient detection process: It integrates the links of nucleic acid extraction, amplification, library construction and sequencing analysis, and the operation process is relatively simple and efficient. (5) Complete evaluation and verification process: The risk of the multiplex PCR method is that the primers in each group may affect each other, or the detection effectiveness of some primer systems may be insufficient. The present invention conducts detailed verification and evaluation of all primer pairs using reference substances, ensuring the effectiveness and accuracy of its entire primer system. (6) Optimization design of each step: For example, standardized reaction systems and conditions can significantly shorten the entire detection cycle, providing timely test results for disease diagnosis and prevention and control in a relatively short period of time, meeting the urgent needs of clinical and epidemic prevention and control for detection timeliness, and facilitating the rapid implementation of targeted prevention and treatment measures.

[0018] Most of the tNGS products currently on the market target dozens or even hundreds of common clinical pathogens and can typically detect over 95% of infectious pathogens. When designing primers for specific pathogenic species, the design process typically involves selecting a reference gene unique to that species. Sometimes, up to two pairs of reference gene primers may be used. This results in the final primer mix often containing far more primers than the required number of species. Excessive primers may compete with each other in the reaction system, affecting amplification and reducing the accuracy and stability of the test results. Nonspecific binding may also occur between primers, resulting in false-positive results and interfering with clinical diagnosis. The present invention, however, uses a minimal number of primers to cover a greater proportion of specific pathogenic microorganisms, offering the following advantages: Cost control: This reduces the human, material, and time costs of primer synthesis, screening, and optimization, lowering product production costs and facilitating the promotion of the technology in large-scale screening and primary healthcare institutions, benefiting more people. Detection efficiency and accuracy: Avoid competition between primers that affects the amplification effect, reduce false positive results caused by non-specific binding, improve the accuracy and stability of test results, and provide a more reliable basis for clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0020] Figure 1 Schematic diagram of the Mock plasmid sequence in Example 2; Figure 2 This is a schematic diagram of agarose gel electrophoresis detection of the first-round PCR products in Example 2; Figure 3 This is a flow chart of the magnetic bead sorting operation in Example 2; Figure 4 This is a schematic diagram of agarose gel electrophoresis detection of the products after the first round of PCR purification in Example 2; Figure 5 This is a schematic diagram of agarose gel electrophoresis detection of the second-round PCR products in Example 2; Figure 6 Schematic diagram of agarose gel electrophoresis detection of the products after the second round of PCR purification in Example 2. DETAILED DESCRIPTION

[0021] The present invention provides a primer set for simultaneously detecting multiple pathogenic microorganisms, the nucleotide sequences of the primer set being shown in SEQ ID NOs: 1 to 122. In a specific embodiment, the pathogenic microorganisms include one or more of: hantavirus, human bocavirus, Lassa fever virus, forest encephalitis virus, Crimean-Congo hemorrhagic fever virus, novel bunyavirus, Langya virus, sandfly fever virus, Yersinia pestis, Leptospira, Borrelia burgdorferi, Q fever Rickettsia, Rickettsia ersi, Orientia tsutsugamushi, Francisella tularensis, Anaplasma phagocytophilum, Rickettsia, Brucella, Bartonella, Trypanosoma, Henipavirus, and Leishmania.

[0022] The present invention also provides a kit for simultaneously detecting multiple pathogenic microorganisms, the kit comprising the above-described primer set and PCR-related reagents. In a specific embodiment, the PCR-related reagents include 5×Multi-PCR Mix, 2×Multiplex PCR Mix, and PCR product purification magnetic beads.

[0023] The present invention also provides the use of the primer set or kit for simultaneously detecting multiple pathogenic microorganisms in preparing a product for detecting pathogenic microorganisms in mice and parasites carried by them. In one embodiment, the parasites include one or more of ticks, sand flies, fleas, and chiggers. It should be noted that the parasites described herein are parasites carried by mice, and it should be understood that any parasite carried by mice is a parasite described herein. It should be understood that the ticks, sand flies, fleas, and chiggers listed herein are merely a few specific embodiments that can achieve the effects of the present invention, but are not limited to these types and are not specifically defined herein.

[0024] The present invention also provides a non-diagnostic method for detecting pathogenic microorganisms in mice and parasites carried by them, comprising the following steps: using DNA and cDNA of a sample to be tested as a template, performing multiplex PCR amplification (i.e., first-round PCR) using the primer set for simultaneously detecting multiple pathogenic microorganisms as described above or the kit for simultaneously detecting multiple pathogenic microorganisms as described above, purifying the multiplex PCR products to obtain purified PCR products, adding sample-specific barcode primers to perform a second-round PCR amplification; constructing a sequencing library using the recovered and purified second-round PCR amplification products, performing high-throughput sequencing on the library of qualified quality, and identifying the types of pathogenic microorganisms through bioinformatics analysis.

[0025] As a specific embodiment, the DNA and cDNA can be obtained from the sample using a commercially available kit. As a specific embodiment, the reaction system for the multiplex PCR amplification, measured in 20 μL, includes: 2-4 μL of template, 4 μL of a 0.1 μM primer set mixture, 4 μL of a 5×Multi-PCR Mix, and ddH2O to make up to 20 μL. As a specific embodiment, the amount of the template used can be 2 μL, 3 μL, or 4 μL. The program for the multiplex PCR amplification is: 95°C for 3 minutes; 95°C for 15 seconds, 58°C for 30 seconds, 72°C for 15 seconds, 36 cycles; 72°C for 3 minutes, and storage at 4°C. As a specific embodiment, the reaction system for the second round of PCR amplification, measured in 50 μL, includes: 3-5 μL of purified PCR product, 2 μL of sample-specific barcode primers, 25 μL of a 2×Multiplex PCR Mix, and ddH2O to make up to 50 μL. In a specific embodiment, the template volume can be 3 μL, 4 μL, or 5 μL. In a specific embodiment, the sample-specific barcode primers are primers containing Illumina Index PCR. In the Illumina sequencing process, the core purpose of the second round of PCR amplification (also known as "Index PCR") is to add sample-specific index sequences and universal adapters to the library, so that the subsequent sequencer can identify different samples and complete cluster generation. In a specific embodiment, the second round of PCR amplification is as follows: 94°C for 2 minutes; 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, 8 cycles; 72°C for 5 minutes, and incubation at 10°C.

[0026] The detection method of the present invention is suitable for screening of pathogens carried by vectors, and is particularly suitable for pathogen detection in public health emergencies and annual surveys of rodent-borne pathogens organized by customs, disease control, etc. In this field, fluorescent PCR is commonly used to detect pathogens carried by vectors, but the fluorescent PCR method can only detect a small number of pathogens at a time. The detection method provided by the present invention has a high throughput and can detect 22 pathogens at a time. At the same time, the operation is simpler than metagenomic sequencing, and the data analysis is simple. The detection method provided by the present invention is not to discover the specific correlation between certain specific and disease or health conditions, nor does it involve any specific analysis, comparison and other diagnostic processes and steps. The information obtained based on the detection method of the present invention cannot directly determine the diagnostic results of the disease or the health status. The information belongs to the "intermediate result", and the corresponding detection method does not belong to the diagnosis method of the disease, and is not a hospital diagnosis and treatment.

[0027] To further illustrate the present invention, the following detailed description of a primer set and a kit for simultaneously detecting multiple pathogenic microorganisms provided by the present invention and their application in detecting mouse-borne pathogenic microorganisms is given in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention.

[0028] Unless otherwise specified, the present invention has no special requirements for the raw materials, and commercially available products known to those skilled in the art can be used.

[0029] Example 1 Primer Design In the present invention, primers for tNGS detection were designed for 22 common pathogenic microorganisms in mice and the parasites they carry (including ticks, sand flies, fleas, chiggers, etc.) (see Table 1 for details).

[0030] Table 1 tNGS detection primer information for 22 pathogenic microorganisms

[0031]

[0032]

[0033]

[0034] Example 2 Construction and testing of "chimera" plasmids 1.Mock plasmid construction Artificially synthesize "chimera" plasmids containing PCR products of each target gene by gene synthesis (mock plasmid, see Figure 1 ): Each mock plasmid, using the puc18 plasmid (source leaf; S12076-20µg) as a vector, was inserted with a synthetic sequence: the sequences for tNGS primer binding were retained at both ends, but the sequence in the middle of the PCR product was replaced with a Lamda DNA (λDNA) sequence of similar length (see Table 2, the λDNA sequence does not contain forward and reverse specific primers) (GC content is also similar), which is used to verify the detection sensitivity of tNGS and its resistance to background genomic DNA interference, and can be used as a positive control in subsequent experiments.

[0035] The purpose of replacing the middle sequence of the target gene PCR product is to avoid potential DNA template contamination in sample testing when the mock plasmid is used as a positive control in the future, which may cause false positives.

[0036] Table 2 λDNA sequence

[0037]

[0038] 2. Template Mixing The mock plasmid synthesized above was linearized using BamHI (Takara; 1010S). After agarose gel extraction and quantification using Qubit 3.0, the linearized plasmids were diluted to 10,000 copies / μL based on DNA concentration and molecular weight. The mock plasmids were then divided into two groups: the first 11 and the last 11, with equal copy numbers as shown in Table 2. The two groups were then combined for subsequent experiments. The linearized plasmids in these two groups were diluted to 1,000 copies / μL, 100 copies / μL, 25 copies / μL, 10 copies / μL, 5 copies / μL, and 0 copies (negative control), respectively.

[0039] After mixing all specific primers, amplify the target gene at 2000 copies / reaction, 200 copies / reaction, 50 copies / reaction, 20 copies / reaction, 10 copies / reaction, 5 copies / reaction, and 0 copies / reaction (add 2 μL of template to each reaction). In addition, 10 ng of mouse genomic DNA was added to the reaction system to simulate interference from host DNA on the amplification of the target band.

[0040] 3. tNGS Amplification of Target Gene PCR system: 2 μL of mouse genomic DNA (5 ng / μL), 2 μL of mock template, 4 μL of Primer Mix (final concentration 0.1 μM, such as the primer mixture shown in SEQ ID NO:1 to SEQ ID NO:122), 4 μL of 5× Multi-PCR Mix, and ddH2O to 20 μL. PCR program: 95°C for 3 min; 36 cycles of 95°C for 15 s, 58°C for 30 s, and 72°C for 15 s; 72°C for 3 min, then store at 4°C.

[0041] Take 3 μL of PCR product and perform electrophoresis on 1.2% agarose gel. Figure 2 As shown in the figure; the marker is DL2000, and the bands from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp, respectively. The loading volume is 3µL, the 750bp band is 30ng / µL, and the remaining bands are 10ng / µL. 1-7 represent the copies of each mock plasmid in each amplification (i.e., first-round PCR) reaction system: 1:2000copies / reaction, 2:200copies / reaction, 3:50copies / reaction, 4:20copies / reaction, 5:10copies / reaction, 6:5copies / reaction, 7:0copies / reaction.

[0042] 4. Magnetic bead separation and recovery The PCR products of each concentration gradient were amplified in two tubes at equal volumes and recovered using magnetic beads (Yisheng; 12601ES08). Figure 3 As shown, the specific steps are as follows: 1) Make up to 100 μL of PCR product with ultrapure water and vortex or invert the beads thoroughly to ensure uniform mixing. 2) Add 80 μL of the first-round sorting magnetic beads to the DNA solution in step 1) and vortex or pipette up and down 10 times to mix thoroughly. 3) Incubate at room temperature for 5 minutes. 4) Briefly centrifuge the tube and place it on a magnetic rack. After the solution has cleared (approximately 5 minutes), carefully transfer the supernatant to a clean centrifuge tube. (When transferring the supernatant, leave 2 μL of liquid at the bottom of the tube. Do not aspirate all of the supernatant to avoid attracting the beads and affecting the sorting efficiency.) 5) Add 25 μL of the second-round sorting magnetic beads to the supernatant. 6) Vortex or pipette up and down 10 times to mix thoroughly. Let it stand at room temperature for 5 minutes. 7) Briefly centrifuge the tube and place it on a magnetic rack. After the solution has cleared (approximately 5 minutes), carefully remove the supernatant. 8) Keep the centrifuge tube in the magnetic rack, add 200μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30s, and carefully remove the supernatant. 9) Repeat step 8). 10) Keep the centrifuge tube in the magnetic rack, open the lid and dry the magnetic beads until cracks just appear (about 5 minutes). 11) Remove the centrifuge tube from the magnetic rack, add an appropriate amount of ddH2O (≥20μL), vortex or use a pipette to gently blow to mix thoroughly, and incubate at room temperature for 5 minutes. 12) Centrifuge the centrifuge tube briefly and place it in a magnetic rack to separate the magnetic beads and liquid. After the solution is clarified (about 5 minutes), carefully pipette the supernatant into a clean tube to complete the sorting. 13) Take 3μL of the recovered PCR product and detect it by 1.2% agarose gel electrophoresis. The results are as follows Figure 4 As shown in the figure; the marker is DL2000, and the bands from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp, respectively. The loading volume is 3µL, the 750bp band is 30ng / µL, and the remaining bands are 10ng / µL; 1-7 represent the copies of each mock plasmid in each amplification reaction system: 1:2000copies / reaction, 2:200copies / reaction, 3:50copies / reaction, 4:20copies / reaction, 5:10copies / reaction, 6:5copies / reaction, 7:0copies / reaction.

[0043] 5. Secondary Amplification PCR amplification system: 3 μL of the purified PCR product was added to 2 μL of primers containing Illumina Index PCR, 25 μL of 2× Multiplex PCR Mix, and made up to 50 μL with nuclease-free ddH2O. PCR program: 94°C for 2 min; 8 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; 72°C for 5 min, and incubation at 10°C.

[0044] In the Illumina sequencing process, the core purpose of the second round of PCR amplification (also known as "Index PCR") is to add sample-specific index sequences and universal adapters to the library so that subsequent sequencers can identify different samples and complete cluster generation.

[0045] Take 3 μL of PCR product and perform electrophoresis on 1.2% agarose gel. Figure 5 As shown; Note: The marker is DL2000, and the bands from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp, the loading volume is 3µL, the 750bp band is 30ng / µL, and the other bands are 10ng / µL; 1-7 represent: 1:2000copies / reaction, 2:200copies / reaction, 3:50copies / reaction, 4:20copies / reaction, 5:10copies / reaction, 6:5copies / reaction, 7:0copies / reaction, respectively.

[0046] 6. Secondary sorting and recycling The recovery steps refer to step 4, with a slightly different amount of magnetic beads added: 80 μL is added in the first step and 20 μL is added in the second step. The remaining steps remain the same.

[0047] Take 3 μL of PCR product and perform electrophoresis on 1.2% agarose gel. Figure 6 As shown; the marker is DL2000, and the bands from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp, the loading volume is 3µL, the 750bp band is 30ng / µL, and the other bands are 10ng / µL; 1-7 represent: 1:2000copies / reaction, 2:200copies / reaction, 3:50copies / reaction, 4:20copies / reaction, 5:10copies / reaction, 6:5copies / reaction, 7:0copies / reaction, respectively.

[0048] 7. Real-Time PCR Quantification The recovered products were quantified by qPCR, and the samples were mixed in an equimolar ratio to complete library construction.

[0049] 8. High-throughput sequencing The library was quantified using Qubit and the length distribution of the library was detected using Qseq400. After the library was judged to be qualified, it was sequenced on the Illumina Novaseq 6000 sequencer using the S4 chip. The sequencing process is as follows: (1) Prepare SBS and cluster generation reagent cartridges: First, the SBS (Sample Buffer Solution) and cluster generation reagent cartridges need to be thawed. (2) Mix the library and denature: Mix the library with ExAmp reagent and denature it. (3) Select the sequencing mode: Select "Sequence" on the software interface and specify a dual flow cell run. (4) Load consumables: Remove the consumables left over from the previous run and load the new consumables required for the current run. (5) Set the run parameters: Set the run parameters in the "Run Setup" screen. (6) Monitor the run: Monitor the run from the "Sequence" screen or use Sequencing Analysis Viewer to monitor the run from a network computer. The data will be transferred to the specified output folder. (7) Cleaning after sequencing is completed: After sequencing is completed, the instrument will automatically start cleaning.

[0050] 9. Data Analysis High-throughput sequencing was performed using the S4 chip model of the Illumina Novaseq 6000. The sequencing results were processed as follows: (1) Quality control was performed using Trimmomatic (version 0.38) software, using a window-based low-quality removal method. The specific operation was as follows: for a 50-bp window, if the average quality value within the window was less than 20, the back-end bases were truncated from the window, and reads less than 50 bp after quality control were filtered; (2) Splicing was performed using FLASH (version 1.2.11) software based on the overlap relationship between PE reads, merging paired reads into a single sequence. The minimum overlap length was 10 bp, and the maximum mismatch ratio allowed in the overlap region of the spliced ​​sequence was 0.2. Incompatible sequences were removed. (3) Blastn (version 2.9.0+) was used to align with a synthetic mock plasmid sequence library with the parameter evalue = 0.00001 and the screening conditions pident>80 and qcovs>80. The number of sequences for each microorganism was counted.

[0051] 10. Result Interpretation: Users can interpret results based on sequence counts. For example, in initial screening, a positive result can be determined by detecting a certain number of sequences. For confirmatory testing, a positive result can be determined by detecting a certain number of sequences. For example, if multiple fragments of the same pathogen are detected, or if the number of sequences detected for a single fragment exceeds 100, the result can be determined as positive.

[0052] Table 3 Pathogen names corresponding to mock fragments and statistics of sequence numbers detected by tNGS under different mock plasmid template concentration gradients

[0053] tNGS Assay Sensitivity Analysis: Table 3 shows that in each PCR assay, all 22 pathogen mock plasmids were detected at template concentrations of 20 copies or greater, with no missed detections. This sensitivity rivals that of many pathogen PCR assays. With the exception of hantavirus, a significant number of sequences (75 to 2233) were detected for the other 20 pathogens at concentrations as low as 10 copies / PCR reaction. Detection of mock plasmid templates for pathogens at concentrations as low as 5 copies / PCR reaction ranged from 1 to 884 sequences. Eight pathogen mock plasmid templates were not detected. In the negative control (0 copies), no pathogens were detected, indicating no cross-contamination and good specificity. These results demonstrate the excellent tNGS primer design and amplification efficiency.

[0054] As shown, in each PCR detection system of the present invention, all 22 pathogenic microorganism mock plasmids were detected when the template amount reached a concentration of 20 copies or above, demonstrating that the primer sets of the present invention can effectively detect pathogenic microorganisms in mice and their parasites, with detection sensitivity comparable to that of PCR. Furthermore, the detection method of the present invention is rapid and efficient, low-cost, and has a wide and accurate detection range, meeting the requirements of high sensitivity and low sample volume.

[0055] Example 3 Port sample testing Twenty samples of mice captured at the port were collected. 20 mg of each of the liver, kidney, lung, and pancreas tissues were placed in a 1.5 mL centrifuge tube. Buffer was added, the tissues were thoroughly ground, and the samples were divided into two parts for DNA and RNA extraction. The extraction reagents used were the DNeasy Blood & Tissue Kit (QIAGEN) and the RNeasy Blood & Tissue Kit (QIAGEN), respectively. For specific steps, refer to the kit instructions. PCR was used to detect Bartonella ( Bartonella ), Yersinia pestis ( Yersinia pestis ), Borrelia burgdorferi ( Borrelia burgdorferi ), Leptospira ( Leptospira ), Trypanosomes ( Trypanosoma ), using nested PCR to detect Anaplasma phagocytophilum in liver, kidney, lung and pancreas tissues ( Anaplasma phagocytophilum ), Orientia tsutsugamushi ( Orientiatsutsugamushi ) and Bocavirus ( Bocavirus ), and the hantavirus in lung tissue was detected by nested reverse transcription PCR (RT-PCR) method ( Hantavirus ).

[0056] At the same time, 10 μL of DNA and RNA extracted from the visceral tissue of each sample were mixed together and tested using the method of Example 2 of the present invention. The test results were compared with the PCR results.

[0057] Table 4 Comparison of PCR results with the method of the present invention

[0058] From the results in Table 4, it can be seen that for specific pathogens, the detection results of the present invention are consistent with those of the traditional PCR method.

[0059] Example 4 Port sample testing 72 samples of port-caught mouse viscera and 20 samples of port-caught mice were collected. 20 mg each of liver, kidney, lung, and pancreas tissues were placed in a 1.5 mL centrifuge tube, buffer was added, and the samples were thoroughly ground. DNA and RNA were then extracted from the two samples separately. The DNeasy Blood & Tissue Kit (QIAGEN) and RNeasy Blood & Tissue Kit (QIAGEN) were used for extraction, respectively. For specific procedures, refer to the kit instructions. The extracted DNA and RNA were mixed and tested using the method of Example 2 of the present invention.

[0060] Table 5 Positive results of port sample testing

[0061] As can be seen from Table 5, pathogens can be directly detected in port samples using this method. The detected pathogens can be determined based on the number of reads, and the pathogen content can also be roughly determined based on the number of reads.

[0062] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A primer set for simultaneously detecting multiple pathogenic microorganisms, characterized in that: The nucleotide sequences of the primer set are shown in SEQ ID NO: 1 to SEQ ID NO:

122.

2. The primer set according to claim 1, characterized in that The pathogenic microorganisms include: one or more of: Hantavirus, human bocavirus, Lassa fever virus, forest encephalitis virus, Crimean-Congo hemorrhagic fever virus, new Bunyavirus, Langya virus, sand fly fever virus, plague bacillus, Leptospira, Borrelia burgdorferi, Q fever Rickettsia, Ehrlichia, Orientia tsutsugamushi, Francisella tularensis, Anaplasma phagocytophilum, Rickettsia, Brucella, Bartonella, Trypanosoma, Henipavirus and Leishmania.

3. A kit for simultaneously detecting multiple pathogenic microorganisms, characterized in that: The kit comprises the primer set according to claim 1 and PCR-related reagents.

4. The kit according to claim 3, wherein The PCR-related reagents include 5×Multi-PCR Mix, 2×Multiplex PCR Mix and PCR product purification magnetic beads.

5. Use of the primer set according to claim 1 or 2 or the kit according to claim 3 or 4 in preparing a product for detecting pathogenic microorganisms in mice and parasites carried by them.

6. The use according to claim 5, characterized in that The parasites include one or more of ticks, sand flies, fleas and chiggers.

7. A method for detecting pathogenic microorganisms in mice and parasites carried by mice for non-diagnostic purposes, characterized in that: The following steps are involved: Using the DNA and cDNA of the sample to be tested as a template, multiplex PCR amplification is performed using the primer set of claim 1 or 2 or the kit of claim 3 or 4, the multiplex PCR products are purified to obtain the purified PCR products, and sample-specific barcode primers are added to perform a second round of PCR amplification; The recovered and purified second-round PCR amplification products were used to construct sequencing libraries. The libraries with qualified quality were subjected to high-throughput sequencing, and the types of pathogenic microorganisms were identified through bioinformatics analysis.

8. The detection method according to claim 7, characterized in that The multiplex PCR amplification reaction system is 20 μL, including: 2-4 μL of template, 4 μL of 0.1 μM primer set mixture, 4 μL of 5×Multi-PCR Mix, and ddH2O to make up to 20 μL; The program of the multiplex PCR amplification was as follows: 95° C. for 3 min; 95° C. for 15 s, 58° C. for 30 s, 72° C. for 15 s, 36 cycles; 72° C. for 3 min, and storage at 4° C.

9. The detection method according to claim 7, characterized in that The reaction system of the second round of PCR amplification is 50 μL, including: 3-5 μL of purified PCR product, 2 μL of sample-specific barcode primer, 25 μL of 2× Multiplex PCR Mix, and ddH2O to make up to 50 μL.

10. The detection method according to claim 7, characterized in that: The program of the second round of PCR amplification was as follows: 94°C for 2 min; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 8 cycles; 72°C for 5 min, and insulation at 10°C.

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