A high-throughput multiplex real-time fluorescent quantitative detection method suitable for 25 tick-borne pathogens and application

Abstract

The application discloses a high-throughput multiplex real-time fluorescent quantitative detection method and application suitable for 25 kinds of tick-borne pathogens, and the method comprises primer probe design, fluorescent quantitative PCR reaction condition and performance and sample nucleic acid extraction method. The high-throughput multiplex real-time fluorescent quantitative detection method and application suitable for 25 kinds of tick-borne pathogens construct a multiplex real-time fluorescent quantitative PCR detection system covering 25 kinds of main tick-borne pathogens. According to extensive epidemiological investigation and fine experimental optimization, the system scientifically distributes the pathogens into 8 independent and non-interfering multiplex reaction units, successfully solves the problems of competitive inhibition and non-specific amplification which are prone to occur in multiplex TaqMan qPCR through systematic optimization of primer probe combination and reaction condition, and realizes one-off, high-throughput and high-sensitivity pathogen broad-spectrum screening.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a high-throughput multiplex real-time fluorescence quantitative detection method and its application applicable to 25 tick-borne pathogens. Background Technology

[0002] Tick-borne diseases (TBDs) are a group of zoonotic diseases transmitted by tick bites. In recent years, with global ecological changes and increased human outdoor activities, the distribution range of tick-borne pathogens has been expanding, and new and emerging tick-borne infectious diseases have frequently occurred. The existing spectrum of tick-borne pathogens is extremely complex, encompassing not only tick-borne encephalitis virus (TBEV) and Lyme disease spirochetes (… Borrelia burgdorferi Traditional pathogens include Severe fever with thrombocytopenia syndrome virus (SFTSV), Alongshan virus (ALSV), and Wetland virus (WELV), among other emerging pathogens.

[0003] Clinically, early symptoms of tick-borne diseases often manifest as fever, headache, and muscle aches, lacking specificity and difficult to distinguish from other febrile illnesses, easily leading to misdiagnosis. Traditional pathogen detection methods have many limitations: pathogen culture is time-consuming and has a low positive rate; serological testing has a window period and is prone to cross-reactivity. Although singleton PCR technology has high sensitivity, testing each tick-borne pathogen individually is not only costly and inefficient, but also requires a large number of samples, given the wide variety of tick-borne pathogens, often with mixed infections. While next-generation sequencing (NGS) technology has a wide range of applications, its high cost and complex bioinformatics analysis limit its application in primary healthcare and large-scale infectious disease screening.

[0004] Therefore, in order to address the challenges of the diversity of tick-borne pathogens, the difficulty of clinical diagnosis, and the limitations of existing detection technologies, there is an urgent need to develop a high-throughput nucleic acid detection method that can rapidly, sensitively, specifically, and simultaneously detect multiple major tick-borne pathogens (TBBs) applicable to the three major categories of viruses, bacteria, and parasites, in order to meet the needs of clinical differential diagnosis and large-scale epidemiological surveillance in the public health field. Summary of the Invention

[0005] Based on the technical problems existing in the prior art, the present invention provides a high-throughput multiplex real-time fluorescence quantitative detection method and application applicable to 25 tick-borne pathogens, which relates to a pathogenic microorganism nucleic acid detection composition and method, specifically to a high-throughput multiplex real-time fluorescence quantitative PCR (Multiplex Real-time PCR) detection technology applicable to 25 major tick-borne pathogens.

[0006] According to a first aspect of the present invention, a high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens is provided, comprising the following steps: Step S1: Establish a high-throughput multiplex detection system applicable to 25 major tick-borne pathogens; In step S2, the concentrations of primers and probes were systematically optimized, and universal thermal cycling conditions were established to obtain positive plasmid standards for each target. Step S3 establishes standardized pretreatment and nucleic acid extraction procedures for different types of clinical and field samples; Step S4: Using the target positive plasmid standards prepared in step S2, prepare plasmids with concentrations suitable for a range of 10 using a 10-fold serial dilution method. 4 Up to 10 8 Five orders of magnitude series of standards were prepared in copies / mL. The standards at each concentration were amplified and detected according to the established multiplex qPCR reaction system and procedure. Three replicate wells were set for each concentration gradient to eliminate sample loading and operation errors. Step S5: Select 60 pathogen nucleic acid samples preserved in the laboratory for rigorous exclusion testing; Step S6: Select two matrices, pathogen-free tick pool and negative human EDTA whole blood, to determine LOD, in order to simulate the real clinical and field sample environment; Step S7: Evaluate the stability of high-throughput multiplex real-time fluorescence quantitative PCR for 25 tick-borne pathogens under different detection environments; Step S8 is a positive verification of the high-throughput multiplex real-time fluorescence quantitative detection of 25 tick-borne pathogens in real samples.

[0007] Preferably, step S1 includes designing and screening strategies and primer evaluation and optimization. The design and screening strategies are based on the pathogen whole genome sequences published in the NCBI GenBank database, and highly conserved regions of each pathogen are identified through bioinformatics analysis. Multiple sets of candidate primers and probes are designed and screened in the highly conserved regions using Primer Express 3.0 software. After evaluation and optimization, sequences with severe hairpin structures, primer dimers, and non-specific binding are eliminated. Finally, the optimal primer-probe combination is selected by specificity comparison using BLASTn in the NCBI database.

[0008] Preferably, in step S1, the grouping scheme of the eight reaction units and the primer and probe sequences of each target are determined. Step S2 further includes the preparation of plasmid standards, the construction of the reaction system, the determination of the reaction procedure, and the optimization of the reaction system. Preferably, step S3 further includes sample grouping and cleaning, and nucleic acid extraction. Preferably, in step S5, 154 pathogen test samples stored in the laboratory test sample library are selected for exclusion testing, and all experimental operations involving pathogens are completed in the biosafety level 2 laboratory of this laboratory.

[0009] Furthermore, in step S6, two matrices, pathogen-free tick pools and negative human EDTA whole blood, are selected to determine the LOD (Level of Detection) to simulate real clinical and field sample environments. Even further, in step S7, two concentrations of simulated positive whole blood samples are selected as test subjects. For intra-batch precision, 20 repeated tests are performed on each of the two concentrations within a single experimental batch. For inter-batch precision, two different operators perform a total of 20 repeated tests on three different experimental dates using different batches of reagent kits.

[0010] According to a second aspect of the present invention, a kit is provided for simultaneously detecting primer and probe combinations for BJNV, JMTV, and XCV in a sample using a real-time fluorescence PCR method. The kit employs the aforementioned high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens. The primer and probe combination includes primers and probes for BJNV, JMTV, and XCV. Specifically, the upstream primer gene sequence for BJNV is SEQ ID NO.1, the downstream primer gene sequence is SEQ ID NO.2, and the probe gene sequence is SEQ ID NO.3; the upstream primer gene sequence for JMTV is SEQ ID NO.4, the downstream primer gene sequence is SEQ ID NO.5, and the probe gene sequence is SEQ ID NO.6; and the upstream primer gene sequence for XCV is SEQ ID NO.7, the downstream primer gene sequence is SEQ ID NO.8, and the probe gene sequence is SEQ ID NO.9.

[0011] According to a third aspect of the present invention, a method for simultaneously detecting [a substance] in a sample using quantitative real-time PCR is provided. Bartonella spp.、 Borrelia A kit containing primers and probes for spp., TBEV, and YEZV, employing the aforementioned high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens, wherein the primer and probe combinations include those targeting spp., TBEV, and YEZV. Bartonella Primers and probes for spp., targeting BorreliaPrimers and probes for spp., primers and probes for TBEV, and primers and probes for YEZV; among which, primers and probes for spp. Bartonella The upstream primer gene sequence of spp. is SEQ ID NO. 10, the downstream primer gene sequence is SEQ ID NO. 11, and the probe gene sequence is SEQ ID NO. 12; targeting Borrelia The upstream primer gene sequence for spp. is SEQ ID NO.13, the downstream primer gene sequence is SEQ ID NO.14, and the probe gene sequence is SEQ ID NO.15; the upstream primer gene sequence for TBEV is SEQ ID NO.16, the downstream primer gene sequence is SEQ ID NO.17, and the probe gene sequence is SEQ ID NO.18; the upstream primer gene sequence for YEZV is SEQ ID NO.19, the downstream primer gene sequence is SEQ ID NO.20, and the probe gene sequence is SEQ ID NO.21.

[0012] According to a third aspect of the present invention, a method for simultaneously detecting ALSV in a sample using quantitative real-time PCR is provided. F . tularensis A kit containing primers and probes for SFTSV, employing the aforementioned high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens, wherein the primer and probe combination includes primers and probes targeting ALSV, and primers and probes targeting SFTSV. F . tularensis Primers and probes for ALSV and for SFTSV; wherein, the upstream primer gene sequence for ALSV is SEQ ID NO.22, the downstream primer gene sequence is SEQ ID NO.23, and the probe gene sequence is SEQ ID NO.24; for ALSV... F . tularensis The upstream primer gene sequence is SEQ ID NO.25, the downstream primer gene sequence is SEQ ID NO.26, and the probe gene sequence is SEQ ID NO.27; the upstream primer gene sequence for SFTSV is SEQ ID NO.28, the downstream primer gene sequence is SEQ ID NO.29, and the probe gene sequence is SEQ ID NO.30.

[0013] According to a fourth aspect of the present invention, a method for simultaneously detecting [a substance] in a sample using quantitative real-time PCR is provided. A . phagocytophilum CCHFV and Rickettsia A kit containing primers and probes for spp., employing the aforementioned high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens, wherein the primer and probe combination includes those targeting A. phagocytophilumPrimers and probes for CCHFV, primers and probes for CCHFV, and primers and probes for CCHFV. Rickettsia Primers and probes for spp.; among which, those targeting A . phagocytophilum The upstream primer gene sequence is SEQ ID NO.31, the downstream primer gene sequence is SEQ ID NO.32, and the probe gene sequence is SEQ ID NO.33; the upstream primer gene sequence for CCHFV is SEQ ID NO.34, the downstream primer gene sequence is SEQ ID NO.35, and the probe gene sequence is SEQ ID NO.36; for... Rickettsia The upstream primer gene sequence of spp. is SEQ ID NO.37, the downstream primer gene sequence is SEQ ID NO.38, and the probe gene sequence is SEQ ID NO.39.

[0014] According to a fifth aspect of the present invention, a method for simultaneously detecting [a substance] in a sample using quantitative real-time PCR is provided. C . burnetii , Ehrlichia spp. and T . gondii A primer and probe kit is provided, employing the aforementioned high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens. The primer and probe combination includes primers and probes targeting *C. burnetii*, and primers and probes targeting... Ehrlichia Primers and probes for spp. and targeting T . gondii Primers and probes; among them, those targeting C . burnetii The upstream primer gene sequence is SEQ ID NO.40, the downstream primer gene sequence is SEQ ID NO.41, and the probe gene sequence is SEQ ID NO.42; targeting Ehrlichia The upstream primer gene sequence of spp. is SEQ ID NO.43, the downstream primer gene sequence is SEQ ID NO.44, and the probe gene sequence is SEQ ID NO.45; targeting T . gondii The upstream primer gene sequence is SEQ ID NO.46, the downstream primer gene sequence is SEQ ID NO.47, and the probe gene sequence is SEQ ID NO.48.

[0015] According to a sixth aspect of the present invention, a method for simultaneously detecting SGLV in a sample using quantitative real-time PCR is provided. Theileria spp. / HepatozoonA kit containing primers and probes for spp. and internal control of MS2 phage, employing the aforementioned high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens, wherein the primer and probe combination includes primers and probes targeting SGLV, and primers and probes targeting MS2 phage. Theileria spp. / Hepatozoon Primers and probes for spp. and for the internal control MS2; wherein, the upstream primer gene sequence for SGLV is SEQ ID NO.49, the downstream primer gene sequence is SEQ ID NO.50, and the probe gene sequence is SEQ ID NO.51; for Theileria spp. / Hepatozoon The upstream primer gene sequence for spp. is SEQ ID NO.52, the downstream primer gene sequence is SEQ ID NO.53, and the probe and gene sequence is SEQ ID NO.54; the upstream primer and gene sequence for internal control MS2 is SEQ ID NO.55, the downstream primer and gene sequence is SEQ ID NO.56, and the probe and gene sequence is SEQ ID NO.57.

[0016] According to the seventh aspect of the present invention, a kit is provided for simultaneously detecting NSDV, TAMV, and WELV in a sample using a real-time fluorescence PCR method. The kit employs the aforementioned high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens. The primer and probe combination includes primers and probes for NSDV, primers and probes for TAMV, and primers and probes for WELV. Specifically, the upstream primer and gene sequence for NSDV is SEQ ID NO. 58, the downstream primer and gene sequence is SEQ ID NO. 59, and the probe and gene sequence is SEQ ID NO. 60; the upstream primer gene sequence for TAMV is SEQ ID NO. 61, the downstream primer gene sequence is SEQ ID NO. 62, and the probe gene sequence is SEQ ID NO. 63; and the upstream primer gene sequence for WELV is SEQ ID NO. 64, the downstream primer gene sequence is SEQ ID NO. 65, and the probe gene sequence is SEQ ID NO. 66.

[0017] According to the eighth aspect of the present invention, a method for simultaneously detecting [a substance] in a sample using quantitative real-time PCR is provided. Babesia spp.、 B . duncani , B . microti A kit containing primers and probes for NOMV and TcTV-1, employing the aforementioned high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens, wherein the primer and probe combinations include those targeting NOMV and TcTV-1.Babesia Primers and probes for spp., targeting B . duncani Primers and probes targeting B . microti Primers and probes for NOMV, primers and probes for TcTV-1; among which, primers and probes for TcTV-1... Babesia The upstream primer gene sequence of spp is SEQ ID NO. 67, the downstream primer gene sequence is SEQ ID NO. 68, and the probe gene sequence is SEQ ID NO. 69; targeting B . duncani The upstream primer gene sequence is SEQ ID NO.70, the downstream primer gene sequence is SEQ ID NO.71, and the probe gene sequence is SEQ ID NO.72; targeting B . microti The upstream primer gene sequence is SEQ ID NO.73, the downstream primer gene sequence is SEQ ID NO.74, and the probe gene sequence is SEQ ID NO.75; the upstream primer gene sequence for NOMV is SEQ ID NO.76, the downstream primer gene sequence is SEQ ID NO.77, and the probe gene sequence is SEQ ID NO.78; the upstream primer gene sequence for TcTV-1 is SEQ ID NO.79, the downstream primer gene sequence is SEQ ID NO.80, and the probe gene sequence is SEQ ID NO.81.

[0018] Compared with existing technologies, the high-throughput multiplex real-time fluorescence quantitative detection method and its application applicable to 25 tick-borne pathogens of the present invention have the following technical advantages: 1. This invention, through meticulous design of primer and probe sequences and systematic optimization of the reaction system, effectively overcomes competitive inhibition in multiplex reactions. Experimental data show that the limit of detection (LOD) for each target pathogen can reach 10²-10³ copies per milliliter of blood or 10³-10⁴ copies per single tick pool, effectively detecting samples with low viral loads and reducing the false negative rate of tick-borne pathogens.

[0019] 2. Compared with existing diagnostic technologies for tick-borne pathogens, the multiplex real-time quantitative PCR detection system and kit provided by this invention have the following significant advantages: This invention integrates the detection of 25 identified tick-borne pathogens (covering viruses, bacteria, and parasites) into 8 reaction units. The detection spectrum is widely applicable and highly targeted, encompassing not only key monitored pathogens such as fever with thrombocytopenia syndrome virus, tick-borne encephalitis virus, and rickettsiae, but also newly emerging viruses closely related to human febrile diseases, such as wetland viruses and Syracuse viruses. Compared with traditional singleton qPCR or ordinary PCR, this system greatly reduces the sample volume, reagent consumables, and operation time required for detection, making it particularly suitable for large-scale epidemiological screening.

[0020] 3. The primer-probe combination and kit described in this invention can simultaneously perform rapid and sensitive broad-spectrum screening of multiple tick-borne viruses, bacteria and parasites in one detection process, and is suitable for clinical auxiliary diagnosis and large-scale epidemiological monitoring of tick-borne diseases. Attached Figure Description

[0021] Figure 1 The real-time fluorescence amplification curves of the reaction unit 1 (Assay 1) of the present invention for the simultaneous detection of three targets are shown.

[0022] Figure 2 The real-time fluorescence amplification curves of the reaction unit 2 (Assay 2) of the present invention for the simultaneous detection of four targets are shown.

[0023] Figure 3 The real-time fluorescence amplification curves of the reaction unit 3 (Assay 3) of the present invention for the simultaneous detection of three targets are shown.

[0024] Figure 4 The real-time fluorescence amplification curves of the reaction unit 4 (Assay 4) of the present invention for the simultaneous detection of three targets are shown.

[0025] Figure 5 The real-time fluorescence amplification curves of the reaction unit 5 (Assay 5) of the present invention for the simultaneous detection of three targets are shown.

[0026] Figure 6 The real-time fluorescence amplification curves of reaction unit 6 (Assay 6) of the present invention for simultaneous detection of three targets are shown.

[0027] Figure 7 The real-time fluorescence amplification curves of the reaction unit 7 (Assay 7) of the present invention for the simultaneous detection of three targets are shown.

[0028] Figure 8The real-time fluorescence amplification curves of the reaction unit 8 (Assay 8) of the present invention for the simultaneous detection of five targets are shown. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] This invention provides a systematic solution to the technical bottlenecks of existing detection technologies, such as the wide variety of tick-borne diseases (TBDs) pathogens, the common occurrence of mixed infections, the poor specificity of clinical symptoms, low throughput, high cost, and long processing time.

[0032] This invention presents a high-throughput multiplex real-time quantitative PCR detection method and application for 25 tick-borne pathogens. A multiplex real-time quantitative PCR detection system covering 25 major tick-borne pathogens was constructed. Based on extensive epidemiological surveys and meticulous experimental optimization, the pathogens were scientifically allocated into eight independent and non-interfering multiplex reaction units (Assays). Through systematic optimization of primer-probe combinations and reaction conditions, the competitive inhibition and non-specific amplification problems that are prone to occur in multiplex TaqManqPCR were successfully solved, achieving one-time, high-throughput, and highly sensitive broad-spectrum pathogen screening.

[0033] The 25 major tick-borne pathogens that can be detected by this invention are selected from one or more of the following pathogens: Viruses include Alongshan virus (ALSV), Beiji-nairovirus (BJNV), Crimean-Congo hemorrhagic fever virus (CCHFV), Jingmen tick virus (JMTV), Nuomin virus (NOMV), Nairobi sheep disease virus (NSDV), Severefever with thrombocytopenia syndrome virus (SFTSV), Songling virus (SGLV), Tamdy virus (TAMV), Tick-borne encephalitis virus (TBEV), Tacheng Tick Virus-1 (TcTV-1), Wetland virus (WELV), Xue-Cheng virus (XCV), and Yezovirus (Yezovirus). Virus, YEZV; bacteria including anaplasmophilic anaplasm ( Anaplasma phagocytophilum Bartonoidea ( Bartonella spp.), genus Spirochetes ( Borrelia spp.), Benacox bodies ( Coxiella burnetii ), Ehrlich body ( Ehrlichia spp.), Tulafrancsis ( Francisella tularensis ), Rickettsia spp. ( Rickettsia spp.); parasites include the genus Babesia ( Babesia spp.), Duncan-Babesia ( Babesia duncani ) tiny babesi ( Babesia microti ), Toxoplasma gondii ( Toxoplasma gondii ), Theileria ( Theileria spp.), genus *Hepatella* ( Hepatozoon spp.).

[0034] In a first aspect, the present invention provides a primer and probe combination for the simultaneous detection of BJNV, JMTV, and XCV in a sample using a real-time fluorescence PCR method, employing the high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens described in this application. The combination includes primers and probes for BJNV, primers and probes for JMTV, and primers and probes for XCV; wherein the upstream primer gene sequence for BJNV is SEQ ID NO.1, the downstream primer gene sequence is SEQ ID NO.2, and the probe gene sequence is SEQ ID NO.3; the upstream primer gene sequence for JMTV is SEQ ID NO.4, the downstream primer gene sequence is SEQ ID NO.5, and the probe gene sequence is SEQ ID NO.6; and the upstream primer gene sequence for XCV is SEQ ID NO.7, the downstream primer gene sequence is SEQ ID NO.8, and the probe gene sequence is SEQ ID NO.9.

[0035] Secondly, the present invention provides a method for simultaneously detecting [a substance] in a sample using a quantitative real-time PCR method. Bartonella spp.、 Borrelia Primer and probe combinations for spp., TBEV, and YEZV are used in accordance with the high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens described in this invention application. The combinations include those targeting spp., TBEV, and YEZV. Bartonella Primers and probes for spp., targeting Borrelia Primers and probes for spp., primers and probes for TBEV, and primers and probes for YEZV; among which, primers and probes for spp. Bartonella The upstream primer gene sequence of spp. is SEQ ID NO. 10, the downstream primer gene sequence is SEQ ID NO. 11, and the probe gene sequence is SEQ ID NO. 12; targeting Borrelia The upstream primer gene sequence for .spp. is SEQ ID NO.13, the downstream primer gene sequence is SEQ ID NO.14, and the probe gene sequence is SEQ ID NO.15; the upstream primer gene sequence for TBEV is SEQ ID NO.16, the downstream primer gene sequence is SEQ ID NO.17, and the probe gene sequence is SEQ ID NO.18; the upstream primer gene sequence for YEZV is SEQ ID NO.19, the downstream primer gene sequence is SEQ ID NO.20, and the probe gene sequence is SEQ ID NO.21.

[0036] Thirdly, the present invention provides a method for simultaneously detecting ALSV in a sample using a real-time fluorescence PCR method. F . tularensisThe primer and probe combination for SFTSV employs the high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens described in this invention application. The combination includes primers and probes targeting ALSV, and primers and probes targeting SFTSV. F . tularensis Primers and probes for ALSV and for SFTSV; wherein, the upstream primer gene sequence for ALSV is SEQ ID NO.22, the downstream primer gene sequence is SEQ ID NO.23, and the probe gene sequence is SEQ ID NO.24; for ALSV... F . tularensis The upstream primer gene sequence is SEQ ID NO.25, the downstream primer gene sequence is SEQ ID NO.26, and the probe gene sequence is SEQ ID NO.27; the upstream primer gene sequence for SFTSV is SEQ ID NO.28, the downstream primer gene sequence is SEQ ID NO.29, and the probe gene sequence is SEQ ID NO.30.

[0037] Fourthly, the present invention provides a method for simultaneously detecting [a substance] in a sample using a real-time fluorescence PCR method. A . phagocytophilum CCHFV and Rickettsia The primer and probe combination for *S. spp.* employs the high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens described in this invention application. The combination includes primers and probes targeting... A . phagocytophilum Primers and probes for CCHFV, primers and probes for CCHFV, and primers and probes for CCHFV. Rickettsia Primers and probes for spp.; among which, those targeting A . phagocytophilum The upstream primer gene sequence is SEQ ID NO.31, the downstream primer gene sequence is SEQ ID NO.32, and the probe gene sequence is SEQ ID NO.33; the upstream primer gene sequence for CCHFV is SEQ ID NO.34, the downstream primer gene sequence is SEQ ID NO.35, and the probe gene sequence is SEQ ID NO.36; for... Rickettsia The upstream primer gene sequence of spp. is SEQ ID NO.37, the downstream primer gene sequence is SEQ ID NO.38, and the probe gene sequence is SEQ ID NO.39.

[0038] Fifthly, the present invention provides a method for simultaneously detecting [a substance] in a sample using a real-time fluorescence PCR method. C . burnetii , Ehrlichia spp. and T . gondiiThe primer and probe combination employs the high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens described in this invention application. The combination includes primers and probes targeting... C . burnetii Primers and probes targeting Ehrlichia Primers and probes for spp. and targeting T . gondii Primers and probes; among them, those targeting C . burnetii The upstream primer gene sequence is SEQ ID NO.40, the downstream primer gene sequence is SEQ ID NO.41, and the probe gene sequence is SEQ ID NO.42; targeting Ehrlichia The upstream primer gene sequence of spp. is SEQ ID NO.43, the downstream primer gene sequence is SEQ ID NO.44, and the probe gene sequence is SEQ ID NO.45; targeting T . gondii The upstream primer gene sequence is SEQ ID NO.46, the downstream primer gene sequence is SEQ ID NO.47, and the probe gene sequence is SEQ ID NO.48.

[0039] Sixthly, the present invention provides a method for simultaneously detecting SGLV in a sample using a real-time fluorescence PCR method. Theileria spp.、 Hepatozoon The primer and probe combination for spp. and the internal control MS2 bacteriophage is used in accordance with the high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens described in this application. The combination includes primers and probes targeting SGLV, and primers and probes targeting... Theileria spp. / Hepatozoon Primers and probes for spp., and primers and probes for the internal control MS2; wherein, the upstream primer gene sequence for SGLV is SEQ ID NO.49, the downstream primer gene sequence is SEQ ID NO.50, and the probe gene sequence is SEQ ID NO.51; for Theileria spp. / Hepatozoon The upstream primer gene sequence for spp. is SEQ ID NO.52, the downstream primer gene sequence is SEQ ID NO.53, and the probe gene sequence is SEQ ID NO.54; the upstream primer gene sequence for internal control MS2 is SEQ ID NO.55, the downstream primer gene sequence is SEQ ID NO.56, and the probe gene sequence is SEQ ID NO.57.

[0040] In a seventh aspect, the present invention provides primer and probe combinations for the simultaneous detection of NSDV, TAMV, and WELV in a sample using a real-time fluorescence PCR method, employing the high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens described in this application. The combination includes primers and probes for NSDV, primers and probes for TAMV, and primers and probes for WELV; wherein the upstream primer gene sequence for NSDV is SEQ ID NO. 58, the downstream primer gene sequence is SEQ ID NO. 59, and the probe gene sequence is SEQ ID NO. 60; the upstream primer gene sequence for TAMV is SEQ ID NO. 61, the downstream primer gene sequence is SEQ ID NO. 62, and the probe gene sequence is SEQ ID NO. 63; and the upstream primer gene sequence for WELV is SEQ ID NO. 64, the downstream primer gene sequence is SEQ ID NO. 65, and the probe gene sequence is SEQ ID NO. 66.

[0041] Eighthly, the present invention provides a method for simultaneously detecting [a substance] in a sample using a real-time fluorescence PCR method. Babesia spp.、 B. duncani , B. microti The primer and probe combination for NOMV and TcTV-1 employs the high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens described in this invention application. The combination includes primers and probes targeting NOMV and TcTV-1. Babesia Primers and probes for spp., targeting B. duncani Primers and probes targeting B. microti Primers and probes for NOMV, primers and probes for TcTV-1; among which, primers and probes for TcTV-1... Babesia The upstream primer gene sequence of spp. is SEQ ID NO. 67, the downstream primer gene sequence is SEQ ID NO. 68, and the probe gene sequence is SEQ ID NO. 69; targeting B . duncani The upstream primer gene sequence is SEQ ID NO.70, the downstream primer gene sequence is SEQ ID NO.71, and the probe gene sequence is SEQ ID NO.72; targeting B. microtiThe upstream primer gene sequence is SEQ ID NO.73, the downstream primer gene sequence is SEQ ID NO.74, and the probe gene sequence is SEQ ID NO.75; the upstream primer gene sequence for NOMV is SEQ ID NO.76, the downstream primer gene sequence is SEQ ID NO.77, and the probe gene sequence is SEQ ID NO.78; the upstream primer gene sequence for TcTV-1 is SEQ ID NO.79, the downstream primer gene sequence is SEQ ID NO.80, and the probe gene sequence is SEQ ID NO.81.

[0042] In a ninth aspect, the present invention provides the use of the primer and probe combination described in any one of the first to eighth aspects in the preparation of reagents or kits for diagnosing or assisting in the diagnosis of tick-borne diseases, wherein the reagents or kits employ the high-throughput multiplex real-time fluorescence quantitative detection method applicable to 25 tick-borne pathogens described in this application.

[0043] In a tenth aspect, the present invention provides a method for detecting nucleic acid of tick-borne pathogens in a sample, the method comprising: extracting total nucleic acid from the sample to be tested; amplifying the nucleic acid in a multiplex real-time quantitative PCR system using the primer and probe combinations described in the first to eighth aspects; acquiring fluorescence signals of the FAM, VIC, ROX, and Cy5 channels through a specific thermal cycling program; and analyzing the fluorescence signals in conjunction with the cut-off value determined by the ROC curve to determine whether the sample contains one or more of the tick-borne pathogens.

[0044] The experimental reagents used in the following examples were sourced as follows: Primers, probes, and plasmid standards required for this invention were all custom-synthesized by Sangon Biotech (Shanghai) Co., Ltd. For the construction of the detection system, the real-time PCR enzyme used was qPCR Master Mix manufactured by Nanjing Novizan Biotechnology Co., Ltd.; tick nucleic acid extraction used the Biospin Virus DNA / RNA Extraction Kit from Hangzhou Bori Biotechnology Co., Ltd.; and whole blood sample extraction used the MagaBio Virus DNA / RNA Large-Scale Purification Kit II manufactured by the same company, along with a fully automated nucleic acid extractor for high-throughput processing. Nested PCR (nPCR) amplification reagents were used according to the pathogen type: the AccurSTART OneStep RT-PCR Kit from Nanjing Novizan was used for the first round of amplification targeting RNA, while the 2× Taq Plus Master Mix from Nanjing Novizan was used for the first round of amplification targeting DNA and for the second round of amplification targeting all pathogens.

[0045] The following is in conjunction with the appendix Figures 1 to 8 The present invention patent application is described in detail with reference to the embodiments, wherein...Figure 1 The diagram shows the real-time fluorescence amplification curves of reaction unit 1 (Assay 1) for simultaneous detection of three targets. The horizontal axis represents the cycle number, and the vertical axis represents relative fluorescence units (RFU). Different colored curves correspond to different detection channels and targets: the Cy5 channel corresponds to BJNV, the FAM channel to JMTV, and the VIC channel to XCV. The results show that within the same reaction system, all three sets of fluorescence signals can be amplified independently and efficiently. Figure 2 The diagram shows the real-time fluorescence amplification curves of reaction unit 2 (Assay 2) for simultaneous detection of four targets. The horizontal axis represents the cycle number, and the vertical axis represents the relative fluorescence units. Different colored curves correspond to different detection channels and targets: the FAM channel corresponds to... Bartonella spp., Cy5 channel corresponding Borrelia spp., ROX channel corresponds to TBEV, and VIC channel corresponds to YEZV; the results show that, within the same reaction system, the above four groups of fluorescence signals can be amplified independently and efficiently. Figure 3 The diagram shows the real-time fluorescence amplification curves of reaction unit 3 (Assay 3) for simultaneous detection of three targets. The horizontal axis represents the cycle number, and the vertical axis represents the relative fluorescence units. Different colored curves correspond to different detection channels and targets: the VIC channel corresponds to ALSV, and the Cy5 channel corresponds to... F . tularensis The FAM channel corresponds to SFTSV; the results show that, within the same reaction system, all three groups of fluorescence signals can be amplified independently and efficiently. Figure 4 The diagram shows the real-time fluorescence amplification curves of reaction unit 4 (Assay 4) for simultaneous detection of three targets. The horizontal axis represents the cycle number, and the vertical axis represents the relative fluorescence units. Different colored curves correspond to different detection channels and targets: the VIC channel corresponds to... A . phagocytophilum The Cy5 channel corresponds to CCHFV, and the FAM channel corresponds to... Rickettsia The results showed that, within the same reaction system, all three groups of fluorescence signals could be amplified independently and efficiently. Figure 5 The diagram shows the real-time fluorescence amplification curves of reaction unit 5 (Assay 5) for simultaneous detection of three targets. The horizontal axis represents the cycle number, and the vertical axis represents the relative fluorescence units. Different colored curves correspond to different detection channels and targets: the VIC channel corresponds to... C . burnetii Cy5 channel corresponds to Ehrlichia spp., FAM channel corresponding T . gondiiThe results showed that, within the same reaction system, all three groups of fluorescence signals could be amplified independently and efficiently. Figure 6 The diagram shows the real-time fluorescence amplification curves of reaction unit 6 (Assay 6) for simultaneous detection of three targets. The horizontal axis represents the cycle number, and the vertical axis represents the relative fluorescence units. Different colored curves correspond to different detection channels and targets: the Cy5 channel corresponds to SGLV, and the VIC channel corresponds to... Theileria spp. / Hepatozoon spp., the FAM channel corresponds to the internal control MS2; the results show that, within the same reaction system, the above three groups of fluorescence signals can be amplified independently and efficiently. Figure 7 The diagram shows the real-time fluorescence amplification curves of reaction unit 7 (Assay 7) of the present invention when simultaneously detecting three targets. The horizontal axis represents the cycle number, and the vertical axis represents the relative fluorescence units. Different colored curves correspond to different detection channels and targets: the Cy5 channel corresponds to NSDV, the VIC channel to TAMV, and the FAM channel to WELV. The results show that within the same reaction system, all three sets of fluorescence signals can be amplified independently and efficiently. Figure 8 The diagram shows the real-time fluorescence amplification curves of reaction unit 8 (Assay 8) for simultaneous detection of five targets. The horizontal axis represents the cycle number, and the vertical axis represents the relative fluorescence units. Different colored curves correspond to different detection channels and targets: the FAM channel corresponds to... Babesia spp. and Babesia duncani Corresponding to ROX channels Babesia microti The VIC channel corresponds to NOMV, and the Cy5 channel corresponds to TcTV-1. The results show that, within the same reaction system, all five groups of fluorescence signals can be amplified independently and efficiently.

[0046] Example 1: Design and Screening of Primers and Probes The present invention aims to establish a high-throughput multiplex detection system applicable to 25 major tick-borne pathogens.

[0047] Design and Screening Strategy: This invention first retrieves multi-regional prevalent strain sequences of various pathogens from the NCBI GenBank database. Clustal W algorithm is used for multiple sequence alignment to identify highly conserved regions (such as the L / S gene of viruses, 16S rRNA of bacteria, and 18S rRNA of protozoa). Then, Primer Express 3.0 software is used to design and screen multiple sets of candidate primers and probes within these highly conserved regions. BLAST specificity alignment and experimental verification are performed. BLAST screening is conducted on candidate sequences to ensure coverage of variant strains and eliminate non-specific binding to human or tick genomes. The primer-probe combinations described in Tables 1 and 2 below are selected as the preferred combinations.

[0048] Primer evaluation and optimization: To further ensure the stability of the multiplex system, all candidate sequences were evaluated using the multiplex primer analysis tools of ThermoFisher Scientific and IDT (Integrated DNA Technologies), eliminating sequences with severe hairpin structures, primer dimers, and nonspecific binding. Simultaneously, the selected primer sets were ensured to possess the following characteristics: (1) efficient operation under universal cycling conditions; (2) compatible melting temperatures of primers and probes within the same reaction tube, minimizing primer dimer formation and nonspecific amplification.

[0049] Final Scheme and 96-well Plate Prefabrication System Layout: The final grouping scheme of the 8 reaction units (Assays) and the specific primer and probe sequences for each target are detailed in Tables 1 and 2 of the instruction manual. To further achieve high-throughput rapid screening of clinical and field samples, this invention designs a standardized 96-well plate sample loading array: 8 independent multiplex reaction units (Assay 1 - Assay 8) are pre-placed in the row direction of the 96-well plate, and the samples to be tested are added along the column direction; under this layout, each 96-well plate can simultaneously complete the full-spectrum screening of 10 samples to be tested, 1 negative control, and 1 positive control for all 25 tick-borne pathogens (see Table 3).

[0050] Table 1. Target allocation, target genes, and amplification product length for quantitative real-time PCR detection of tick-borne pathogens.

[0051] Table 2. Sequences of primers and probes used in the invention

[0052]

[0053] Table 3. Preparation of the 96-well PCR plate for the pre-fabricated detection system in Example 1

[0054] Example 2: Establishment and optimization of multiplex qPCR reaction system To address the competitive inhibition problem prevalent in multiplex PCR, this invention systematically optimizes primer and probe concentrations and establishes universal thermal cycling conditions.

[0055] Preparation of plasmid standards: Based on the target gene sequences and amplified fragments of each pathogen described in Table 2 of Example 1, the corresponding nucleic acid sequences were artificially synthesized by Sangon Biotech (Shanghai) Co., Ltd. The target gene fragments of each pathogen were cloned into the pUC57 vector to prepare recombinant plasmids. After verifying the sequence correctness by Sanger sequencing, the concentration was measured using a high-precision spectrophotometer. Based on the total base length of the plasmid (vector length + insert length) and the average molecular weight, the mass concentration was converted to copy number concentration using the following formula: Copy number (copies / µL) = [6.02 × 10⁻⁶]. 23 × Concentration (ng / µL) × 10 ⁻9 [[plasmid length (bp) × 660]]. Subsequently, using 0.1×TE buffer, each high-concentration plasmid standard was serially diluted 10-fold to prepare plasmids with concentrations ranging from 10 to 10. 8 copies / mL to 10 1 A series of standards were prepared in copies / mL. The diluted standards were aliquoted and stored at -80°C for subsequent sensitivity validation and standard curve plotting for single and multiplex qPCR reactions.

[0056] Reaction system: This invention uses a 10µL reaction system for amplification, which includes 2µL template, primers and probes of optimal concentration for each reaction unit (Assay), and a fluorescent quantitative enzyme.

[0057] Reaction procedure: The present invention adopts the following general "one-step" thermal cycling procedure: reverse transcription: 55℃, 15 min (this step is used for cDNA synthesis of RNA virus and does not affect the subsequent amplification of DNA target in template); pre-denaturation: 95℃, 30s; PCR amplification (45 cycles): 95℃, 10s; 60℃, 60s (fluorescence signal is collected at this stage).

[0058] Reaction system optimization: To determine the optimal reaction conditions, this invention first independently optimized each singleton qPCR assay and systematically tested the effects of different primer and probe concentration combinations on amplification performance. The primer concentration range was 400 nM–1000 nM, and the probe concentration range was 150 nM–400 nM. Simultaneously, to determine universal thermal cycling conditions, parallel tests were performed on the above concentration combinations at different annealing temperatures (58°C–62°C). Then, the combinations were grouped into multiplex qPCR assays, and the Ct values ​​of singleton and multiplex assays were compared to determine the optimal multiplex qPCR concentration. All reactions were run on an Applied Biosystems 7500 real-time quantitative PCR system (Thermo Fisher Scientific, Waltham, MA, USA), and each condition was performed in triplicate to ensure data reliability. The optimal primer and probe formulations for each assay are detailed in Table 4.

[0059] Table 4. Optimization of reaction conditions for quantitative real-time PCR detection of tick-borne pathogens

[0060] Example 3: Sample Pretreatment and Nucleic Acid Extraction Methods To ensure the accuracy and reproducibility of test results, this invention establishes a standardized pretreatment and nucleic acid extraction process for different types of clinical and field samples.

[0061] 1. Tick Sample Classification: To ensure the accuracy of test results and minimize the potential inhibition of qPCR reactions by host blood, this embodiment establishes strict sample classification and pooling principles. First, the collected samples are morphologically identified, and the species is determined in conjunction with the collection time and location. Then, following the standard of "same species, same collection location, same collection time," differentiated pooling is performed according to the degree of blood feeding: free ticks / unengaged ticks are pooled in groups of 15 to improve throughput; while for parasitic ticks or engorged ticks collected from the host's body surface, considering that they contain a large amount of host blood and protein which may lead to a decrease in nucleic acid extraction efficiency or PCR inhibition, a low-throughput pooling strategy is adopted, with only 1–5 ticks grouped into a pooled sample. For particularly large engorged adults (such as Haemaphysalis longicornis), individual grinding is performed.

[0062] 2. Tick Sample Grinding Procedure: To eliminate exogenous contamination and ensure sufficient release of nucleic acids, the following pretreatment steps are employed: Using sterile forceps, the separated ticks are placed into a sterile culture dish and rinsed three times with phosphate-buffered saline (PBS) to thoroughly remove environmental impurities and potential microbial contamination from the sample surface. Subsequently, the cleaned ticks are placed in a mortar pre-cooled with liquid nitrogen and repeatedly ground under liquid nitrogen until the sample is completely pulverized (liquid nitrogen is replenished as needed to maintain a low-temperature brittle state). Finally, the powder is transferred to an enzyme-free sterile centrifuge tube using a pre-cooled spatula, and 400 µL of lysis buffer and 20 µL of proteinase K are added (the amount of lysis buffer can be increased proportionally if the powder volume is large). After vortexing for 30 seconds, the mixture is centrifuged at 12,000 rpm for 5 minutes, and the supernatant is transferred to a new centrifuge tube for later use.

[0063] 3. Tick Nucleic Acid Extraction: Take the supernatant after the above treatment and extract total nucleic acid using the Biospin Viral DNA / RNA Extraction Kit, strictly following these steps: First, place the centrifuge tube containing the tick sample, along with the positive and negative control tubes, in a constant temperature water bath and incubate at 56°C for 15 minutes. After incubation, centrifuge the sample rapidly, then add 500µL of anhydrous ethanol to the tube, vortex thoroughly to mix, and centrifuge again. Next, transfer the mixture to a purification column and centrifuge at 10,000 rpm for 1 minute to bind the nucleic acid to the membrane; if there is too much liquid in the tube, this step can be performed twice. Then discard the filtrate in the tube, add 500µL of protein removal wash buffer to the purification column, and centrifuge at 10,000 rpm for 1 minute. Discard the filtrate again, add 500µL of wash buffer, and centrifuge at 10,000 rpm for 1 minute; if the adsorption membrane is still not clean, this washing step can be repeated once. Subsequently, the filtrate was discarded, and the purification column was returned to the collection tube. The column was centrifuged at 10,000 rpm for 2 minutes to completely remove residual ethanol. Finally, the dried purification column was transferred to a new enzyme-free centrifuge tube, and 100 µL of elution buffer was added to the center of the adsorption membrane. After standing at room temperature for 2 minutes, the column was centrifuged at 12,000 rpm for 2 minutes to elute. The eluted nucleic acid extract was promptly used for downstream experiments.

[0064] 4. Whole Blood Nucleic Acid Extraction: This study used the MagaBio Viral DNA / RNA High-Throughput Purification Kit II for high-throughput extraction. The specific procedures were as follows: First, remove the plastic seal from the deep-well plate of the kit and manually shake the plate three times to remove any adhering droplets. Then, under strictly partitioned operating conditions, add 100 µL of proteinase K, 200 µL of whole blood release agent, 2 µL of exogenous internal control MS2, and 2000 µL of thawed and mixed whole blood sample sequentially to the wells. Place the deep-well plate with the sample added into a nucleic acid extractor with the magnetic rod inserted, and automatically complete the lysis, binding, rinsing, and elution steps according to the standard extraction program in Table 5. After the program is complete, aspirate all the nucleic acid eluent from the elution wells and transfer it to a 1.5 mL centrifuge tube.

[0065] Table 5. Standard Extraction Procedure for Whole Blood Nucleic Acid

[0066] Example 4: Linear dynamic range and amplification efficiency analysis To comprehensively evaluate the performance of the multiplex detection system described in this invention, this embodiment and subsequent embodiments strictly followed the relevant requirements of the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines, and systematically verified the linear dynamic range, amplification efficiency (E), specificity, limit of detection (LOD), and precision.

[0067] To evaluate the quantitative performance and amplification kinetics of the multiplex detection system of this invention over a wide concentration range, a standard curve model was established in this embodiment.

[0068] The specific operation is as follows: First, using the target positive plasmid standards prepared in Example 2, a concentration range suitable for 10 is prepared by a 10-fold serial dilution method. 4 Up to 10 8 Five series of standards at concentrations of copies / mL were prepared. Subsequently, amplification and detection of the standards at each concentration were performed strictly according to the established multiplex qPCR reaction system and procedure, with three replicate wells for each concentration gradient to eliminate sample loading and operational errors. After the experiment, linear regression analysis was performed using statistical software, with the logarithm of the standard concentration as the x-axis and the measured cycle threshold (Ct) as the y-axis, and a standard curve was plotted. The coefficient of determination (Ct) was calculated based on the regression equation. R 2 This is used to evaluate the linear fit of the standard curve and calculate the amplification efficiency of each target based on the slope value.

[0069] Detailed experimental statistical results are shown in Table 6. The data shows that the 25 pathogen targets covered in the 1-8 reaction units (Assays) of this invention are within 10... 4 Up to 10 8 Excellent linearity was observed across a wide dynamic range of copies / mL, with linear correlation coefficients for all targets ( R 2 All values ​​were greater than 0.99. Furthermore, the amplification efficiency of each target remained consistently within the ideal range of 90% to 105%. This result strongly demonstrates that although this invention employs a multiplex reaction system, no significant competitive inhibition occurred between the primer-probe combinations, and the reaction system can support efficient simultaneous amplification of multiple targets with good quantitative accuracy.

[0070] Table 6. Linear dynamic range and amplification efficiency of pathogen targets in quantitative real-time PCR detection of tick-borne pathogens.

[0071] Example 5, Specificity Analysis To verify whether nonspecific amplification or cross-reaction exists in this invention, this embodiment selected 154 pathogen test samples stored in the laboratory test sample library for rigorous exclusion testing. All experimental operations involving pathogens were completed in the biosafety level 2 laboratory of this laboratory. The test sample library (see Table 7 for details) extensively covers many potential sources of interference, including tick-borne pathogens (such as BJNV, etc.). Babesia microti The above samples were tested using the reaction units 1-8 of this invention. The judgment criteria were set as follows: if the Ct value was >40 or there was no amplification curve, and the internal control MS2 signal was normal, then it was determined that there was no cross-reaction.

[0072] Experimental results show that the combination of reaction units 1-8 of the present invention did not produce non-specific amplification curves (Ct values ​​were all undetermined) for the above-mentioned pathogen samples, and only showed typical S-shaped amplification curves when detecting the corresponding target. This confirms that the kit of the present invention has extremely high specificity, can accurately distinguish between target tick-borne pathogens and background microorganisms, and effectively avoids the generation of false positive results in clinical practice.

[0073] Table 7. Nucleic acid of non-target pathogens for specificity validation of the quantitative real-time PCR detection method for tick-borne pathogens.

[0074] Example 6, Sensitivity Analysis To accurately evaluate the detection performance of reaction units 1-8 of this invention in complex samples, two matrices were selected for LOD determination: a pathogen-free tick pool (15 adult ticks) and negative human EDTA whole blood, to simulate real clinical and field sample environments. Specifically, based on the approximate detection limit range determined in previous preliminary experiments, to achieve precise quantification of LOD, this embodiment established a specific low-concentration gradient near the estimated LOD threshold: firstly, plasmid standards for each pathogen target were incorporated into the aforementioned negative matrix; for the human whole blood matrix, a concentration range of 10... 2 Up to 10 3 A series of simulated samples were constructed using copies / mL; for tick-borne substrates, concentrations ranging from 10 to 10 were used. 3 Up to 10 4 A series of simulated samples were prepared using copies / pools. For each concentration point, eight complete nucleic acid extraction and purification operations were performed independently (i.e., eight full-process biological replicates). The elution buffers from each extraction were then used for qPCR detection strictly according to the system configuration and amplification procedure described in Example 2. After the experiment, the amplification percentage (detection rate) for each concentration was calculated. Probit probability unit regression analysis was performed using IBM SPSS Statistics 26 software to fit the amplification ratio and the detected copy number. The x-axis value of the fitted curve y=0.99 (i.e., 99% detection probability) was selected as the LOD of that target.

[0075] The results, as shown in Table 8, indicate that the blood matrix LOD distribution of reaction units 1-8 in this invention is within 10. 2 Up to 10 3 Between copies / mL, the LOD distribution of the tick-borne matrix was between 10. 3 Up to 10 4 The sensitivity is between copies / pool. This is close to that of conventional multiplex qPCR on the market. Therefore, the above data proves that even in complex matrices containing PCR inhibitors (such as heme in blood or impurities in tick body fluids), the present invention still has high sensitivity and can effectively avoid missed detections in low-load samples.

[0076] Table 8. Sensitivity analysis of the quantitative real-time PCR detection method for tick-borne pathogens

[0077] Example 7, Precision Analysis To evaluate the stability of the kit under different detection environments, this embodiment designed a rigorous repeatability and reproducibility verification experiment. First, two concentrations (10... 6 copies / mL and 10 5Simulated positive whole blood samples (copies / mL) were used as test subjects. For intra-assay, 20 replicate tests were performed on samples of the two concentrations within a single experimental batch. For inter-assay, 20 replicate tests were performed by two different operators on three different experimental dates using different batches of the kit. The Ct value for each test was recorded, and the coefficient of variation was calculated using the formula CV = (SD / Mean) × 100%.

[0078] The results (Table 9) show that the CV values ​​of all test groups are less than 2%, indicating that the kit of the present invention has extremely high precision and can effectively resist interference caused by experimental operation, instrument fluctuations and batch-to-batch differences, ensuring the stability and reliability of the test results.

[0079] Table 9. Coefficients of variation for repeatability and reproducibility (precision) of quantitative real-time PCR detection methods for tick-borne pathogens

[0080] Example 8: Positive confirmation of tick-borne pathogens detection method in real samples using real samples. To objectively verify the accuracy of the positive results generated by this invention (with Ct ≤ 35.0 as the cutoff value) in real samples, this embodiment designed a confirmatory experiment (nPCR + Sanger sequencing) for positive samples. First, a validation set was constructed from a large field sample library containing 1635 ticks collected in the field, divided into 178 tick pools based on sampling location, sampling time, and tick species. From this sample library, 30 nucleic acid samples from each tick pool were randomly selected (a total of 750 tests) for all 25 detection targets covered by this invention as the validation set.

[0081] The detection process of this invention is as follows: Nucleic acid extracts from the above 750 tests were analyzed. Nucleic acid extraction was strictly performed according to the method described in Example 3. qPCR amplification used the 10µL reaction system and the "one-step" general thermal cycling procedure described in Example 2. The result judgment criteria were strictly set as follows: when the detection Ct value ≤ 35.0 and the amplification curve shows a typical S-shape, it was judged as "qPCR positive"; when the Ct value > 35.0 or there was no amplification, it was judged as "qPCR negative". Subsequently, for the samples judged as qPCR positive, the gold standard verification process was initiated.

[0082] The specific operating procedure for the gold standard is as follows: The first round of amplification uses a 25µL reaction system, with 2µL of extracted nucleic acid as a template, and 1µL of each primer (10µM) added. The amplification reagents are selected according to the pathogen type: AccurSTART OneStep RT-PCR Kit (for RNA targets) or 2×Taq Plus Master Mix (for DNA targets). All reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd. The second round of amplification also uses a 25µL system, with 2µL of the first round amplification product as a template, and 2×Taq Plus Master Mix is ​​used for the reaction. The primer concentration is the same as in the first round. Specific thermal cycling conditions optimized for each target are detailed in Table 8. All nPCR positive products are subjected to Sanger bidirectional sequencing; only those with identical sequence alignment are considered true positives.

[0083] The validation results (see Table 9) show that a total of 75 qPCR positive samples were screened in this validation set. Confirmed by nPCR + sequencing, 74 of these samples were true positives. The overall positive concordance rate was 98.7%. This result confirms that the present invention, using Ct35 as the cutoff value, has an extremely high positive predictive value, indicating that the positive results it screens are highly reliable and have extremely high credibility, fully meeting the needs of epidemiological surveillance.

[0084] Table 10. Nested / semi-nested primer sequences and thermal cycling conditions for tick-borne pathogens

[0085] Table 11. Comparison between the present invention's real-time PCR method and the nested PCR + sequencing method

[0086] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A primer-probe composition for detecting 25 tick-borne pathogens, characterized in that: The primer and probe composition for detecting 25 tick-borne pathogens includes a primer and probe combination for simultaneously detecting Arctic Nairobi virus (BJNV), Jingmen tick virus (JMTV), and Xuecheng virus (XCV) in samples. The primer and probe combination includes primers and probes for Arctic Nairobi virus (BJNV), Jingmen tick virus (JMTV), and Xuecheng virus (XCV). The upstream primer gene sequence for Arctic Nairobi virus (BJNV) is SEQ ID NO.1, the downstream primer gene sequence is SEQ ID NO.2, and the probe gene sequence is SEQ ID NO.

3. The upstream primer gene sequence for Jingmen tick virus (JMTV) is SEQ ID NO.4, the downstream primer gene sequence is SEQ ID NO.5, and the probe gene sequence is SEQ ID NO.

6. The upstream primer gene sequence for Xuecheng virus (XCV) is SEQ ID NO.7, the downstream primer gene sequence is SEQ ID NO.8, and the probe gene sequence is SEQ ID NO.

9. The primer probe composition for detecting 25 kinds of tick-borne pathogens further comprises a primer and probe set for simultaneously detecting Bartonella spp. Bartonella , Borrelia spp. Borrelia , Forest encephalitis virus TBEV and Yezo virus YEZV, comprising a primer and probe for Bartonella spp. , a primer and probe for Spirochetes belong to the genus Borrelia spp. , a primer and probe for Forest encephalitis virus TBEV and a primer and probe for Yezo virus YEZV; against Bartonella spp. The upstream primer gene sequence is SEQ ID NO.10, the downstream primer gene sequence is SEQ ID NO.11, and the probe gene sequence is SEQ ID NO.12; targeting Spiroleum Borrelia spp The upstream primer gene sequence for the target virus is SEQ ID NO.13, the downstream primer gene sequence is SEQ ID NO.14, and the probe gene sequence is SEQ ID NO.15; the upstream primer gene sequence for the target virus against tick-borne encephalitis virus (TBEV) is SEQ ID NO.16, the downstream primer gene sequence is SEQ ID NO.17, and the probe gene sequence is SEQ ID NO.18; the upstream primer gene sequence for the target virus against YEZV is SEQ ID NO.19, the downstream primer gene sequence is SEQ ID NO.20, and the probe gene sequence is SEQ ID NO.

21. The primer and probe composition for detecting 25 tick-borne pathogens further includes simultaneous detection of Arungshan virus (ALSV) and Tulafrancsis in the sample. F . tularensis Primer and probe combinations for SFTSV (Severe Fever with Thrombocytopenia Syndrome Virus), wherein the primer and probe combinations include primers and probes for ALSV (Arunson Virus), and primers and probes for... Tulafo Rancis bacterium F. tularensis Primers and probes for SFTSV (severe fever with thrombocytopenia syndrome virus); the upstream primer gene sequence for ALSV (Alonshan virus) is SEQ ID NO.22, the downstream primer gene sequence is SEQ ID NO.23, and the probe gene sequence is SEQ ID NO.24; for F. tularensis The upstream primer gene sequence is SEQ ID NO.25, the downstream primer gene sequence is SEQ ID NO.26, and the probe gene sequence is SEQ ID NO.27; the upstream primer gene sequence for SFTSV (severe fever with thrombocytopenia syndrome virus) is SEQ ID NO.28, the downstream primer gene sequence is SEQ ID NO.29, and the probe gene sequence is SEQ ID NO.

30. The primer-probe composition for detecting 25 tick-borne pathogens further includes simultaneous detection of anaplasmophilic phagocytosins in the sample. A . phagocytophilum Crimean-Congo hemorrhagic fever virus (CCHFV) and Rickettsia genus Rickettsia Primer and probe combinations for *Anaphagocytophilum* spp., comprising primers and probes for *Anaphagocytophilum* phagocytophilum, primers and probes for Crimean-Congo hemorrhagic fever virus (CCHFV), and primers and probes for *Rickettsia* spp. Rickettsia Primers and probes for spp.; against Phagocytophilum (A. phagocytophilum) The upstream primer gene sequence is SEQ ID NO.31, the downstream primer gene sequence is SEQ ID NO.32, and the probe gene sequence is SEQ ID NO.33; the upstream primer gene sequence for Crimean-Congo hemorrhagic fever virus (CCHFV) is SEQ ID NO.34, the downstream primer gene sequence is SEQ ID NO.35, and the probe gene sequence is SEQ ID NO.36; for Rickettsia genus... Rickettsia The upstream primer gene sequence of spp. is SEQ ID NO.37, the downstream primer gene sequence is SEQ ID NO.38, and the probe gene sequence is SEQ ID NO.39; The primer-probe composition for detecting 25 tick-borne pathogens further includes simultaneous detection of *Benacoxiella* in the sample. C . burnetii Ehrlich body Ehrlichia spp. and Toxoplasma gondii T . gondii The primer and probe combination includes primers and probes targeting *C. burnetii* and *C. erechylus*. Ehrlichia Primers and probes for *S. spp.* and targeting *Toxoplasma gondii*. T . gondii Primers and probes; For Benacox body C . burnetii The upstream primer gene sequence is SEQ ID NO.40, the downstream primer gene sequence is SEQ ID NO.41, and the probe gene sequence is SEQ ID NO.42; targeting Ehrlich assemblage. Ehrlichia The upstream primer gene sequence of *S. spp.* is SEQ ID NO. 43, the downstream primer gene sequence is SEQ ID NO. 44, and the probe gene sequence is SEQ ID NO. 45; targeting *Toxoplasma gondii*. T . gondii The upstream primer gene sequence is SEQ ID NO.46, the downstream primer gene sequence is SEQ ID NO.47, and the probe gene sequence is SEQ ID NO.48; The primer-probe composition for detecting 25 tick-borne pathogens further includes simultaneous detection of Pineridge Virus SGLV and Theileria species in the sample. Theileria spp. and Hepatella genus Hepatozoon spp. and primer and probe combinations for the internal control of MS2 bacteriophage, the primer and probe combinations including primers and probes for Pine Ridge virus SGLV and for Theileria genus. Theileria spp. and the genus *Hepatella* Hepatozoon Primers and probes for *S. spp.* and for internal control MS2; the upstream primer gene sequence for *S. spp.* is SEQ ID NO. 49, the downstream primer gene sequence is SEQ ID NO. 50, and the probe gene sequence is SEQ ID NO. 51; for *Theileria* genus... Theileria spp. and the genus *Hepatella* Hepatozoon The upstream primer gene sequence for spp. is SEQ ID NO.52, the downstream primer gene sequence is SEQ ID NO.53, and the probe and gene sequence is SEQ ID NO.54; the upstream primer and gene sequence for internal control MS2 is SEQ ID NO.55, the downstream primer and gene sequence is SEQ ID NO.56, and the probe and gene sequence is SEQ ID NO.

57. The primer and probe composition for detecting 25 tick-borne pathogens further comprises primer and probe combinations for simultaneously detecting Nairobi sheep disease virus (NSDV), tamd virus (TAMV), and wetland virus (WELV) in samples. The primer and probe combinations include primers and probes for Nairobi sheep disease virus (NSDV), tamd virus (TAMV), and wetland virus (WELV). The upstream primer and gene sequence for Nairobi sheep disease virus (NSDV) are SEQ ID NO. 58, the downstream primer and gene sequence are SEQ ID NO. 59, and the probe and gene sequence are SEQ ID NO.

60. The upstream primer gene sequence for tamd virus (TAMV) is SEQ ID NO. 61, the downstream primer gene sequence is SEQ ID NO. 62, and the probe gene sequence is SEQ ID NO.

63. The upstream primer gene sequence for wetland virus (WELV) is SEQ ID NO. 64, the downstream primer gene sequence is SEQ ID NO. 65, and the probe gene sequence is SEQ ID NO.

66. The primer-probe composition for detecting 25 tick-borne pathogens further includes simultaneous detection of Babesia spp. in the sample. Babesia spp., Duncan-Babesia worm B . duncani Microbabesi B . microti Primer and probe combinations for Nominogen viremia (NOMV) and Tacheng tick virus type I (TcTV-1), wherein the primer and probe combinations include those targeting Babesia genus. Babesia Primers and probes for *S. spp.* targeting *Duncan-Babesia*. B . duncani Primers and probes targeting Babesia microsporum B . microti Primers and probes for [specific virus name], primers and probes for Nominosis virus (NOMV), and primers and probes for Tacheng tick virus type I (TcTV-1); primers and probes for Babesia species. Babesia The upstream primer gene sequence of spp is SEQ ID NO. 67, the downstream primer gene sequence is SEQ ID NO. 68, and the probe gene sequence is SEQ ID NO. 69; targeting Duncan-Babesia. B . duncani The upstream primer gene sequence is SEQ ID NO.70, the downstream primer gene sequence is SEQ ID NO.71, and the probe gene sequence is SEQ ID NO.72; targeting Babesia microsporum. B . microti The upstream primer gene sequence for the target virus is SEQ ID NO.73, the downstream primer gene sequence is SEQ ID NO.74, and the probe gene sequence is SEQ ID NO.75; the upstream primer gene sequence for the target virus NOMV is SEQ ID NO.76, the downstream primer gene sequence is SEQ ID NO.77, and the probe gene sequence is SEQ ID NO.78; the upstream primer gene sequence for the target virus type I TcTV-1 is SEQ ID NO.79, the downstream primer gene sequence is SEQ ID NO.80, and the probe gene sequence is SEQ ID NO.81.

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