A primer set and kit for simultaneous detection of multiple pathogenic microorganisms and their application in the detection of rodent-borne pathogenic microorganisms.

Abstract

This invention belongs to the field of microbial detection technology, specifically relating to a primer set and kit for simultaneously detecting multiple pathogenic microorganisms, and its application in detecting rodent-borne pathogenic microorganisms. The nucleotide sequences of the primer set are shown in SEQ ID NO:1~SEQ ID NO:122. In each PCR detection system of this invention, when the template concentration reaches 20 copies or more, all 22 pathogenic microorganism mock plasmids can be detected, indicating that the primer set of this invention can effectively detect pathogenic microorganisms in mice and their parasites, and the detection sensitivity is comparable to that of PCR. Furthermore, the detection method of this invention has the advantages of being rapid and efficient, low-cost, having a wide and accurate detection range, and meeting the requirements of high sensitivity and low sample volume.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to a primer set and kit for simultaneously detecting multiple pathogenic microorganisms and its application in detecting rodent-borne pathogenic microorganisms. Background Technology

[0002] Rats are a significant species of disease vector, serving as hosts or vectors for numerous pathogens. More than 50 diseases are known to be directly or indirectly transmitted to humans, among which plague, hemorrhagic fever with relapse, leptospirosis, typhus, and tick-borne relapsing fever have a particularly large impact. Rats transmit diseases through three main routes: 1. Ectoparasites on rats act as vectors, transmitting pathogens to humans during bites; 2. Rats carrying pathogenic microorganisms contaminate food or water sources through their activity or feces, causing illness in humans who consume these products; 3. Infection occurs through direct bites from rats or through pathogens entering via external wounds. Therefore, accurate detection of rodent-borne pathogens is crucial for the prevention and control of infectious diseases, serving as an important means of assessing the risk of rodent-borne pathogens in a specific region and a vital component of port-of-entry vector-borne infectious disease risk control.

[0003] For rodent-borne diseases, the most commonly used method is PCR. However, PCR has limited throughput, and can only detect one or a few specific pathogens at a time, making it insufficient for detecting mixed infections or infections with unknown pathogens. Metagenomic sequencing (mNGS) is expensive, involves complex bioinformatics analysis, is difficult to interpret results, is difficult to operate, and has a long detection time, which limits its application in field laboratories and large-scale screening.

[0004] Therefore, there is an urgent need to provide a rapid, efficient, low-cost, wide-ranging, 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 this invention is to provide a primer set and kit for the simultaneous detection of multiple pathogenic microorganisms and its application in the detection of rodent-borne pathogenic microorganisms. The primer set and kit of this invention can achieve effective detection of rodent-borne pathogens, and the detection sensitivity is comparable to that of PCR.

[0006] This 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~SEQ ID NO:122.

[0007] As a preferred embodiment, the pathogenic microorganisms include one or more of the following: Hantavirus, human bocavirus, Lassa fever virus, tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, novel Bunyavirus, Langya virus, sandfly fever virus, Yersinia pestis, Leptospira, Borrelia burgdorferi, Q fever Rickettsia, Ehrlich's Rickettsia, Orientia scrub typhus, Francisella tularensis, Anaplasma phagocytophilum, Rickettsia, Brucella, Bartonella, Trypanosoma, Hennipa virus, and Leishmania.

[0008] The present invention also provides a kit for the simultaneous detection of multiple pathogenic microorganisms, the kit comprising the primer set and PCR-related reagents as described above.

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

[0010] The present invention also provides the application of the primer set for simultaneous detection of multiple pathogenic microorganisms or the kit for simultaneous detection of multiple pathogenic microorganisms in the preparation of pathogenic microorganism detection products for mice and their parasites.

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

[0012] This invention also provides a non-diagnostic method for detecting pathogens in mice and their parasites, comprising the following steps: using DNA and cDNA of the sample to be tested as templates, performing multiplex PCR amplification using primer sets for simultaneous detection of multiple pathogens as described above or kits for simultaneous detection of multiple pathogens as described above; purifying the multiplex PCR products to obtain purified PCR products; adding sample-specific barcode primers for 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 qualified libraries; and identifying the types of pathogens through bioinformatics analysis.

[0013] As a preferred embodiment, the multiplex PCR amplification reaction system, in 20 μL increments, comprises: 2-4 μL template, 4 μL of 0.1 μM primer mixture, 4 μL of 5×Multi-PCR Mix, and ddH2O to a final volume of 20 μL. The multiplex PCR amplification program is as follows: 95℃ for 3 min; 95℃ for 15 s, 58℃ for 30 s, 72℃ for 15 s, repeated 36 times; 72℃ for 3 min, and stored at 4℃.

[0014] As a preferred embodiment, the reaction system for the second round of PCR amplification, in 50 μL increments, includes: 3-5 μL of purified PCR product, 2 μL of sample-specific barcode primers, 25 μL of 2×Multiplex PCR Mix, and ddH2O to bring the total to 50 μL.

[0015] As a preferred method, the program for the second round of PCR amplification is as follows: 94℃ for 2 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, for 8 cycles; 72℃ for 5 min, and incubation at 10℃.

[0016] Beneficial Effects: This 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~SEQ ID NO:122. In each PCR detection system of this invention, when the template concentration reaches 20 copies or more, all 22 pathogenic microorganism mock plasmids can be detected, indicating that the primer set of this invention can effectively detect pathogenic microorganisms in mice and their parasites, and the detection sensitivity is comparable to that of PCR. Furthermore, the detection method of this invention has the advantages of being rapid and efficient, low-cost, having a wide and accurate detection range, and meeting the requirements of high sensitivity and low sample volume.

[0017] This invention utilizes tNGS technology to demonstrate several 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 once, such as Yersinia pestis, Hantavirus, and Borrelia burgdorferi, covering different types of pathogens such as bacteria, viruses, and fungi, and has the ability to effectively detect both common and rare pathogenic microorganisms. Through carefully designed primer sets, it can accurately identify and amplify the specific regions of the target pathogenic microorganisms. For example, primers are designed for the conserved nucleic acid sequences of different pathogens to ensure the accuracy and specificity of the detection, avoid non-specific amplification and false positive results, and provide strong support for a comprehensive understanding of the pathogen spectrum transmitted by mice and their parasites. (2) High sensitivity and low sample volume requirement: It can still achieve efficient detection with limited sample volume. Only a small amount of nucleic acid from mouse and parasite samples, such as nucleic acid in a small amount of blood, tissue fluid, or tissue grinding material, is needed to enrich the nucleic acid of the target pathogenic microorganisms through multiplex PCR amplification technology, which significantly improves 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 the sample. It is of great significance in early disease screening and monitoring. (3) Efficient human nucleic acid exclusion and cost control: In the process of library construction, the target pathogen nucleic acid is amplified by using specific primers, which greatly reduces the amplification of a large number of human nucleic acids in the sample and nucleic acids introduced by library construction reagents, and effectively reduces the proportion of human nucleic acids in the sequencing data. Compared with metagenomic sequencing (mNGS), there is no need to excessively increase the amount of sample sequencing data in order 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 nucleic acid extraction, amplification, library construction and sequencing analysis, and the operation process is relatively simple and efficient. (5) Comprehensive evaluation and verification process: The risk of multiplex PCR is that the primers in different groups may interfere with each other, or that some primer systems may not be effective enough. This invention has conducted detailed verification and evaluation of all primer pairs through reference materials, ensuring the effectiveness and accuracy of the entire primer system. (6) Optimized design of each step: Standardized reaction system and conditions significantly shorten the entire detection cycle, enabling timely detection results for disease diagnosis and prevention in a short period of time, meeting the urgent needs of clinical and epidemic prevention and control for timely detection, and helping to quickly take targeted prevention and treatment measures.

[0018] Currently available tNGS products mostly target dozens or even hundreds of clinically common pathogens, typically detecting over 95% of infectious pathogens. For primer design of specific pathogen species, the design generally uses species-specific internal control genes, sometimes increasing to two pairs of internal control gene primers. This often results in a primer mix with a number of primers far exceeding 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 detection results. Non-specific binding between primers may also occur, producing false positive results and interfering with clinical diagnosis. This invention uses as few primers as possible to cover a larger proportion of specific pathogens, offering the following advantages: Cost control: Reduces the manpower, material resources, and time costs of primer synthesis, screening, and optimization, lowering product production costs and facilitating the technology's widespread adoption in large-scale screening and primary healthcare institutions, benefiting more people. Detection efficiency and accuracy: Avoid competition between primers that may affect amplification, reduce false positive results caused by nonspecific binding, improve the accuracy and stability of detection results, and provide a more reliable basis for clinical diagnosis. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 This is a schematic diagram of the Mock plasmid sequence in Example 2;

[0021] Figure 2 This is a schematic diagram of the agarose gel electrophoresis detection of the first round of PCR products in Example 2;

[0022] Figure 3 This is a flowchart of the magnetic bead sorting operation in Example 2;

[0023] Figure 4 This is a schematic diagram of agarose gel electrophoresis detection of the first-round PCR purification product in Example 2;

[0024] Figure 5 This is a schematic diagram of the agarose gel electrophoresis detection of the second round of PCR products in Example 2;

[0025] Figure 6 This is a schematic diagram of the agarose gel electrophoresis detection of the purified product after the second round of PCR in Example 2. Detailed Implementation

[0026] This 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~SEQ ID NO:122. As a specific embodiment, the pathogenic microorganisms include one or more of the following: Hantavirus, human bocavirus, Lassa fever virus, tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, novel Bunyavirus, Langya virus, sandfly fever virus, Yersinia pestis, Leptospira, Borrelia burgdorferi, Q fever Rickettsia, Ehrlich's Rickettsia, Orientia scrub typhus, Francisella tularensis, Anaplasma phagocytophilum, Rickettsia, Brucella, Bartonella, Trypanosoma, Hennipa virus, and Leishmania.

[0027] This invention also provides a kit for the simultaneous detection of multiple pathogenic microorganisms, the kit comprising the primer set and PCR-related reagents as described above. As one specific embodiment, the PCR-related reagents include 5×Multi-PCRMix, 2×Multiplex PCR Mix, and magnetic beads for PCR product purification.

[0028] This invention also provides the application of the primer set for simultaneous detection of multiple pathogenic microorganisms or the kit for simultaneous detection of multiple pathogenic microorganisms in the preparation of pathogenic microorganism detection products for mice and their parasites. As a specific embodiment, the parasites include one or more of ticks, sand flies, fleas, and chiggers. It should be noted that the parasites described in this invention are parasites carried by mice; it can be understood that any parasite carried by a mouse is a parasite described in this invention. It can also be understood that the ticks, sand flies, fleas, and chiggers listed in this invention are only several specific embodiments that can achieve the effects of this invention, but are not limited to these types, and are not specifically limited here.

[0029] This invention also provides a non-diagnostic method for detecting pathogenic microorganisms in mice and their parasites, comprising the following steps: using DNA and cDNA of the sample to be tested as templates, performing multiplex PCR amplification (i.e., the first round of PCR) using primer sets for simultaneous detection of multiple pathogenic microorganisms as described above or kits for simultaneous detection of multiple pathogenic microorganisms as described above; purifying the multiplex PCR products to obtain purified PCR products; adding sample-specific barcode primers for the 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 qualified libraries; and identifying the types of pathogenic microorganisms through bioinformatics analysis.

[0030] In one specific embodiment, the DNA and cDNA can be obtained from the sample using commercially available kits. In another specific embodiment, the multiplex PCR amplification reaction system, in 20 μL increments, includes: 2-4 μL template, 4 μL of 0.1 μM primer mixture, 4 μL of 5×Multi-PCR Mix, and ddH2O to a final volume of 20 μL. In yet another specific embodiment, the amount of template used can be 2 μL, 3 μL, or 4 μL. The multiplex PCR amplification program is: 95℃ for 3 min; 95℃ for 15 s, 58℃ for 30 s, 72℃ for 15 s, repeated 36 times; 72℃ for 3 min, and stored at 4℃. In yet another specific embodiment, the second round of PCR amplification reaction system, in 50 μL increments, includes: 3-5 μL purified PCR product, 2 μL of sample-specific barcode primers, 25 μL of 2×Multiplex PCR Mix, and ddH2O to a final volume of 50 μL. In one specific implementation, the amount of template used can be 3 μL, 4 μL, or 5 μL. In another specific implementation, the sample-specific barcode primers are primers containing Illumina Index PCR. In the Illumina sequencing pipeline, 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 sequencer can subsequently identify different samples and complete cluster generation. In one specific implementation, the program for the second round of PCR amplification is: 94℃ for 2 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, repeated 8 times; 72℃ for 5 min, then incubated at 10℃.

[0031] The detection method described in this invention is suitable for screening vector-borne pathogens, especially for pathogen detection during public health emergencies and for annual rodent pathogen surveys organized by customs, disease control, and other organizations. Fluorescent PCR is commonly used in this field to detect vector-borne pathogens, but it can only detect a limited number of pathogens at a time. The detection method provided by this invention has high throughput, capable of detecting 22 pathogens simultaneously, and is simpler to operate and analyze than metagenomic sequencing. The detection method provided by this invention does not discover specific correlations between certain diseases or health conditions, nor does it involve any specific analytical, comparative, or other diagnostic processes or steps. The information obtained based on the detection method described in this invention cannot directly determine the diagnosis of a disease or the health condition; this information is an "intermediate result," and the corresponding detection method is not a diagnostic method for diseases, nor is it a hospital treatment.

[0032] To further illustrate the present invention, the primer set and kit for simultaneous detection of multiple pathogenic microorganisms provided by the present invention, as well as their application in detecting rodent-borne pathogenic microorganisms, are described in detail below with reference to embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0033] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0034] Example 1 Primer Design

[0035] This invention designs primers for tNGS detection targeting 22 common pathogenic microorganisms in mice and their parasites (including ticks, sand flies, fleas, chiggers, etc.) (see Table 1 for details).

[0036] Table 1. Primer information for tNGS detection of 22 pathogenic microorganisms

[0037]

[0038]

[0039]

[0040]

[0041] Example 2: Construction and Testing of "Chimeric" Plasmids

[0042] 1. Mock plasmid construction

[0043] Chimeric plasmids (mock plasmids) containing PCR products of various target genes are artificially synthesized through gene synthesis. Figure 1 For each mock plasmid, a synthetic sequence was inserted using puc18 plasmid (source leaf; S12076-20µg) as a vector: the sequences binding to tNGS primers 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) (the GC content is also similar). This was used to verify the detection sensitivity of tNGS and its resistance to background genomic DNA interference, and could also be used as a positive control in subsequent experiments.

[0044] The intermediate sequence of the target gene PCR product was replaced to avoid potential DNA template contamination and false positives when the mock plasmid was used as a positive control in the future.

[0045] Table 2. λDNA Sequence

[0046]

[0047]

[0048] 2. Template Mixing

[0049] The synthesized mock plasmids were 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 according to their DNA concentration and molecular weight. The mock plasmids were then divided into two groups: the first 11 and the second 11, with equal copy numbers, as shown in Table 2, and these were mixed into two groups for subsequent experiments. The two groups of linearized plasmids were diluted to 1000 copies / μL, 100 copies / μL, 25 copies / μL, 10 copies / μL, 5 copies / μL, and 0 copies (negative control), respectively.

[0050] After mixing all the specific primers, amplification was performed according to the following copy numbers for each target gene: 2000 copies / reaction, 200 copies / reaction, 50 copies / reaction, 20 copies / reaction, 10 copies / reaction, 5 copies / reaction, and 0 copies / reaction (2 μL template added 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 bands.

[0051] 3. tNGS amplification of the target gene

[0052] PCR system: 2 μL mouse genomic DNA (5 ng / μL), 2 μL mock template, 4 μL primer mix (primer mixture with a final concentration of 0.1 μM, as shown in SEQ ID NO:1~SEQ ID NO:122), 4 μL 5×Multi-PCRMix, and ddH2O to a final volume of 20 μL. PCR program: 95℃ for 3 min; 95℃ for 15 s, 58℃ for 30 s, 72℃ for 15 s, 36 cycles; 72℃ for 3 min, store at 4℃.

[0053] Take 3 μL of PCR product and perform electrophoresis on a 1.2% agarose gel. The results are as follows: Figure 2As shown; where the marker is DL2000, 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 the copy numbers of each mock plasmid in each primary amplification (i.e. the first round of PCR) reaction system: 1: 2000 copies / reaction, 2: 200 copies / reaction, 3: 50 copies / reaction, 4: 20 copies / reaction, 5: 10 copies / reaction, 6: 5 copies / reaction, 7: 0 copies / reaction.

[0054] 4. Magnetic bead sorting and recycling

[0055] Two tubes of primary amplification PCR products from each concentration gradient were mixed in equal volumes and recovered using magnetic beads (Yisheng; 12601ES08). The magnetic bead sorting procedure is as follows: Figure 3 The specific steps are as follows: 1) Add ultrapure water to the PCR product to a final volume of 100 μL, vortex or invert the magnetic beads thoroughly to ensure homogeneity. 2) Add 80 μL of the first round of sorting magnetic beads to the DNA solution from step 1), vortex or pipette 10 times to mix. 3) Incubate at room temperature for 5 min. 4) Briefly centrifuge the centrifuge tube and place it on a magnetic rack. After the solution has clarified (approximately 5 min), 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 aspirating the magnetic beads and affecting the sorting effect). 5) Add 25 μL of the second round of sorting magnetic beads to the supernatant. 6) Vortex or pipette 10 times to mix, and let stand at room temperature for 5 min. 7) Briefly centrifuge the centrifuge tube and place it on a magnetic rack. After the solution has clarified (approximately 5 min), carefully remove the supernatant. 8) Keep the centrifuge tube in the magnetic rack at all times. Add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 seconds, then carefully remove the supernatant. 9) Repeat step 8). 10) Keep the centrifuge tube in the magnetic rack at all times. Open the cap and dry the magnetic beads until they just begin to crack (approximately 5 minutes). 11) Remove the centrifuge tube from the magnetic rack, add an appropriate amount of ddH2O (≥20 μL), vortex or gently pipette to mix thoroughly, and incubate at room temperature for 5 minutes. 12) Briefly centrifuge the centrifuge tube and place it in the magnetic rack to separate the magnetic beads and liquid. After the solution becomes clear (approximately 5 minutes), carefully aspirate the supernatant into a clean tube. This completes the sorting. 13) Take 3 μL of the recovered PCR product and perform electrophoresis on a 1.2% agarose gel. The results are as follows: Figure 4As shown; where the marker is DL2000, the bands from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp, the loading amount is 3µL, the 750bp band is 30ng / µL, and the other bands are 10ng / µL; 1-7 represent the copy numbers of each mock plasmid in each primary expansion reaction system: 1: 2000 copies / reaction, 2: 200 copies / reaction, 3: 50 copies / reaction, 4: 20 copies / reaction, 5: 10 copies / reaction, 6: 5 copies / reaction, 7: 0 copies / reaction.

[0056] 5. Secondary amplification

[0057] PCR amplification system: 3 μL of sorted and purified PCR product, 2 μL of primers containing Illumina Index PCR, 25 μL of 2×Multiplex PCR Mix, and Nuclease-free ddH2O to a final volume of 50 μL. PCR program: 94℃ for 2 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, 8 cycles; 72℃ for 5 min, then incubate at 10℃.

[0058] In the Illumina sequencing pipeline, 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 sequencer can identify different samples and complete cluster generation.

[0059] Take 3 μL of PCR product and perform electrophoresis on a 1.2% agarose gel. The results are as follows: Figure 5 As shown; where, note: the marker is DL2000, the bands from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp, the sample loading amount is 3µL, the 750bp band is 30ng / µL, and the other bands are 10ng / µL; 1-7 represent: 1: 2000 copies / reaction, 2: 200 copies / reaction, 3: 50 copies / reaction, 4: 20 copies / reaction, 5: 10 copies / reaction, 6: 5 copies / reaction, 7: 0 copies / reaction.

[0060] 6. Secondary sorting and recycling

[0061] The recycling steps are the same as in step 4, except that the amount of magnetic beads added is slightly different. The first step is to add 80 μL, and the second step is to add 20 μL. The rest of the steps are the same.

[0062] Take 3 μL of PCR product and perform electrophoresis on a 1.2% agarose gel. The results are as follows: Figure 6 As shown; where the marker is DL2000, the bands from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp, the sample loading amount is 3µL, the 750bp band is 30ng / µL, and the other bands are 10ng / µL; 1-7 represent: 1: 2000 copies / reaction, 2: 200 copies / reaction, 3: 50 copies / reaction, 4: 20 copies / reaction, 5: 10 copies / reaction, 6: 5 copies / reaction, 7: 0 copies / reaction.

[0063] 7. Real-Time PCR Quantification

[0064] The recovered products were quantified using qPCR. The samples were mixed in equimolar ratios to complete library construction.

[0065] 8. High-throughput sequencing

[0066] The library was quantified using Qubit and its length distribution was detected using Qseq400. After the library was deemed qualified, it was sequenced using an Illumina Novaseq 6000 sequencer with an S4 chip. The sequencing process was 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 and denature the library: Mix the library with ExAmp reagents and denature it. (3) Select sequencing mode: Select "Sequence" in the software interface and then specify dual flow cell operation. (4) Load consumables: Remove the consumables left over from the previous run and load the new consumables required for the current run. (5) Set running parameters: Set the running parameters in the "Run Setup" screen. (6) Monitor the run: Monitor the run from the "Sequence" screen or use SequencingAnalysis Viewer to monitor the run from a network computer. Data will be transferred to the specified output folder. (7) Cleaning after sequencing: After sequencing is completed, the instrument will automatically start cleaning.

[0067] 9. Data Analysis

[0068] High-throughput sequencing was performed using the Illumina Novaseq 6000 S4 chip. The sequencing results were processed as follows: (1) Quality control was performed using Trimmomatic (version 0.38) software. Low-quality reads were removed by windowing. Specifically, for a 50bp window, if the average quality value within the window was less than 20, the back end bases were removed from the window. Reads below 50bp after quality control were filtered out. (2) Assembly was performed using FLASH (version 1.2.11) software. Based on the overlap relationship between PE reads, paired reads were assembled into a single sequence. The minimum overlap length was 10bp. The maximum mismatch ratio allowed in the overlap region of the assembled sequence was 0.2. Non-compliant sequences were removed. (3) The sequence was aligned with the artificially synthesized mock plasmid sequence library using blastn (version 2.9.0+). The parameter evalue was 0.00001. The screening conditions were pident>80 and qcovs>80. The sequence count for each microorganism was recorded.

[0069] 10. Result Interpretation: Users can interpret the results based on the number of sequences. For example, during initial screening, the detection of a certain number of sequences is sufficient to determine a positive result. For confirmatory experiments, a certain number of sequences need to be detected. For example, if multiple fragments of the same pathogen are detected or the number of sequences detected for a single fragment exceeds 100, it can be interpreted as a positive result.

[0070] Table 3. Pathogen names corresponding to mock fragments and the number of sequences detected by tNGS under different mock plasmid template concentration gradients.

[0071]

[0072] tNGS detection sensitivity analysis: As shown in Table 3, in each PCR detection system, when the template concentration reached 20 copies or more, all 22 pathogenic microorganism mock plasmids could be detected without any missed detections. This detection sensitivity is comparable to that of many pathogenic microorganism PCR detection reagents. Except for Hantavirus, a large number of sequences (75 to 2233 sequences) could still be detected in the other 20 pathogenic bacteria at a PCR reaction system as low as 10 copies / PCR reaction. Even at a PCR reaction system as low as 5 copies / PCR reaction, the pathogenic microorganism mock plasmid templates could still be detected, with the number of detected sequences ranging from 1 to 884. The mock plasmid templates of 8 pathogenic microorganisms were not detected. The negative control (0 copies) showed no detection of any pathogens, indicating no cross-contamination and good specificity. These results demonstrate the excellent design and amplification efficiency of tNGS primers.

[0073] Therefore, it can be seen that in each PCR detection system of this invention, when the template concentration reaches 20 copies or more, all 22 pathogenic microorganism mock plasmids can be detected. This indicates that the primer set described in this invention can effectively detect pathogenic microorganisms in mice and their parasites, and the detection sensitivity is comparable to that of PCR. Furthermore, the detection method of this invention has the advantages of being rapid and efficient, low-cost, having a wide and accurate detection range, and meeting the requirements of high sensitivity and low sample volume.

[0074] Example 3: Port Sample Testing

[0075] Twenty mouse samples captured at the port were collected. 20 mg of each of liver, kidney, lung, and pancreas tissues were placed in 1.5 mL centrifuge tubes, buffer was added, and the samples were thoroughly ground and divided into two portions for DNA and RNA extraction, respectively. The extraction reagents used were the DNeasy Blood & Tissue Kit (QIAGEN) and the RNeasy Blood & Tissue Kit (QIAGEN), respectively; specific steps were described in the kit instructions. Bartonella (Baltonella) was detected in the liver, kidney, lung, and pancreas tissues using PCR. Bartonella Yersinia pestis (Yersinia pestis) Yersinia pestis Borrelia burgdorferi () Borrelia burgdorferi ), Hooked spirochete ( Leptospira Trypanosoma ( ) Trypanosoma Nested PCR was used to detect anaplasmosis (APA) in liver, kidney, lung, and pancreas tissues. Anaplasma phagocytophilum Orientia scrub typhus ( Orientiatsutsugamushi ) and Bocavirus ( Bocavirus Hantavirus in lung tissue was detected using nested reverse transcription PCR (RT-PCR). Hantavirus ).

[0076] Simultaneously, 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 this invention. The test results were compared with the PCR results.

[0077] Table 4 Comparison of PCR results with the method of this invention

[0078]

[0079] The results in Table 4 show that, for specific pathogens, the detection results of this invention are consistent with those of the traditional PCR method.

[0080] Example 4: Port Sample Testing

[0081] Seventy-two visceral samples from mice captured at ports of entry were collected, along with 20 samples from mice captured at ports of entry. 20 mg each of liver, kidney, lung, and pancreas tissues were placed in 1.5 mL centrifuge tubes, buffer solution was added, and the samples were thoroughly ground. The mixtures were then divided into two fractions for DNA and RNA extraction. The extraction reagents used were the DNeasy Blood & Tissue Kit (QIAGEN) and the RNeasy Blood & Tissue Kit (QIAGEN), respectively; specific steps are detailed in the kit instructions. The extracted nucleic acid DNA and RNA were mixed together and detected using the method described in Example 2 of this invention.

[0082] Table 5 Positive results of port sample testing

[0083]

[0084] As can be seen from Table 5, pathogens can be directly detected in port samples using this method. The detected pathogens can be roughly determined based on the number of Reads.

[0085] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A primer set for simultaneous detection of multiple pathogenic microorganisms, characterized in that, The nucleotide sequences of the primer set are shown in SEQ ID NO:1~SEQ ID NO:122; The pathogenic microorganisms mentioned are Hantavirus, human bocavirus, Lassa fever virus, tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, novel Bunyavirus, Langya virus, sandfly fever virus, Yersinia pestis, Leptospira, Borrelia burgdorferi, Q fever Rickettsia, Ehrlichtonus, Orientia scrub typhus, Francisella tularensis, Anaplasma phagocytophilum, Rickettsia, Brucella, Bartonella, Trypanosoma, Hennipa virus, and Leishmania.

2. A kit for simultaneously detecting multiple pathogenic microorganisms, characterized in that, The kit includes the primer set and PCR-related reagents as described in claim 1; The pathogenic microorganisms mentioned are Hantavirus, human bocavirus, Lassa fever virus, tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, novel Bunyavirus, Langya virus, sandfly fever virus, Yersinia pestis, Leptospira, Borrelia burgdorferi, Q fever Rickettsia, Ehrlichtonus, Orientia scrub typhus, Francisella tularensis, Anaplasma phagocytophilum, Rickettsia, Brucella, Bartonella, Trypanosoma, Hennipa virus, and Leishmania.

3. The reagent kit according to claim 2, characterized in that, The PCR-related reagents include 5×Multi-PCRMix, 2×Multiplex PCR Mix, and magnetic beads for PCR product purification.

4. The application of the primer set according to claim 1 or the kit according to claim 2 or 3 in the preparation of detection products for pathogenic microorganisms carried by mice and their parasites; The pathogenic microorganisms mentioned are Hantavirus, human bocavirus, Lassa fever virus, tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, novel Bunyavirus, Langya virus, sandfly fever virus, Yersinia pestis, Leptospira, Borrelia burgdorferi, Q fever Rickettsia, Ehrlichtonus, Orientia scrub typhus, Francisella tularensis, Anaplasma phagocytophilum, Rickettsia, Brucella, Bartonella, Trypanosoma, Hennipa virus, and Leishmania.

5. The application according to claim 4, characterized in that, The parasites include one or more of ticks, sand flies, fleas, and chiggers.

6. A method for detecting pathogenic microorganisms in mice and their parasites for non-diagnostic purposes, characterized in that, The pathogenic microorganisms mentioned are Hantavirus, human bocavirus, Lassa fever virus, tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, novel Bunyavirus, Langya virus, sandfly fever virus, Yersinia pestis, Leptospira, Borrelia burgdorferi, Q fever Rickettsia, Ehrlichtonus, Orientia scrub typhus, Francisella tularensis, Anaplasma phagocytophilum, Rickettsia, Brucella, Bartonella, Trypanosoma, Hennipa virus, and Leishmania. The detection method includes the following steps: Using the DNA and cDNA of the sample to be tested as templates, multiplex PCR amplification was performed using the primer set described in claim 1 or the kit described in claim 2 or 3. The multiplex PCR products were purified to obtain purified PCR products. Sample-specific barcode primers were added for a second round of PCR amplification. Sequencing libraries were constructed using the recovered and purified second-round PCR amplification products. High-throughput sequencing was performed on qualified libraries, and the types of pathogenic microorganisms were identified through bioinformatics analysis.

7. The detection method according to claim 6, characterized in that, The reaction system for the multiplex PCR amplification, in 20 μL increments, includes: 2-4 μL template, 4 μL primer mixture (0.1 μM), 4 μL 5×Multi-PCR Mix, and ddH2O to bring the total volume to 20 μL. The multiplex PCR amplification program is as follows: 95℃ for 3 min; 95℃ for 15 s, 58℃ for 30 s, 72℃ for 15 s, for 36 cycles; 72℃ for 3 min, and store at 4℃.

8. The detection method according to claim 6, characterized in that, The reaction system for the second round of PCR amplification is 50 μL, including: 3-5 μL of purified PCR product, 2 μL of sample-specific barcode primers, 25 μL of 2×Multiplex PCR Mix, and ddH2O to make up to 50 μL.

9. The detection method according to claim 6, characterized in that, The second round of PCR amplification was performed as follows: 94℃ for 2 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, for 8 cycles; 72℃ for 5 min, and then incubated at 10℃.

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