Compositions, kits, and applications for triple real-time fluorescence PCR detection of pathogenic Yersinia pestis based on TaqMan probes.

Abstract

This invention provides a composition, kit, and application for triple real-time fluorescence PCR detection of pathogenic Yersinia pestis based on TaqMan probes. Specific primers and TaqMan probes are designed targeting the caf1 gene of Yersinia pestis, the foxA gene of Yersinia enterocolitica, and the inv gene of Yersinia pseudotuberculosis, enabling simultaneous detection of these three pathogens in a single reaction system. The optimized annealing temperature is 54℃, and the final probe concentration is 0.3 μM. This method exhibits good linearity (R²>0.99), with detection limits of 5×10² copies / μL for I9 and C4, and 1×10¹ copies / μL for F4. Intra-assay and inter-assay coefficients of variation are mostly below 2%, and there is no cross-reactivity with non-target bacteria. It demonstrates good sensitivity, specificity, and repeatability, and can be used for rapid detection of pathogenic Yersinia pestis in food, environmental, clinical, and animal-derived samples.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to a composition, kit, and application of triple real-time fluorescence PCR for detecting pathogenic Yersinia based on TaqMan probes. Background Technology

[0002] Yersinia spp. are important zoonotic pathogens. Among them, Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are three major pathogens that can cause diseases such as plague, pseudotuberculosis, and Yersinia enterocolitica, respectively, posing a significant threat to public health, food safety, and animal health.

[0003] Studies have shown that all three pathogenic Yersinia species can be maintained in the ecological environment for extended periods through natural hosts such as rodents. Therefore, the simultaneous detection and identification of these three pathogens in animal-derived, food, and environmental samples is of great significance for disease surveillance, epidemic early warning, and public health control.

[0004] Currently, detection methods for pathogenic Yersinia pestis mainly include bacterial isolation and culture, serological detection, and PCR molecular detection technology. Among these, real-time quantitative PCR (qPCR) is widely used in pathogen detection due to its advantages such as high sensitivity, strong specificity, and fast detection speed. However, existing detection methods mostly focus on single or dual pathogen detection, and there is still a lack of a triple real-time fluorescence PCR method that can simultaneously and rapidly detect and identify Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica in a single reaction system.

[0005] Therefore, developing a triple real-time fluorescent PCR detection system capable of simultaneously detecting and identifying three pathogenic Yersinia species is of great significance for improving detection efficiency, reducing detection costs, increasing detection throughput, and enhancing on-site rapid screening capabilities. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a composition, kit and application for the detection of pathogenic Yersinia pestis using triple real-time fluorescence PCR based on TaqMan probes, so as to achieve simultaneous, rapid, specific and sensitive detection of Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis in a single tube.

[0007] This invention provides a composition for triple real-time fluorescence PCR detection of pathogenic Yersinia pestis based on TaqMan probes, comprising primer pairs and TaqMan probes targeting the caf1 gene of Yersinia pestis, the foxA gene of Yersinia enterocolitica, and the inv gene of Yersinia pseudotuberculosis, respectively; wherein:

[0008] The primer pairs for the caf1 gene are shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe sequence is shown in SEQ ID NO:3;

[0009] The primer pairs for the foxA gene are shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe sequence is shown in SEQ ID NO:6.

[0010] The primer pairs for the inv gene are shown in SEQ ID NO:7 and SEQ ID NO:8, and the probe sequence is shown in SEQ ID NO:9.

[0011] Optionally, the forward primer sequence of the caf1 gene primer pair is SEQ ID NO:1, and the reverse primer sequence of the caf1 gene primer pair is SEQ ID NO:2; the forward primer sequence of the foxA gene primer pair is SEQ ID NO:4, and the reverse primer sequence of the foxA gene primer pair is SEQ ID NO:5; the forward primer sequence of the inv gene primer pair is SEQ ID NO:7, and the reverse primer sequence of the inv gene primer pair is SEQ ID NO:8.

[0012] Optionally, the TaqMan probe is labeled with different fluorescent reporter groups and quencher groups:

[0013] caf1 probe label: ROX / BHQ-2;

[0014] foxA probe label: CY5 / BHQ-3;

[0015] inv probe label: FAM / BHQ-1.

[0016] Optionally, the primers for the caf1 gene primer pair, the foxA gene primer pair, and the inv gene primer pair are C4, F4, and I9, respectively.

[0017] Optionally, the caf1 gene primer pair, the foxA gene primer pair, and the inv gene primer pair are amplified and then cloned into the pESI-T vector to obtain recombinant plasmids pESI-T-I9, pESI-T-C4, and pESI-T-F4, respectively.

[0018] This invention provides a kit for detecting pathogenic Yersinia pestis using triple real-time fluorescence PCR based on TaqMan probes, comprising the detection composition described above, and further comprising qPCR premix, positive control plasmid, negative control, and enzyme-free ultrapure water.

[0019] The present invention also provides the use of any of the above-described compositions or the above-described kits in the preparation of pathogenic Yersinia pestis detection products.

[0020] Optionally, the pathogenic Yersinia species are: Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) Using a single-tube three-channel synchronous detection mode, the identification and detection of three pathogens, Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis, can be completed in one PCR reaction without the need for separate single reactions, which greatly shortens the detection time and increases the detection throughput, making it suitable for large-scale sample screening.

[0023] (2) Primers and probes are designed for specific conserved genes of the three pathogens. Combined with probes with different fluorescent labels, specific recognition is achieved without cross-reaction. This can effectively distinguish the target pathogen from other common pathogens and avoid missed detection and false detection. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 PCR identification of recombinant bacterial cultures (M. Marker; 2. pESI-T-I9; 4. pESI-T-C4; 6. pESI-T-F4; 1, 3, 5 negative controls).

[0026] Figure 2 These are the preliminary amplification results of triple real-time quantitative PCR;

[0027] Figure 3 Optimization of annealing temperature for triple TaqMan real-time quantitative PCR detection; (Amplification curves of three target genes (inv[I9], caf1[C4], and foxA[F4]) at different annealing temperatures (52-58°C). The experiment used a series of 10-fold dilutions (10... 6 -104 (Copies / μL) were used as templates. The optimal annealing temperature was determined based on amplification efficiency and curve characteristics.

[0028] Figure 4 The amplification curves for triple real-time fluorescence PCR detection (for targets I9(A), C4(B), and F4(C)) were generated using a series of ten-fold dilutions of the recombinant plasmid. 1×10 10 -1×10 1 (copies / μL));

[0029] Figure 5 The standard curves for triplet real-time fluorescence PCR detection (for targets I9(A), C4(B), and F4(C)) were established using serial 10-fold dilutions of the recombinant plasmid. 1×10 10 -1×10 1 (copies / μL));

[0030] Figure 6 Sensitivity assays for inv, caf1, and foxA using conventional PCR methods;

[0031] Figure 7 For sensitivity analysis of triple real-time quantitative PCR;

[0032] Figure 8 This is for the specificity analysis of triple real-time quantitative PCR. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0034] Example 1 Test Method

[0035] (1) Primer and probe design

[0036] Reference sequences of different genotypes of the inv, foxA, and caf1 genes were downloaded from the GenBank database of NCBI (Table 1). Multiple sequence alignment analysis was performed on the target sequences using SnapGene software to screen highly conserved regions of each gene as target fragments. Sequences with <70% homology to non-target organisms and <60% query coverage were retained. Specific primers and TaqMan probes were designed using Primer Express 3.0 software. The designed primer sequences (Table 2) were submitted to the NCBI database for BLAST homology comparison to verify their cross-reactivity with non-target genes and ensure primer specificity. The primer and probe sequences were synthesized by Sangon Biotech Co., Ltd., and the synthesized products were processed using enzyme-free ultrapure water (…). Dilute to 10 μmol / L, dispense into containers, and store at 4 ℃ for later use. Avoid repeated freeze-thaw cycles to prevent affecting its activity.

[0037] Table 1. Genomic strain information used for primer design of TaqMan probes

[0038]

[0039] Table 2 Primers and probes

[0040]

[0041] (2) Sample processing and nucleic acid extraction

[0042] Genomic DNA was extracted using a commercial bacterial DNA extraction kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions. The samples were then stored... Until the analysis.

[0043] The target gene fragments (inv, caf1, and foxA) were amplified and cloned into the pESI-T vector to obtain recombinant plasmids, namely pESI-T-I9, pESI-T-C4, and pESI-T-F4.

[0044] Specifically, the national reference samples of Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica were allowed to thaw naturally at room temperature. After complete thawing, they were gently inverted and mixed to obtain samples for nucleic acid extraction. Genomic DNA of the target strains (the three Yersinia species mentioned above) and non-target strains was extracted using the TIANAMP Genomic DNA kit. Before the operation, the specified volume of anhydrous ethanol was added to the GD buffer and PW wash buffer according to the kit instructions, and the mixture was thoroughly mixed before proceeding with the nucleic acid extraction operation as follows:

[0045] (a) Take 0.5 mL of the target strain and non-target strain into centrifuge tubes, centrifuge at 12000 rpm / min for 1 min, and extract DNA from the precipitate.

[0046] (b) Add 200 μL of buffer GA to the precipitate and shake until completely suspended.

[0047] (c) Add 20 μL of proteinase K solution to the suspension and mix well.

[0048] (d) Add 200 μL of buffer GB to the tube, mix thoroughly by inverting, and place in a 70 °C water bath for 10 min to allow the solution to become clear. Briefly centrifuge to remove water droplets from the tube wall.

[0049] (e) Add 200 μL of anhydrous ethanol to the tube, shake well for 15 sec, and briefly centrifuge to remove water droplets from the tube wall.

[0050] (f) Add the liquid obtained in step (5) into the CB3 adsorption column, centrifuge at 12000 rpm for 30 sec, and discard the waste liquid. Return the adsorption column to the collection tube.

[0051] (g) Add 500 μL of buffer GD to the adsorption column, centrifuge at 12000 rpm for 30 sec, discard the waste liquid, and put the adsorption column back into the collection tube.

[0052] (h) Add 600 μL of washing solution to the adsorption column, centrifuge at 12000 rpm for 30 sec, discard the waste liquid, and put the adsorption column back into the collection tube.

[0053] (g) Repeat step (h).

[0054] (h) Place the adsorption column back into the collection tube, centrifuge at 12,000 rpm for 2 min, discard the waste liquid, and place the adsorption column at room temperature for 10 min to thoroughly dry the residual bleach and anhydrous ethanol in the adsorption material.

[0055] (i) Place the adsorption column in a new centrifuge tube, add 50 μL-200 μL of elution buffer TE in the air, incubate at room temperature for 2-5 min, centrifuge at 12000 rpm for 2 min, and collect the DNA extract into the centrifuge tube for testing.

[0056] (j) The purity and concentration were measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the extracted DNA was stored in a refrigerator at -20°C.

[0057] (3) Amplification of the target gene

[0058] Using extracted nucleic acids from the inv, caf1, and foxA genes as templates, conventional PCR amplification was performed using designed TaqMan real-time quantitative PCR specific primers. Reaction conditions: pre-denaturation 94 ℃ for 30 s; 94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 30 s; 30 cycles, 72 ℃ for 2 min.

[0059] Table 3. Standard PCR Reaction System

[0060]

[0061] (4) PCR product recovery and purification

[0062] The PCR products were loaded onto a 2% agarose gel for electrophoresis at 120 V for 30 min. After electrophoresis, the target band was excised and purified according to the DNA gel extraction kit instructions. The purified product was immediately ligated and transformed.

[0063] (5) Preparation of recombinant plasmid standards

[0064] The recovered gel products were TA-linked using the pESI-T support to prepare a 10 μL reaction system (Table 4).

[0065] The above ligation system was placed in a metal bath at 25 °C for 5 min. After the reaction, DH5α competent cells were immediately transformed according to the instructions. The transformed competent cells were plated on LB medium containing Amp antibiotic and cultured at 37 °C for 12 h. After culture, single colonies were picked and inoculated into LB liquid medium and cultured overnight at 37 °C and 250 rpm. The overnight culture was used to extract recombinant plasmids using a high-purity plasmid DNA mini-extraction kit according to the instructions and sent to Sangon Biotech for sequencing. The concentration of successfully ligated and sequenced recombinant plasmids was determined using a nucleic acid protein analyzer, copies were calculated, and 10-fold serial dilutions were performed. The diluted plasmids were stored as positive standards at -20 °C for later use. Copies calculation formula:

[0066] 6.02×10 14 × plasmid concentration (ng / μL) / MW (average molecular weight) = copies / μL

[0067] For dsDNA, MW = number of bases × 650 (Dalton / number of bases).

[0068] Table 4 Connection System

[0069]

[0070] (6) TaqMan triple real-time quantitative PCR amplification

[0071] Using specific primers and probes, and positive standards as templates, TaqMan triplet real-time quantitative PCR amplification was performed according to the recommended reaction system and conditions in the reagent instructions. This preliminarily verified primer specificity and provided a basis for subsequent optimization. Reaction conditions: 95 ℃ for 30 s; 95 ℃ for 5 s, 55 ℃ for 30 s, for a total of 45 cycles.

[0072] Table 5 Triple Real-Time Quantitative PCR Reaction System

[0073]

[0074] (7) Optimization of TaqMan triple real-time quantitative PCR reaction system and reaction conditions

[0075] Based on the above reaction system and conditions, the system was optimized using a gradient annealing temperature combined with a matrix method for probe concentration. Referring to similar studies, the final primer concentration was fixed at 0.2 μM; the final probe concentrations were set at five gradients: 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, and 0.5 μM; and the annealing temperature gradients were set at 58 ℃, 56 ℃, 54 ℃, and 52 ℃. Under the premise of consistent template concentration, the lowest Ct value was used as the core screening criterion; if the Ct values ​​were similar under different conditions, a higher annealing temperature was preferred to improve reaction specificity and reduce non-specific amplification.

[0076] (8) Establishment of TaqMan triple real-time quantitative PCR standard curve

[0077] After a 10-fold serial dilution, the number of copies was 10. 10 -10 1 1.5 μL of positive standard (copies / μL) was added to the optimized reaction system as template, and ddH2O was used as the negative control template. Triple real-time PCR amplification was performed according to the optimized reaction conditions.

[0078] (9) TaqMan triple real-time quantitative PCR sensitivity experiment

[0079] After a 10-fold serial dilution, the number of copies was 10. 8 -10 0 Using 1.5 μL of positive standard (copies / μL) as template and dd H2O as template for negative control, TaqMan triple real-time quantitative PCR and conventional PCR amplification were performed separately to compare the sensitivity of the two methods. Furthermore, TaqMan triple real-time quantitative PCR was used to detect the positive standard separately and simultaneously, repeated three times, to further evaluate the impact of the co-existence of multiple pathogens on sensitivity.

[0080] (10) TaqMan triple real-time quantitative PCR specificity experiment

[0081] Using extracted positive standards and nucleic acids from *Francis tularensis*, *Brucella*, *Bacillus anthracis*, *Vibrio cholerae*, *Salmonella typhi*, and *Shigella* as templates, TaqMan triple real-time quantitative PCR amplification was performed according to the optimized reaction system and conditions to verify the specificity of the established TaqMan triple real-time quantitative PCR method.

[0082] (11) TaqMan triple real-time quantitative PCR repeatability experiment

[0083] After a 10-fold serial dilution, the number of copies was 10. 6 -10 4 Using positive standards (copies / μL) as templates, three replicate tests were performed using the established TaqMan triplet real-time quantitative PCR method for intra-group replicate experiments. The same positive standards were then used for three replicate tests every week under the same reaction conditions for inter-group replicate experiments. The intra- and inter-group coefficients of variation were calculated to verify the reproducibility of the established TaqMan triplet real-time quantitative PCR method.

[0084] Example 2 Test Results

[0085] (1) Identification of recombinant plasmids

[0086] By bacterial culture PCR identification, the recombinant plasmids pESI-T-I9, pESI-T-C4, and pESI-T-F4 were amplified to produce three target bands of approximately 148 bp, 150 bp, and 185 bp, respectively. Figure 1 Sequencing analysis of the bacterial cultures that tested positive showed 100% homology between the sequencing results and the target gene sequence via BLAST alignment, indicating successful construction of the recombinant plasmid.

[0087] (2) Preparation of positive standards

[0088] The extracted plasmids were named inv-9, caf1-4, and foxA-4, respectively. Their concentrations and A260 / A280 ratios were measured using a nucleic acid protein analyzer, and the corresponding copies were calculated (Table 6). All of the above plasmids were serially diluted 10-fold and stored at -20 ℃ for later use.

[0089] Table 6. Results of positive plasmid preparation

[0090]

[0091] (3) Preliminary amplification results of TaqMan triple real-time quantitative PCR

[0092] The three extracted positive templates were added to the same reaction system for preliminary amplification using TaqMan triple real-time quantitative PCR. Figure 2 The results showed that the negative control was valid, and all three positive templates exhibited typical amplification curves, indicating that the primers and probes had good specificity and the reaction system could be further optimized.

[0093] (4) Optimization of TaqMan triple real-time quantitative PCR reaction system and reaction conditions

[0094] To optimize the TaqMan triple real-time quantitative PCR reaction system, a systematic comparison was made of different annealing / extension temperatures (52–58 ℃) and probe concentrations (0.1–0.5 μM) (Table 7).

[0095] The results showed that, Figure 3 As shown, at 52℃, amplification specificity was poor, especially with multiple instances of undetectable C4 targets (No Cq), indicating non-specific amplification or insufficient amplification efficiency. With increasing temperature, amplification specificity gradually improved, and the Ct value tended to stabilize. At 54℃, all three targets—I9, F4, and C4—exhibited good amplification performance, with low Ct values ​​and good reproducibility. Further increasing the temperature to 56–58℃ resulted in an overall increase in Ct values, indicating a decrease in amplification efficiency.

[0096] Regarding probe concentration, at 0.1 μM, the amplification signals of some targets (especially C4) were weak or even undetectable; when the concentration was increased to 0.3 μM, the amplification curves of each target were stable, and the signal intensity and repeatability were optimal; further increasing to 0.4-0.5 μM did not significantly improve the amplification efficiency, but instead increased background fluctuations.

[0097] In summary, the optimal conditions for the TaqMan triple real-time quantitative PCR reaction system in this study were determined to be: annealing / extension temperature of 54 ℃ and probe concentration of 0.3 μM. The optimized reaction system is shown in Table 8.

[0098] Table 7 Optimization of Annealing Temperature and Probe Concentration for Triple TaqMan Real-Time Quantitative PCR

[0099]

[0100] Table 8 Optimization results of triple TaqMan real-time quantitative PCR

[0101]

[0102] (5) Establishment of TaqMan triple real-time quantitative PCR standard curve

[0103] Using 10-fold serially diluted plasmid standards as templates, TaqMan triple real-time quantitative PCR amplification curves were obtained. Figure 4 ) and the establishment of a triple TaqMan real-time quantitative PCR standard curve ( Figure 5 The results showed that the Ct values ​​of the three targets, I9, F4, and C4, all exhibited a good linear relationship with the logarithm of template copies. Their linear regression equations were as follows:

[0104] I9 (Yersinia pseudotuberculosis, inv gene):

[0105] C4 (Yersinia pestis, caf1 gene):

[0106] F4 (Yersinia enterocolitica, foxA gene):

[0107] The standard curve results show that this method exhibits excellent linearity over a wide concentration range, with correlation coefficients R² = 0.993–0.996 and amplification efficiencies of 109.6%–114.8%, indicating good amplification efficiency. It meets the technical requirements of real-time quantitative PCR. Although the amplification efficiency is slightly higher than the theoretical optimum of 100%, this phenomenon is common in triple probe qPCR and is within an acceptable range, not affecting detection accuracy and stability.

[0108] Standard curves for the three target genes (caf1, inv, and foxA) were constructed using a tenfold serial dilution of the recombinant plasmid. Figure 5 As shown, the slopes of the standard curves for I9, C4, and F4 are respectively , and The correlation coefficient (R²) is between 0.993 and 0.996, indicating an excellent linear relationship.

[0109] The calculated amplification efficiency ranged from 109.6% to 114.8%, indicating that the triple qPCR detection method has excellent amplification performance.

[0110] (6) TaqMan triple real-time quantitative PCR sensitivity experiment

[0111] like Figure 6 and Figure 7 As shown, the analytical sensitivity of this triple qPCR detection method was determined using serially diluted recombinant plasmids. A positive detection threshold of Ct ≤ 35 was used; the detection limits for I9 and C4 were 5 × 10² copies / μL; and the detection limit for F4 was 1 × 10¹ copies / μL.

[0112] In comparison, the detection limit of conventional PCR is 1×10³ copies / μL, indicating that the sensitivity of triple qPCR is at least 10 times higher.

[0113] No significant differences were observed between individual template detection and mixed template detection, indicating that no interference occurred between multiple targets in the triple detection system.

[0114] (7) TaqMan triple real-time quantitative PCR specificity experiment

[0115] Using extracted positive standards and nucleic acids from *Francis tularensis*, *Brucella*, *Bacillus anthracis*, *Vibrio cholerae*, *Salmonella typhi*, and *Shigella* as templates, TaqMan triple real-time quantitative PCR amplification was performed according to the optimized reaction system and conditions. Figure 8 The results showed that specific amplification signals were observed only in the target genes (I9, C4, and F4), and not in non-target microorganisms or negative controls. The detection of amplified signals in the sample demonstrates the high specificity of this detection method.

[0116] (8) TaqMan triple real-time quantitative PCR repeatability experiment

[0117] Using concentrations respectively , and The reproducibility and reproducibility of the detection method were evaluated using recombinant plasmids at copies / μL. The intra- and inter-batch coefficients of variation (CV) of Ct values ​​were low, mostly below 2%, indicating that the detection method has good reproducibility and stability (Table 9).

[0118] Table 9 Repeatability of Triple Real-Time Quantitative PCR

[0119]

[0120] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A composition for detecting pathogenic Yersinia based on TaqMan probe-based triple real-time fluorescent PCR, characterized in that, This includes primer pairs and TaqMan probes targeting the caf1 gene of Yersinia pestis, the foxA gene of Yersinia enterocolitica, and the inv gene of Yersinia pseudotuberculosis, respectively; among which: The primer pairs for the caf1 gene are shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe sequence is shown in SEQ ID NO:3; The primer pairs for the foxA gene are shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe sequence is shown in SEQ ID NO:

6. The primer pairs for the inv gene are shown in SEQ ID NO:7 and SEQ ID NO:8, and the probe sequence is shown in SEQ ID NO:

9.

2. The composition of claim 1, wherein, The forward primer sequence of the caf1 gene primer pair is SEQ ID NO:1, and the reverse primer sequence of the caf1 gene primer pair is SEQ ID NO:2; the forward primer sequence of the foxA gene primer pair is SEQ ID NO:4, and the reverse primer sequence of the foxA gene primer pair is SEQ ID NO:5; the forward primer sequence of the inv gene primer pair is SEQ ID NO:7, and the reverse primer sequence of the inv gene primer pair is SEQ ID NO:

8.

3. The composition of claim 1, wherein, The TaqMan probes are labeled with different fluorescent reporter groups and quencher groups: caf1 probe label: ROX / BHQ-2; foxA probe label: CY5 / BHQ-3; inv probe label: FAM / BHQ-1.

4. The composition according to any one of claims 1 to 3, characterized in that, The primers for the caf1 gene primer pair, the foxA gene primer pair, and the inv gene primer pair are C4, F4, and I9, respectively.

5. The composition of claim 4, wherein, The caf1 gene primer pair, the foxA gene primer pair, and the inv gene primer pair were amplified and then cloned into the pESI-T vector to obtain recombinant plasmids pESI-T-I9, pESI-T-C4, and pESI-T-F4, respectively.

6. A kit for detecting pathogenic Yersinia based on TaqMan probe-based triple real-time fluorescent PCR, characterized by, The composition comprising any one of claims 1-5 further comprises qPCR premix, positive control plasmid, negative control, and enzyme-free ultrapure water.

7. The use of the composition of any one of claims 1-5 or the kit of claim 6 in the preparation of a pathogenic Yersinia pestis detection product.

8. The application according to claim 7, wherein the pathogenic Yersinia is: Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis.

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