Primer probe pair, microfluidic chip, kit for detecting pathogen of vector-borne disease based on taqman microfluidic chip technology, preparation method and use thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a primer probe pair for detecting a pathogen of a vector-borne infectious disease based on TaqMan microfluidic chip technology, a microfluidic chip, a kit, a preparation method and application thereof. The application provides a detection method for simultaneously detecting 28 kinds of pathogen of a vector-borne infectious disease based on TaqMan microfluidic chip technology, wherein the types of the pathogen of the vector-borne infectious disease involved include viruses, bacteria and parasites, the range is wide, and the difference is large. The detection method has a low detection limit (5-20 copies / μL); when applied to an actual sample, the detection method has high sensitivity, specificity, accuracy and precision, and has good consistency compared with a gold standard method; and compared with a traditional qPCR method, the detection method greatly reduces the time efficiency, manpower and material consumption, is particularly suitable for a scene of disease monitoring and emergency disposal which needs rapid and comprehensive investigation, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to primer-probe pairs, microfluidic chips, reagent kits, preparation methods, and applications for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology. Background Technology

[0002] Vector-borne diseases generally refer to organisms that can transmit human diseases (such as rodents, mosquitoes, ticks, fleas, sandflies, etc.). They can transmit infectious diseases directly through bites, food contamination, and other routes, seriously affecting or disrupting normal life. Currently, 70% of infectious diseases worldwide are vector-borne, such as dengue fever, malaria, yellow fever, and plague, which have always been important public health issues of international concern. Since most vector-borne infectious diseases still lack effective vaccines and specific treatments, the situation regarding vector-borne disease surveillance and control remains very serious. Currently, global vector-borne infectious diseases are characterized by an increasing number of species, expanding epidemic range, and increasing frequency of outbreaks. The new challenges posed by imported vector-borne infectious diseases should not be underestimated.

[0003] Traditional methods for detecting vector-borne infectious disease pathogens mainly include microscopic examination, pathogen culture, conventional PCR, and serological testing. Microscopic examination is simple to perform but has low sensitivity, relies on experience, and is prone to missed detections; pathogen culture provides accurate results but is time-consuming (several days to weeks) and is ineffective against difficult-to-culture pathogens; conventional PCR is cumbersome and has low sensitivity; serological testing is suitable for tracing infection but is susceptible to false positives due to cross-reactivity. These methods generally suffer from low throughput and poor timeliness, making them unsuitable for meeting the needs of rapid screening and large-scale monitoring in sudden outbreaks.

[0004] Real-time quantitative PCR (qPCR) technology combines high sensitivity and specificity, offering significant advantages in rapid and accurate quantification, and has become a primary technique for pathogen detection both domestically and internationally. Currently, many commercially available kits exist, but they primarily target single vector-borne infectious disease pathogens. Methods for simultaneously detecting different vector-borne infectious disease pathogens are less common, hindering the simultaneous screening of multiple pathogens. Furthermore, in multiplex qPCR, the cross-reactivity and competition effects caused by the coexistence of multiple primers and probes reduce sensitivity and specificity, affecting quantitative accuracy. In addition, multiplex qPCR has very strict experimental requirements, making standardization difficult and resulting in poor reproducibility and comparability between different laboratories. Therefore, to address these technical issues and further improve the monitoring and early warning capabilities for vector-borne infectious diseases, and to enhance the disease prevention and control system, it is necessary to establish a simple, rapid, sensitive, and accurate high-throughput vector-borne infectious disease pathogen screening technology system.

[0005] TaqMan microfluidic chip technology integrates real-time quantitative PCR and microfluidic technology, representing an emerging high-throughput pathogen screening technology. Compared to traditional qPCR, it offers advantages such as high throughput, low consumption, and rapid reaction speed. Currently, TaqMan microfluidic chip technology has been applied to the detection of respiratory and intestinal pathogens, but its application to vector-borne infectious disease pathogens has not been reported. Furthermore, vector-borne infectious diseases involve a wide variety of pathogens (bacteria, viruses, and parasites, etc.) and often exhibit similar clinical symptoms, making differentiation difficult. Conventional qPCR requires multiple reaction systems and multiple runs on several real-time quantitative PCR instruments to complete screening, which is time-consuming and labor-intensive, failing to meet the new and demanding requirements of disease monitoring and emergency response. Therefore, developing a simple, rapid, sensitive, and accurate high-throughput method for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology is an urgent problem to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides primer-probe pairs, microfluidic chips, reagent kits, preparation methods, and applications for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology.

[0007] This invention provides primer-probe pairs for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology, comprising the following primers and probes:

[0008] Group 1: Primers as shown in SEQ ID NO. 1-2, and probes as shown in SEQ ID NO. 3;

[0009] The second group consists of primers as shown in SEQ ID NO. 4-5 and probes as shown in SEQ ID NO. 6.

[0010] The third group includes primers as shown in SEQ ID NO. 7-8 and probes as shown in SEQ ID NO. 9.

[0011] Group 4: Primers as shown in SEQ ID NO. 10-11, and probes as shown in SEQ ID NO. 12;

[0012] Group 5: Primers as shown in SEQ ID NO. 13-14, and probes as shown in SEQ ID NO. 15;

[0013] Group 6: Primers as shown in SEQ ID NO. 16-17, and probes as shown in SEQ ID NO. 18;

[0014] Group 7: Primers as shown in SEQ ID NO. 19-20, and probes as shown in SEQ ID NO. 21;

[0015] Group 8: Primers as shown in SEQ ID NO. 28-29, and probes as shown in SEQ ID NO. 30;

[0016] Group 9: Primers as shown in SEQ ID NO. 34-35, and probes as shown in SEQ ID NO. 36;

[0017] Group 10: Primers as shown in SEQ ID NO. 37-38, and probes as shown in SEQ ID NO. 39;

[0018] Group 11: Primers as shown in SEQ ID NO. 40-41, and probes as shown in SEQ ID NO. 42;

[0019] Group 12: Primers as shown in SEQ ID NO. 43-44, and probes as shown in SEQ ID NO. 45;

[0020] Group 13: Primers as shown in SEQ ID NO. 49-50, and probes as shown in SEQ ID NO. 51;

[0021] Group 14: Primers as shown in SEQ ID NO. 52-53, and probes as shown in SEQ ID NO. 54;

[0022] Group 15: Primers as shown in SEQ ID NO. 55-56, and probes as shown in SEQ ID NO. 57;

[0023] Group 16: Primers as shown in SEQ ID NO. 58-59, and probes as shown in SEQ ID NO. 60;

[0024] Group 17: Primers as shown in SEQ ID NO. 61-62, and probes as shown in SEQ ID NO. 63;

[0025] Group 18: Primers as shown in SEQ ID NO. 64-65, and probes as shown in SEQ ID NO. 66;

[0026] Group 19: Primers as shown in SEQ ID NO. 67-68, and probes as shown in SEQ ID NO. 69;

[0027] Group 20: Primers as shown in SEQ ID NO. 70-71, and probes as shown in SEQ ID NO. 72;

[0028] Group 21: Primers as shown in SEQ ID NO. 73-74, and probes as shown in SEQ ID NO. 75;

[0029] Group 22: Primers as shown in SEQ ID NO. 76-77, and probes as shown in SEQ ID NO. 78;

[0030] Group 23: Primers as shown in SEQ ID NO. 79-80, and probes as shown in SEQ ID NO. 81;

[0031] The primers consist of an upstream primer and a downstream primer.

[0032] Furthermore, it also includes the following primers and probes:

[0033] Group 24: Primers as shown in SEQ ID NO. 22-23, and probes as shown in SEQ ID NO. 24;

[0034] Group 25: Primers as shown in SEQ ID NO. 25-26, and probes as shown in SEQ ID NO. 27;

[0035] Group 26: Primers as shown in SEQ ID NO. 31-32, and probes as shown in SEQ ID NO. 33;

[0036] Group 27: Primers as shown in SEQ ID NO. 46-47, and probes as shown in SEQ ID NO. 48;

[0037] Group 28: Primers as shown in SEQ ID NO. 82-83, and probes as shown in SEQ ID NO. 84.

[0038] Furthermore, the molar ratio of the upstream primer, downstream primer, and probe is 1:1:0.5;

[0039] And / or, one end of the probe is modified with a fluorescent reporter group, and the other end is modified with a small groove binding group and a non-fluorescent quencher group.

[0040] This invention provides the use of the primer-probe pair based on TaqMan microfluidic chip technology for detecting vector-borne infectious disease pathogens as described in any of the preceding claims in the preparation of microfluidic chips or kits for detecting vector-borne infectious disease pathogens, wherein the vector-borne infectious disease pathogens include at least one of dengue virus type I, dengue virus type II, dengue virus type III, dengue virus type IV, Zika virus, Japanese encephalitis virus, Hantan virus, Lassa virus, Marburg virus, Rift Valley fever virus, West Nile virus, Sindbis virus, Dabie Bandar virus, Crimean-Congo fever virus, Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, Yersinia pestis, Orientia tsutsugamushi, Leptospira tsutsugamushi, Rickettsia typhus, Anaplasma phagocytophilum, and Bartonella.

[0041] Furthermore, the vector-borne infectious disease pathogens also include at least one of the following: Plasmodium ovale, yellow fever virus, chikungunya virus, Seoul virus, and Tulafrancella.

[0042] Furthermore, the annealing temperature for the PCR reaction of the microfluidic chip or kit is 58-60℃.

[0043] This invention provides a microfluidic chip for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology, wherein each reaction well is modified with a set of primer-probe pairs as described in any of the preceding claims.

[0044] Furthermore, the reaction wells are also provided with internal quality control wells and external quality control wells; the internal quality control wells are modified with primer-probe pairs of the 18S gene and RNase P gene, respectively, and the external quality control wells are modified with primer-probe pairs of the Xeno gene; and / or, the microfluidic chip is a TaqMan array card.

[0045] The present invention provides a method for preparing a microfluidic chip for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology as described in any of the preceding claims, comprising: modifying a set of primer-probe pairs from the primer-probe pair for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology into each reaction well.

[0046] The present invention provides a kit comprising primer-probe pairs for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology as described in any of the preceding claims.

[0047] In this invention, the numerical designations such as "Group 1", "Group 2", ..., "Group 28" serve only to distinguish different terms and do not indicate any priority or order, nor do they limit the scope of the technical features described.

[0048] This invention, based on TaqMan microfluidic chip technology, provides a method for simultaneously detecting 28 vector-borne infectious disease pathogens by optimizing primer-probe pairs, reaction systems, and conditions. The pathogens involved include bacteria, viruses, and parasites, representing a wide range with significant differences. This method achieves simultaneous detection of 28 vector-borne infectious disease pathogens under the same reaction conditions, exhibiting a low detection limit (5-20 copies / μL). When applied to clinical and animal tissue samples, it demonstrates high sensitivity, specificity, accuracy, and precision, with results showing good consistency with the gold standard method. Furthermore, compared to traditional qPCR methods, this method significantly reduces detection time, manpower, and material consumption, making it particularly suitable for scenarios involving unclear pathogen profiles requiring rapid and comprehensive screening, as well as for emergency response to public health events, demonstrating promising application prospects.

[0049] This invention solves the following problems and produces beneficial effects:

[0050] 1. Each reaction plate can simultaneously detect 1-8 samples, and can detect up to 384 target points. Each target point detection is a completely independent reaction unit, which solves the problem of complex primer and probe design.

[0051] 2. Single-well small-volume reaction, combined with TaqMan real-time PCR, is sensitive and accurate, solving the problem of reduced sensitivity and specificity in multiplex qPCR.

[0052] 3. Each reaction well is a single experiment, avoiding the steps of optimizing reagents and cross-contamination in multiple reactions, thus solving the problem of accuracy in multiplex qPCR quantification.

[0053] 4. The experimental operation is highly standardized, the system is stable, effectively eliminates systematic errors, facilitates the comparison of results between different batches or laboratories, and solves the problems of standardization and reproducibility of multiplex qPCR.

[0054] 5. It is easy to operate and can detect 28 vector-borne infectious disease pathogens in one sample within 2 hours, solving the problem of time-consuming and labor-intensive multiplex qPCR.

[0055] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0056] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0057] Figure 1 The layout diagram of the dedicated reaction board for TaqMan Array Card (TAC) is shown in Table 2, where the settings of serial numbers 1-31 are shown in Table 2.

[0058] Figure 2 This is a sample loading pattern diagram for a TAC-specific reaction plate. Detailed Implementation

[0059] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0060] Example 1: A method for detecting 28 vector-borne infectious disease pathogens using Taqman microfluidic chip technology.

[0061] 1. Pathogens being detected

[0062] Dengue virus type 1 (DV-1), Dengue virus type 2 (DV-2), Dengue virus type 3 (DV-3), Dengue virus type 4 (DV-4), Yellow fever virus (YFV), Chikungunya virus (CHIKV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), Hantaan virus (HTNV), Seoul virus (SEOV), Lassa virus (LASV), Marburg virus (MARV), Rift Valley fever virus (RVFV), West Nile virus (WNV), Sindbis virus (SV), Dabie virus (DBV) bandavirus, Crimean-Congo fever virus (CCHFV, Crimean-Congohemorrhagic fever virus), Plasmodium vivax ( Pv , Plasmodium vivax ), Plasmodium falciparum ( Pf , Plasmodium falciparum ), Plasmodium malariae ( PM , Plasmodium malariae ), Plasmodium ovale ( Po , Plasmodium ovale Yersinia pestis (Yersinia pestis) Y. pestis , Yersinia pestis Orientia scrub typhus ( Or , Orient tsutsugamushi ), question mark hook-shaped spiral ( L. interrogans , Leptospira asking ), typhoid rickettsia ( R. mooseri , Rickettsia mooseri ), Phagocytophilic anaplasm ( A. phagocytophilum , Anaplasma phagocytophilum Bartonella ( B.bacilliformis , Bartonella bacilliformis ), Tulafrancsis ( F. tularensis , Francisella tularensis ), totaling 28 types.

[0063] The genomes of the above pathogens span six major categories (+ssRNA, -ssRNA, segmented RNA, parasitic DNA, bacterial dsDNA, and spirochetal DNA), with significant differences in GC content and copy number.

[0064] (1) The pathogens with positive-sense single-stranded RNA (+ssRNA) type are: dengue virus type I–IV (DV-1~4, Flaviviridae, Flavivirus), yellow fever virus (YFV, same as above), Zika virus (ZIKV, same as above), Japanese encephalitis virus (JEV, same as above), West Nile virus (WNV, same as above), Chikungunya virus (CHIKV, Bryophyllidae, Aviridae), and Sindbis virus (SV, same as above).

[0065] (2) The pathogens with negative-sense single-stranded RNA (ssRNA) type are: Lassa virus (LASV, Arenaviridae), Marburg virus (MARV, Filoviridae), Rift Valley fever virus (RVFV, Sandflyvirus, Bunyaviridae), and Crimean-Congo hemorrhagic fever virus (CCHFV, Nairovirus, Bunyaviridae).

[0066] (3) The pathogens with segmented RNA are: Hantan virus (HTNV, Bunyaviridae, Hantavirus, 3 segments), Seoul virus (SEOV, same as above), Dabiebanda virus (DBV, Bunyaviridae, Sandflyvirus, 3 segments), Rift Valley fever virus (RVFV, Bunyaviridae, Sandflyvirus, 3 segments), Crimean-Congo hemorrhagic fever virus (CCHFV, Bunyaviridae, Nairovirus, 3 segments), and Lassa virus (LASV, Arenaviridae, 2 segments).

[0067] (4) The pathogens with parasitic DNA types are: Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, and Plasmodium ovale.

[0068] (5) The pathogens with bacterial dsDNA types are: Yersinia pestis (Proteobacteria, circular dsDNA), Orientia tsutsugamushi (same as above), Rickettsia mooseri (same as above), Anaplasma phagocytophilum (same as above), Bartonella bacilliformis (same as above), and Francisella tularensis (same as above).

[0069] (6) The pathogen with spirochete DNA type is: Leptospira interrogans (spirochete phylum).

[0070] 2. Primer-probe pair

[0071] For 28 vector-borne infectious disease pathogens, multiple sets of candidate primer and probe sequences were designed and generated, and the primer and probe combinations with the best efficiency and specificity were obtained through screening, as shown in Table 1.

[0072] Table 1 Primer and probe sequences

[0073]

[0074]

[0075] Note: Degenerate base Y represents C or T; degenerate base R represents G or A; degenerate base M represents C or A; degenerate base W represents A or T; degenerate base H represents C, A, or T; F represents the upstream primer; R represents the downstream primer; P represents the MGB (minor groove binder) probe. The 5' end of the MGB probe is labeled with a fluorescent group (FAM), and the 3' end is labeled with an MGB group (TaqMan MGB Probe (Applied Biosystems)), namely a non-fluorescent quencher (NFQ) group and a minor groove binder. In each primer-probe combination, the molar ratio of the upstream primer, downstream primer, and probe is 1:1:0.5.

[0076] 3. Microfluidic chip

[0077] The detection primers and probes for the 28 vector-borne infectious disease pathogens shown in Table 1 were immobilized on a TaqMan Array Card (TAC) reaction plate by Applied Biosystems, Inc. The TaqMan microfluidic chip is an eight-channel reaction plate designed based on qPCR reactions. Each channel contains 48 1μL reaction wells, allowing for the simultaneous execution of 384 qPCR reactions. Each reaction well is pre-embedded with the target pathogen detection primer pair and TaqMan probe. The TAC layout is shown in Table 2. Figure 1 As shown in the diagram. The TAC sampling pattern is as follows. Figure 2 As shown.

[0078] Table 2 TAC Layout

[0079]

[0080] Note: TAC assay uses 18S and IC Rnase P as internal quality control genes, and Xeno as an exogenous quality control gene.

[0081] 4. Detection Method

[0082] (1) Detection steps

[0083] The sample to be tested is an animal tissue or a clinical sample.

[0084] Nucleic acid was extracted using a magnetic bead enrichment method: all nucleic acid extraction kits were from Jiangsu ShuoShi Biotechnology Co., Ltd. Animal tissues were extracted using a pathogen total nucleic acid extraction kit, while clinical samples were extracted using a viral DNA / RNA co-extraction kit.

[0085] A one-step reagent-based TAC reaction system was constructed for sample loading. The TAC reaction system and reaction conditions are shown in Table 3. Real-time quantitative PCR was performed using an ABI QUANTSTUDIO DX instrument. Negative control: nuclease-free water; exogenous control: 2 × 10⁻⁶.5 copies / μL, Xeno.

[0086] Table 3 TAC Reaction System and Conditions

[0087]

[0088] Note: The Mix buffer specifically uses commercially available TaqMan Fast 1-step Virus Master Mix (Applied Biosystems).

[0089] (2) Judgment criteria

[0090] Each amplification showed normal results for both the exogenous control and negative control, indicating no contamination; each internal control also showed normal results. Specifically, the exogenous and internal controls exhibited typical S-shaped amplification curves with Ct values ​​< 34, while the negative control showed no amplification curve or a Ct value. This demonstrates the effectiveness of the test.

[0091] The cutoff value is set at 36. A Ct value less than 36 indicates a positive sample; a Ct value between 36 and 38 indicates a suspected sample; and a Ct value greater than 38 indicates a negative sample.

[0092] The method in this embodiment has high accuracy, sensitivity, specificity and precision.

[0093] The technical solution of the present invention will be further explained through experiments below.

[0094] Experimental Example 1: Linearity and Detection Limit

[0095] I. Experimental Methods

[0096] 1. Preparation of positive plasmids

[0097] Positive plasmid standards were prepared for the 28 vector-borne infectious disease pathogens in Example 1. The target fragments were digested with enzymes and ligated into a cloning vector (pUC57-Amp, 2710 bp). Positive plasmid standards were obtained through transformation and screening. The target fragments for each pathogen are shown in Table 4.

[0098] Table 4 Target Fragment Information

[0099]

[0100] 2. Amplification efficiency and linearity

[0101] Positive plasmids of 28 vector-borne infectious disease pathogens were mixed to prepare 10 5 ~10 1Five concentration gradients were set up with copies / μL, and each gradient was repeated three times. A standard curve was plotted. The system was constructed and PCR reaction was performed using the one-step reagents in Table 5. The amplification efficiency and linearity were calculated.

[0102] Table 5 System construction and PCR reaction

[0103]

[0104] Note: The Mix buffer specifically uses commercially available TaqMan Fast 1-step Virus Master Mix (Applied Biosystems).

[0105] Amplification efficiency and linearity: Ct values ​​were obtained using the above PCR procedure. A standard curve was plotted based on the logarithmic values ​​of each sample concentration and the corresponding Ct values ​​to obtain the linear equation, where the slope is k and the correlation coefficient R is [value missing]. 2 For linearity, the amplification efficiency is E = 10^(-1 / k) - 1.

[0106] 3. Detection limit

[0107] Positive plasmids of 28 vector-borne infectious disease pathogens were mixed and prepared into four concentration gradients of 100, 20, 10, and 5 copies / μL. Each concentration gradient was replicated eight times. The lowest concentration level at which no negative results were observed was the limit of detection.

[0108] II. Experimental Results

[0109] The results for linearity, amplification efficiency, and detection limit are shown in Table 6. Linearity R 2 The values ​​ranged from 0.9945 to 1.0000, all greater than 0.99; the amplification efficiencies were all greater than 90%; the detection limits for various pathogens were low, ranging from 5 to 20 copies / μL, demonstrating high sensitivity.

[0110] Table 6 Linearity, Amplification Efficiency, and Detection Limit

[0111]

[0112] Experiment Example 2 Repeatability

[0113] I. Experimental Methods

[0114] The positive plasmids of 28 vector-borne infectious disease pathogens prepared by the method in Example 1 were mixed to prepare 10 4 10 3 10 2There were three concentration gradients of copies / μL. Each concentration gradient was replicated 6 times within the plate and 3 times between the plates. The coefficient of variation was calculated.

[0115] II. Experimental Results

[0116] The results of the intraplate coefficient of variation are shown in Table 7. The intraplate coefficient of variation (CV) is less than 3%.

[0117] Table 7 Intraplate Coefficient of Variation

[0118]

[0119] The results of the inter-plate coefficient of variation are shown in Table 8. The inter-plate coefficient of variation (CV) is less than 3%.

[0120] Table 8. Inter-plate variation coefficient

[0121]

[0122] In summary, the coefficients of variation within and between plates were both less than 10%, indicating good repeatability and a stable method.

[0123] Experiment Example 3 Sample Detection

[0124] I. Experimental Methods

[0125] 1. Experimental Samples and Detection Methods

[0126] A total of 19 clinical specimens (plasma, serum, and whole blood from clinical patients) and 22 animal tissue specimens (mouse lung, liver, spleen, and kidney) were collected from the Chengdu Center for Disease Control and Prevention. They were collected between September 2021 and September 2023 and stored at -80°C. All 41 samples were tested using existing conventional gold standard methods (single-particle real-time quantitative PCR detection or staining microscopy) and the method described in Example 1 of this invention. Of these, 21 were positive (specifically, samples infected with dengue virus, malaria parasites, chikungunya virus, Seoul virus, or Leptospira question mark), and 20 were negative.

[0127] The traditional gold standard method is as follows:

[0128] For dengue virus, chikungunya virus, Seoul virus, and malaria parasites, commercially available single qPCR detection kits were used, and the PCR reaction system and reaction conditions were performed according to the instructions. For malaria parasites (Giemsa staining or Wright staining) and leptospires (Fontana staining), staining and microscopic examination were used.

[0129] 2. Performance evaluation indicators

[0130] The accuracy, sensitivity, and specificity of the TAC method were evaluated using traditional detection methods as the gold standard. The accuracy, sensitivity, and specificity were calculated as follows: A 2×2 contingency table was constructed, with the gold standard as the column and the method of this invention as the row, to count true positives (TP), false positives (FP), false negatives (FN), and true negatives (TN). The performance index was calculated using the following formula:

[0131] Sensitivity = TP / (TP + FN)

[0132] Specificity = TN / (FP + TN)

[0133] Accuracy = (TP + TN) / Total number of cases

[0134] Results are expressed as percentages, with 95% confidence intervals provided.

[0135] 3. Consistency check

[0136] The Kappa coefficient was calculated using GraphPad Prism statistical software to test the consistency. The criteria for the Kappa coefficient were: 0.0-0.20, very low consistency; 0.21-0.40, moderate consistency; 0.41-0.60, moderate consistency; 0.61-0.80, high consistency; 0.81-1.0, almost perfect consistency. A p-value < 0.05 was considered statistically significant.

[0137] II. Experimental Results

[0138] Compared with the gold standard method, the detection results of the detection method of the present invention are shown in Table 9. The accuracy is 97.56% (95% CI: 87.14%, 99.94%), the sensitivity is 100.00% (95% CI: 89.39%, 100%), and the specificity is 95.00% (95% CI: 75.13%, 99.87%), indicating that the detection method of the present invention has high accuracy, sensitivity and specificity.

[0139] Table 9 Performance Evaluation Indicators

[0140]

[0141] Note: "TAC+" indicates a positive TAC test result, and "TAC-" indicates a negative TAC test result.

[0142] The consistency test results are shown in Table 10. The Kappa coefficients are all greater than 0.81, indicating good consistency.

[0143] Table 10 Consistency Test Results

[0144]

[0145] The above results demonstrate that the method of the present invention exhibits high accuracy, sensitivity, specificity, and precision when used for actual detection of animal and clinical samples. Furthermore, the detection results show good consistency with traditional gold standard methods, meeting the needs of practical testing.

[0146] Currently, the traditional qPCR method has the following disadvantages:

[0147] (1) Challenges in detecting low-copy-number samples: Accurate detection of samples with low copy numbers, such as viruses, presents certain difficulties. When the initial template amount is extremely low, the PCR reaction may be affected by random factors, such as the uneven distribution of a small number of template molecules and fluctuations in amplification efficiency during early cycles. This may lead to poor reproducibility of results and make it difficult to accurately quantify the initial template concentration. For example, in the early detection of novel coronavirus infection, when patients are in the early stage of infection and the viral load is very low, real-time quantitative PCR may not be able to detect the virus in a timely and accurate manner, requiring multiple tests for diagnosis.

[0148] (2) Challenges in primer and probe design: Primer and probe design is crucial for detection specificity. If primers have high homology with non-target sequences, non-specific amplification may occur. For example, in the detection of complex microbial communities, the designed primers may partially match the gene sequences of multiple microorganisms, leading to the amplification of other microorganisms besides the target microorganism, thus interfering with the detection results. Polymorphism in gene sequences also poses difficulties for primer and probe design. The same gene in different individuals or strains may have variations such as single nucleotide polymorphisms (SNPs), which may cause primers and probes to fail to bind effectively, resulting in false negative results, or bind to unexpected sites, resulting in false positive results. For multiplex PCR reaction systems, the coexistence of multiple primers and probes can easily lead to non-specific amplification and cross-reaction, affecting the accuracy of the results, and optimization is difficult, complex, and time-consuming.

[0149] (3) Cross-reaction in multiplex PCR: When performing multiplex PCR (simultaneous detection of multiple target genes), different primers and probes may interact and produce cross-reactions. In multiplex PCR systems for detecting multiple pathogens, primers for different pathogens may form primer dimers, consuming primers and reaction substrates and reducing the amplification efficiency of the target gene.

[0150] (4) Reduced sensitivity and specificity: In multiplex PCR reaction systems, multiple target sequences compete for PCR reagents (such as Taq enzymes and dNTPs) in the same reaction system, which may reduce the amplification efficiency of some target sequences, thereby affecting sensitivity and specificity; spectral overlap between different fluorescent dyes may cause signal interference and affect quantitative accuracy.

[0151] (5) Quantitative accuracy issues: Multiplex qPCR requires the establishment of a standard curve for each target gene, which becomes more complex and cumbersome in multiplex detection; the amplification efficiency of different target genes may be different, which affects the accuracy of quantitative results.

[0152] (6) Standardization and reproducibility issues: Multiplex qPCR has very strict requirements for experimental conditions, and slight deviations may lead to inconsistent results; moreover, there are differences in reproducibility and comparability between different laboratories, making it difficult to achieve standardization and high reproducibility.

[0153] To address the aforementioned shortcomings, this invention, based on TaqMan microfluidic chip technology, provides a method for simultaneously detecting 28 vector-borne infectious disease pathogens by optimizing primer-probe pairs, reaction systems, and conditions. These pathogens encompass a wide range of bacteria, viruses, and parasites, exhibiting significant variability. This method enables the simultaneous detection of 28 vector-borne infectious disease pathogens under the same reaction conditions, possessing a low detection limit (5-20 copies / μL). When applied to clinical and animal tissue samples, it demonstrates high sensitivity, specificity, accuracy, and precision, with results showing good consistency with the gold standard method. Furthermore, compared to traditional qPCR methods, this method significantly reduces detection time, manpower, and material consumption, making it particularly suitable for scenarios involving unclear pathogen profiles requiring rapid and comprehensive screening, as well as for emergency response to public health events, demonstrating promising application prospects.

Claims

1. A primer-probe pair for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology, characterized in that, It includes the following primers and probes: Group 1: Primers as shown in SEQ ID NO. 1-2, and probes as shown in SEQ ID NO. 3; The second group consists of primers as shown in SEQ ID NO. 4-5 and probes as shown in SEQ ID NO.

6. The third group includes primers as shown in SEQ ID NO. 7-8 and probes as shown in SEQ ID NO.

9. Group 4: Primers as shown in SEQ ID NO. 10-11, and probes as shown in SEQ ID NO. 12; Group 5: Primers as shown in SEQ ID NO. 13-14, and probes as shown in SEQ ID NO. 15; Group 6: Primers as shown in SEQ ID NO. 16-17, and probes as shown in SEQ ID NO. 18; Group 7: Primers as shown in SEQ ID NO. 19-20, and probes as shown in SEQ ID NO. 21; Group 8: Primers as shown in SEQ ID NO. 28-29, and probes as shown in SEQ ID NO. 30; Group 9: Primers as shown in SEQ ID NO. 34-35, and probes as shown in SEQ ID NO. 36; Group 10: Primers as shown in SEQ ID NO. 37-38, and probes as shown in SEQ ID NO. 39; Group 11: Primers as shown in SEQ ID NO. 40-41, and probes as shown in SEQ ID NO. 42; Group 12: Primers as shown in SEQ ID NO. 43-44, and probes as shown in SEQ ID NO. 45; Group 13: Primers as shown in SEQ ID NO. 49-50, and probes as shown in SEQ ID NO. 51; Group 14: Primers as shown in SEQ ID NO. 52-53, and probes as shown in SEQ ID NO. 54; Group 15: Primers as shown in SEQ ID NO. 55-56, and probes as shown in SEQ ID NO. 57; Group 16: Primers as shown in SEQ ID NO. 58-59, and probes as shown in SEQ ID NO. 60; Group 17: Primers as shown in SEQ ID NO. 61-62, and probes as shown in SEQ ID NO. 63; Group 18: Primers as shown in SEQ ID NO. 64-65, and probes as shown in SEQ ID NO. 66; Group 19: Primers as shown in SEQ ID NO. 67-68, and probes as shown in SEQ ID NO. 69; Group 20: Primers as shown in SEQ ID NO. 70-71, and probes as shown in SEQ ID NO. 72; Group 21: Primers as shown in SEQ ID NO. 73-74, and probes as shown in SEQ ID NO. 75; Group 22: Primers as shown in SEQ ID NO. 76-77, and probes as shown in SEQ ID NO. 78; Group 23: Primers as shown in SEQ ID NO. 79-80, and probes as shown in SEQ ID NO. 81; The primers are a combination of upstream and downstream primers.

2. The primer-probe pair for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology according to claim 1, characterized in that, It also includes the following primers and probes: Group 24: Primers as shown in SEQ ID NO. 22-23, and probes as shown in SEQ ID NO. 24; Group 25: Primers as shown in SEQ ID NO. 25-26, and probes as shown in SEQ ID NO. 27; Group 26: Primers as shown in SEQ ID NO. 31-32, and probes as shown in SEQ ID NO. 33; Group 27: Primers as shown in SEQ ID NO. 46-47, and probes as shown in SEQ ID NO. 48; Group 28: Primers as shown in SEQ ID NO. 82-83, and probes as shown in SEQ ID NO.

84.

3. The primer-probe pair for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology according to claim 1, characterized in that: The molar ratio of the upstream primer, downstream primer, and probe is 1:1:0.5; And / or, one end of the probe is modified with a fluorescent reporter group, and the other end is modified with a small groove binding group and a non-fluorescent quencher group.

4. The use of the primer-probe pair based on TaqMan microfluidic chip technology for detecting vector-borne infectious disease pathogens as described in claim 2 in the preparation of microfluidic chips or kits for detecting vector-borne infectious disease pathogens, characterized in that: The vector-borne infectious disease pathogens include at least one of the following: dengue virus type I, dengue virus type II, dengue virus type III, dengue virus type IV, Zika virus, Japanese encephalitis virus, Hantan virus, Lassa virus, Marburg virus, Rift Valley fever virus, West Nile virus, Sindbis virus, Dabie Bandar virus, Crimean-Congo fever virus, Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, Yersinia pestis, Orientia tsutsugamushi, Leptospira question mark, Rickettsia typhi, Anaplasma phagocytophilum, Bartonella, Plasmodium ovale, yellow fever virus, Chikungunya virus, Seoul virus, and Tula Francisella.

5. A microfluidic chip for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology, characterized in that: Each reaction well is modified with a set of primer-probe pairs according to any one of claims 1-3 for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology.

6. The microfluidic chip for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology according to claim 5, characterized in that: The reaction wells also include internal quality control wells and external quality control wells; the internal quality control wells are modified with primer-probe pairs of the 18S gene and RNase P gene, respectively, and the external quality control wells are modified with primer-probe pairs of the Xeno gene; and / or, the microfluidic chip is a TaqMan array card.

7. The method for preparing a microfluidic chip for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology as described in claim 5 or 6, characterized in that, It includes: In each reaction well, one set of primer-probe pairs from the primer-probe pair for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology is modified.

8. A reagent kit, characterized in that: It includes the primer-probe pair for detecting vector-borne infectious disease pathogens based on TaqMan microfluidic chip technology as described in any one of claims 1-3.

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