Yersinia pestis rpa-lfs detection primer probe set, kit and application thereof

Abstract

The present application relates to the technical field of biology, and discloses an RPA primer probe set for detecting Yersinia pestis, which comprises a first primer probe set targeting the Caf1 gene of Yersinia pestis, a second primer probe set targeting the YPO0392 gene of Yersinia pestis, and a third primer probe set targeting the YPO2088 gene of Yersinia pestis.The RPA primer probe set of the present application realizes the three-target joint detection of Yersinia pestis in samples, especially environmental samples, by synchronously targeting three specific genes (Caf1, YPO0392 and YPO2088) of Yersinia pestis, significantly improves the detection sensitivity and the accuracy of the detection result, and can be used for rapidly, quickly and highly credibly detecting Yersinia pestis on site, and has a wide application prospect in scenes with limited resources such as primary disease control, field emergency and port quarantine.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a primer and probe set, kit, and application for detecting Yersinia pestis RPA-LFS. Background Technology

[0002] Plague is caused by Yersinia pestis (Yersinia pestis) Yersinia pestis Plague is a Class A highly contagious disease caused by pneumonic plague, characterized by its high pathogenicity, rapid spread, and high mortality rate. It is a zoonotic disease, primarily affecting rodents (such as marmots and ground squirrels) and spreading through flea bites, contact with infected animals, or inhalation of droplets from infected individuals. Pneumonic plague, in particular, can be transmitted from person to person via respiratory droplets and has an extremely high mortality rate. Globally, plague occurs sporadically or endemicly in countries with natural foci in Africa (Madagascar, Uganda), Asia (India, Mongolia), and the Americas (Peru, the United States). In my country, plague foci are mainly distributed in the Northwest, Southwest, and North China regions. Qinghai Province, due to the widespread and ecologically stable host of the Himalayan marmot foci in the Qinghai-Tibet Plateau, has become a typical endemic area. Tibet, Gansu, and Inner Mongolia are also key areas for prevention and control due to different types of foci (such as those of the long-clawed gerbil and yellow-breasted rat).

[0003] The spread of plague relies on a complex "host-vector-human" transmission chain, making rapid and accurate detection of Yersinia pestis crucial for epidemic control. Currently, real-time quantitative PCR (qPCR) is the mainstream detection technology for this bacterium. While it boasts high sensitivity and mature technology, it suffers from issues such as strong instrument dependence, complex operation, and long detection time, making it difficult to meet the efficient detection needs of grassroots field operations and emergency scenarios. Its limitations are specifically manifested in: the entire detection process takes several hours, resulting in insufficient response speed; reliance on expensive qPCR instruments and other sophisticated equipment leads to high purchase and maintenance costs, limiting accessibility in resource-scarce areas; and the operation requires specialized technicians, demanding high skill levels and hindering large-scale promotion and use.

[0004] Recombinase polymerase amplification (RPA) technology can rapidly amplify target genes under isothermal conditions of 37–42 °C without relying on sophisticated instruments. Combined with lateralflow strips (LFS), RPA-LFS technology allows for short-term visual detection without specialized equipment or technicians. As a novel isothermal amplification nucleic acid detection technology, RPA-LFS has been widely used in the detection of viruses, bacteria, fungi, and parasites due to its advantages of ease of operation and low time cost. However, current RPA detection for plague specimens is mostly single-target detection, posing a risk of false negatives; furthermore, most products lack multi-gene synergistic validation, potentially leading to insufficient inclusion in the differential diagnosis of complex variants of Yersinia pestis. Summary of the Invention

[0005] This invention provides a primer and probe set, a kit, and their applications for detecting Yersinia pestis RPA-LFS, in order to solve the problems of high false negative rate, insufficient sensitivity, and insufficient specificity.

[0006] In a first aspect, the present invention provides an RPA primer and probe set for the detection of Yersinia pestis, the RPA primer and probe set comprising a first primer and probe set targeting the Caf1 gene of Yersinia pestis, a second primer and probe set targeting the YPO0392 gene of Yersinia pestis, and a third primer and probe set targeting the YPO2088 gene of Yersinia pestis.

[0007] In one optional embodiment, the first primer-probe set includes a first upstream primer, a first downstream primer, and a first RPA probe; the nucleotide sequence of the first upstream primer is shown in SEQ ID NO.1; the nucleotide sequence of the first downstream primer is shown in SEQ ID NO.2; and the nucleotide sequence of the first RPA probe is shown in SEQ ID NO.3.

[0008] In one alternative embodiment, a cleavage site for a nuclease is inserted at the middle position of the first RPA probe; a blocking group is attached to the 3' end of the first RPA probe; and biotin is modified to the 5' end of the first downstream primer.

[0009] In one alternative embodiment, the middle position of the first RPA probe forms a cleavage site for a nuclease by replacing one base with tetrahydrofuran (THF); the fluorescent group modified at the 5' end of the first RPA probe includes at least one of FITC and FAM; and the blocking group attached to the 3' end of the first RPA probe includes a C3 spacer.

[0010] In one optional embodiment, the second primer-probe set includes a second upstream primer, a second downstream primer, and a second RPA probe; the nucleotide sequence of the second upstream primer is shown in SEQ ID NO.4; the nucleotide sequence of the second downstream primer is shown in SEQ ID NO.5; and the nucleotide sequence of the second RPA probe is shown in SEQ ID NO.6.

[0011] In one alternative embodiment, a cleavage site for an endonuclease is inserted at the middle position of the second RPA probe; a blocking group is attached to the 3' end of the second RPA probe; and biotin is modified to the 5' end of the second downstream primer.

[0012] In one alternative embodiment, the middle position of the second RPA probe forms a cleavage site for a nuclease by replacing one base with tetrahydrofuran (THF); the fluorescent group modified at the 5' end of the second RPA probe includes at least one of FITC and FAM; and the blocking group attached to the 3' end of the second RPA probe includes a C3 spacer.

[0013] In one optional embodiment, the third primer-probe set includes a third upstream primer, a third downstream primer, and a third RPA probe; the nucleotide sequence of the third upstream primer is shown in SEQ ID NO.7; the nucleotide sequence of the third downstream primer is shown in SEQ ID NO.8; and the nucleotide sequence of the third RPA probe is shown in SEQ ID NO.9.

[0014] In one optional embodiment, a nuclease cleavage site is inserted at the middle position of the third RPA probe; a blocking group is attached to the 3' end of the third RPA probe; and biotin is modified to the 5' end of the third downstream primer.

[0015] In one alternative embodiment, the middle position of the third RPA probe forms a cleavage site for a nuclease by replacing one base with tetrahydrofuran (THF); the fluorescent group modified at the 5' end of the third RPA probe includes at least one of FITC and FAM; and the blocking group attached to the 3' end of the third RPA probe includes a C3 spacer.

[0016] Secondly, the present invention also provides a kit for detecting Yersinia pestis, the kit comprising the aforementioned RPA primer and probe set.

[0017] In one alternative embodiment, the kit further includes RPA lyophilized microspheres, an activator, a reconstitution buffer, a product diluent, a chromatography strip, a positive control, and / or a negative control.

[0018] In one optional embodiment, the RPA lyophilized microspheres comprise recombinase, recombinase coenzyme, single-strand binding protein, strand displacement DNA polymerase, and endonuclease IV.

[0019] In one alternative embodiment, the activator comprises Mg 2+ .

[0020] In one alternative embodiment, the reconstitution buffer comprises polyethylene glycol 35000.

[0021] In one alternative embodiment, the product diluent comprises PBS buffer, sodium chloride, and Tween 20.

[0022] Thirdly, the present invention also provides a method for detecting Yersinia pestis, wherein the detection method is not for the purpose of disease diagnosis and treatment, and the kit is used to detect the sample to be tested.

[0023] In one optional implementation, the detection method includes the following steps: Step 1: Extract genomic DNA from the sample to be tested; Step 2: Mix the reconstitution buffer and the above RPA primer and probe set to obtain a solution; use the solution to dissolve the RPA lyophilized microspheres to obtain an RPA reconstitution solution; add an activator to the RPA reconstitution solution to obtain the RPA reaction system; Step 3: Using the genomic DNA obtained in Step 1 as a template, the template is amplified at an isothermal temperature in the RPA reaction system to obtain the amplification product; Step 4: Dilute the amplification product obtained in Step 3 with product diluent, then perform chromatography on a chromatographic test strip and develop the color; determine whether Yersinia pestis is present in the sample based on the color development results.

[0024] In one optional implementation, in step 3, the temperature of the isothermal amplification is 39~41℃.

[0025] In one optional implementation, in step 3, the isothermal amplification time is 10-30 min.

[0026] In one optional implementation, in step 3, the isothermal amplification temperature is 41°C, and the isothermal amplification time is 30 min.

[0027] In one optional implementation, in step 4, the chromatography and color development time on the chromatographic test strip is 5-15 minutes.

[0028] In one optional implementation, in step 4, the chromatography and color development time on the chromatographic test strip is 5 minutes.

[0029] In one optional implementation, when the target gene is the Caf1 gene, the isothermal amplification temperature is 41°C and the isothermal amplification time is 10~30 min.

[0030] In one optional implementation, when the target gene is the Caf1 gene, the isothermal amplification temperature is 41°C and the isothermal amplification time is 30 min.

[0031] In one optional implementation, when the target gene is the YPO0392 gene, the isothermal amplification temperature is 39~41℃ and the isothermal amplification time is 15~30 min.

[0032] In one optional implementation, when the target gene is the YPO0392 gene, the isothermal amplification temperature is 41°C and the isothermal amplification time is 30 min.

[0033] In one optional implementation, when the target gene is the YPO2088 gene, the isothermal amplification temperature is 39~41℃ and the isothermal amplification time is 15~30 min.

[0034] In one alternative implementation, the isothermal amplification temperature for targeting the YPO2088 gene is 41°C, and the isothermal amplification time is 30 min.

[0035] In one optional implementation, in step 4, the judgment result includes: a positive result is indicated by clear red bands appearing at both the T and C bands of the test strip; a negative result is indicated by clear red bands appearing only at the C band; and an invalid test is indicated by no red band at the C band, regardless of whether a red band appears at the T band, and the test should be repeated once.

[0036] In one optional implementation, step 4 includes the following judgment results: using the three-target result co-interpretation rule, the color development of any target gene (T line + C line) is judged as positive; only when all three genes are negative is it judged as negative; if the C line is missing, the result is invalid and needs to be retested.

[0037] Fourthly, the present invention also provides the application of the RPA primer-probe set, the kit, or the method described herein in the detection of Yersinia pestis, wherein the application is not for the purpose of disease diagnosis and treatment.

[0038] The technical solution of this invention has the following advantages: 1. This invention provides an RPA primer and probe set for the detection of Yersinia pestis, comprising a first primer and probe set targeting the Caf1 gene of Yersinia pestis, a second primer and probe set targeting the YPO0392 gene of Yersinia pestis, and a third primer and probe set targeting the YPO2088 gene of Yersinia pestis. By simultaneously targeting three specific genes (Caf1, YPO0392, and YPO2088), this invention achieves joint detection of three targets of Yersinia pestis in samples and environmental samples, significantly improving sensitivity and accuracy.

[0039] Furthermore, using the RPA primer and probe set provided by this invention for detection, at low concentrations of genomic nucleic acid diluted to 1-10 fg / μL, the positive detection rate of the RPA primer and probe set provided by this invention is superior to that of qPCR. The RPA primer and probe set provided by this invention can achieve a detection sensitivity of 10 copies / μL for the Yersinia pestis Caf1 gene, YPO0392 gene, and YPO2088 gene.

[0040] 2. This invention also provides a kit for detecting Yersinia pestis, the kit comprising the aforementioned RPA primer and probe set. Detection using the kit provided by this invention exhibits high sensitivity and specificity, enabling simultaneous detection of three specific genes (Caf1, YPO0392, and YPO2088) for Yersinia pestis in both physical and environmental samples, significantly improving the reliability of the detection results.

[0041] 3. The present invention provides a method for detecting Yersinia pestis, the method being for non-disease diagnosis and treatment purposes, the method comprising the following steps: Step 1, extracting genomic DNA from the sample to be tested; Step 2: Dissolve RPA lyophilized microspheres in reconstitution buffer, add activator, and prepare the RPA reaction system; Step 3: Using the genomic DNA obtained in Step 1 as a template, perform isothermal amplification in the RPA reaction system using the described RPA primer and probe set to obtain the amplification product; Step 4: Dilute the amplification product obtained in Step 3 with product dilution buffer, perform chromatography on a test strip, and determine the colorimetric result. The method described in this invention simultaneously detects three key genes of Yersinia pestis, achieving simultaneous and efficient amplification of multiple targets and enhancing the stability of the detection.

[0042] Furthermore, the method described in this invention improves detection efficiency and sensitivity by independently optimizing the reaction system, reaction temperature, and reaction time for each target gene. By combining RPA amplification and chromatographic test strips, this method establishes an integrated process and detection standard, completing the entire process from sample extraction to detection results in as little as 35 minutes, overcoming the time bottleneck of qPCR and facilitating rapid on-site detection. Simultaneously, the method establishes a three-target result collaborative interpretation mechanism, enhancing detection confidence through multi-target cross-verification and avoiding false negatives or false positives. Therefore, the detection method provided by this invention can be used for rapid and highly reliable on-site detection of Yersinia pestis, and has broad application prospects in resource-constrained scenarios such as grassroots disease control, field emergency response, and port quarantine. Attached Figure Description

[0043] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0044] Figure 1 This is a technical roadmap of the present invention; Figure 2 This is a schematic diagram illustrating the interpretation of chromatographic test strip results; Figure 3 The image shows the results of RPA-LFS amplification temperature optimization; (A) Caf1 gene; (B) YPO0392 gene; (C) YPO2088 gene; Figure 4 The diagram shows the results of RPA-LFS amplification time optimization; (A) Caf1 gene; (B) YPO0392 gene; (C) YPO2088 gene; Figure 5 The results are from the sensitivity test; (A) Caf1 gene; (B) YPO0392 gene; (C) YPO2088 gene; Figure 6 These are the test results for Yersinia pestis samples; (A) Caf1 gene; (B) YPO0392 gene; (C) YPO2088 gene; Figure 7 It is a nucleic acid sample diluted to 10. -6 Figures showing the results of qPCR detection of the Caf1 gene at different times; all figures have consistent horizontal and vertical axes. Figure 8 It is a nucleic acid sample diluted to 10. -6Figure 1: YPO0392 gene qPCR detection results at 1000°C; all graphs have consistent horizontal and vertical axes. Figure 9 It is a nucleic acid sample diluted to 10. -6 Figures showing the YPO2088 gene qPCR detection results over time; all figures have consistent horizontal and vertical axes. Figure 10 It is a nucleic acid sample diluted to 10. -6 The RPA-LFS detection results at 1000x speed; Figure 10 In the text, (A) Caf1 gene; (B) YPO0392 gene; (C) YPO2088 gene; Figure 11 It is a nucleic acid sample diluted to 10. -7 Figures showing the results of qPCR detection of the Caf1 gene at different times; all figures have consistent horizontal and vertical axes. Figure 12 It is a nucleic acid sample diluted to 10. -7 Figure 1: YPO0392 gene qPCR detection results at 1000°C; all graphs have consistent horizontal and vertical axes. Figure 13 It is a nucleic acid sample diluted to 10. -7 Figures showing the YPO2088 gene qPCR detection results over time; all figures have consistent horizontal and vertical axes. Figure 14 It is a nucleic acid sample diluted to 10. -7 The RPA-LFS detection results at 1000x speed; Figure 14 In the text, (A) Caf1 gene; (B) YPO0392 gene; (C) YPO2088 gene; Figure 15 This is a graph showing the specificity detection results of the RPA primer and probe set. Detailed Implementation

[0045] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0046] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0047] The sources of the materials and reagents involved in the embodiments of this invention are as follows: 1. Reagents and materials (1) The RPA freeze-dried microspheres were purchased from Suzhou Xianda Gene Technology Co., Ltd. The components of the RPA freeze-dried microspheres include recombinase, recombinase coenzyme, single-strand binding protein, strand displacement DNA polymerase, endonuclease IV and energy supply substances, etc. For specific components and concentrations, please refer to the instruction manual. (2) Reconstitution buffer: 20% (g / 100 mL) PEG35000, solvent is double-distilled water; (3) Activator: 350 mM magnesium acetate, solvent is double-distilled water; (4) Product dilution buffer: 0.02 MPBS buffer containing 0.75% (g / 100 mL) sodium chloride and 0.1% (g / 100 mL) Tween 20, pH 7.4.

[0048] 2. Preparation of chromatographic test strips: Colloidal gold was prepared by sodium citrate reduction method (for details of sodium citrate reduction method, please refer to the literature "St. John, AS, Price, CP, & Tovey, J. (2016). Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay. Scientificreports, 6(1), 37732."). Anti-FITC antibody (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was labeled on the colloidal gold and sprayed onto the conjugate pad. Capture molecules (streptavidin antibody and secondary antibody) were coated on the nitrocellulose membrane, and the detection line was coated with streptavidin antibody (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and the control line was coated with goat anti-mouse IgG antibody (purchased from Sangon Biotech (Shanghai) Co., Ltd.). Finally, the nitrocellulose membrane, absorbent pad, conjugate pad, and sample pad were sequentially pasted onto the base plate. The width of each test strip was 2.8 mm (for details of the preparation method of chromatographic test strips, please refer to the literature "St. John, AS, Price, & Tovey, J. (2016). Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay. Scientificreports, 6(1), 37732."). CP,&Tovey, J. (2016). Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay. Scientific reports, 6(1), 37732.").

[0049] 3. Primers, probes, and positive recombinant plasmids were synthesized by BGI Genomics Co., Ltd. in Shenzhen. The preparation process of the positive recombinant plasmids was as follows: the Caf1 gene, YPO0392 gene, and YPO2088 gene were inserted into the LacZ site of the pUC57 plasmid, respectively, to obtain the Caf1 gene plasmid, YPO0392 gene plasmid, and YPO2088 gene plasmid (the Caf1 gene plasmid, YPO0392 gene plasmid, and YPO2088 gene plasmid were synthesized by BGI Genomics Co., Ltd. in Shenzhen; the accession number corresponding to the Caf1 gene is NC_017169.1, and the accession numbers corresponding to the YPO0392 gene and YPO2088 gene are NC_003143.1).

[0050] 4. Samples of Yersinia pestis and Yersinia pestis and Bacillus anthracis ( Bacillus anthraci ), bovine brucellosis ( Brucell abovis ), Bacillus cereus ( Bacillus cereus ), Tulafrancsis ( Francisella tularensis Salmonella typhimurium ( ) Salmonella typhimurium Bacillus subtilis ( Bacillus subtilis ), Escherichia coli ( Escherichia coli Staphylococcus aureus ( Staphylococcus aureus Aeromonas hydrophila ( ) Aeromonas hydrophila ) and Listeria monocytogenes ( Listeria monocytogenes The genomic nucleic acids of all samples were preserved in the laboratory.

[0051] Example 1: An RPA primer and probe set for the detection of Yersinia pestis This embodiment provides an RPA primer and probe set for the detection of Yersinia pestis. The RPA primer and probe set consists of a first primer and probe set targeting the Caf1 gene of Yersinia pestis, a second primer and probe set targeting the YPO0392 gene of Yersinia pestis, and a third primer and probe set targeting the YPO2088 gene of Yersinia pestis. The first primer and probe set consists of a first upstream primer, a first downstream primer, and a first RPA probe. The nucleotide sequence of the first upstream primer is shown in SEQ ID NO.1. The nucleotide sequence of the first downstream primer is shown in SEQ ID NO.2. The nucleotide sequence of the first RPA probe is shown in SEQ ID NO.3. The second primer and probe set consists of a second upstream primer, a second downstream primer, and a second RPA probe. The nucleotide sequence of the second upstream primer is shown in SEQ ID NO.4. The nucleotide sequence of the second downstream primer is shown in SEQ ID NO.5. The nucleotide sequence of the second RPA probe is shown in SEQ ID NO.5. As shown in ID NO. 6; the third primer-probe set consists of a third upstream primer, a third downstream primer, and a third RPA probe; the nucleotide sequence of the third upstream primer is shown in SEQ ID NO. 7; the nucleotide sequence of the third downstream primer is shown in SEQ ID NO. 8; the nucleotide sequence of the third RPA probe is shown in SEQ ID NO. 9; the 5' ends of the first, second, and third RPA probes are modified with the fluorescent group FITC; the middle positions of the first, second, and third RPA probes form endonuclease cleavage sites by replacing one base with tetrahydrofuran (THF); the 3' ends of the first, second, and third RPA probes are connected with the blocking group C3 spacer; the 5' ends of the first, second, and third downstream primers are modified with biotin. The nucleotide sequences of the RPA primer-probe set are shown in Table 1. The target sequences of the primer-probe set are the Caf1 gene with accession number NC_017169.1 and the YPO0392 and YPO2088 genes with accession number NC_003143.1.

[0052] Table 1 Primer and probe sequence

[0053] Example 2: A kit for detecting Yersinia pestis This embodiment provides a kit for detecting Yersinia pestis, which consists of RPA primer and probe set (as shown in Example 1), RPA lyophilized microspheres, activator, reconstitution buffer, product dilution solution, chromatography test strip, positive control and negative control group; The RPA lyophilized microspheres were purchased from Suzhou Xianda Gene Technology Co., Ltd.; the reconstitution buffer was 20% (g / 100 mL) PEG35000; the activator was 350 mM magnesium acetate; the positive control was a positive recombinant plasmid containing target fragments of the Caf1 gene, YPO0392 gene, and YPO2088 gene; and the negative control was enzyme-free water.

[0054] Example 3: A method for detecting Yersinia pestis This embodiment provides a method for detecting Yersinia pestis. The detection method uses the kit described in Example 2 to detect Yersinia pestis in the test sample, and specifically includes the following steps (technical roadmap as shown in Figure 2). Figure 1 (as shown) 1. Nucleic acid extraction Nucleic acid was extracted from the sample using a magnetic bead extraction kit (purchased from Suzhou Zhongke Sujing Biotechnology Co., Ltd.). The specific process is as follows: 1) Add 200 μL of lysis buffer to the sample and incubate at 70℃ for 10 min; 2) Add 300 μL of isopropanol and 15 μL of magnetic beads to the centrifuge tube and let it stand at room temperature for 10 min; 3) Let the magnetic rack stand for 1 minute until the solution becomes clear, then discard the cap and the supernatant in the tube; 4) Add 500 μL of diluted wash solution 1 to the centrifuge tube, shake to mix, let stand for 1 min or until the solution is clear, and discard the cap and the supernatant in the tube. 5) Add 300 μL of diluted washing solution 2 to the centrifuge tube, shake to mix, let stand for 1 min or until the solution is clear, and discard the cap and the supernatant in the tube. 6) Allow the centrifuge tubes to air dry at room temperature, then add 50 μL of elution buffer, vortex to mix for 1 min, and incubate at 56°C for 10 min. 7) Place the centrifuge tube on a magnetic rack and magnetically attract it for 1 minute until the solution is clear. Transfer the supernatant to a new centrifuge tube and store at -80°C for later use.

[0055] 2. RPA amplification (1) Reaction system preparation: The reconstitution buffer and primer probe described in Example 2 were mixed according to the systems in Tables 2 to 4 to obtain the solution; the RPA lyophilized microspheres were dissolved in the solution to obtain the RPA reconstitution solution (one test corresponds to one RPA lyophilized microsphere). (2) Addition of activator: Shake the RPA reconstituted solution to mix well, centrifuge briefly, and transfer it to an eight-tube strip; add the corresponding volume of activator for each target gene detection system to the tube cap (refer to Tables 2-4); gently cap the tube and transfer the eight-tube strip to the sample preparation room to obtain the RPA reaction system; (3) Add nucleic acid samples: Use a pipette to take 2 μL of the nucleic acid template, negative control or positive control extracted in step 1 and add them to the pre-allocated eight-tube strips; add DEPC water to make up the volume of each reaction to 25 μL; where the negative control is NTC (using enzyme-free water instead of nucleic acid sample), and the positive control is the positive recombinant plasmid carrying each target gene. (4) Reaction amplification: After adding the sample, the eight tubes were briefly centrifuged (so that the activator was mixed into the reaction system and the liquid was concentrated at the bottom of the tube). Then, the system was quickly mixed with a shaker and briefly centrifuged again. After completion, the reaction tubes were immediately placed in a 41°C constant temperature device for 30 min of amplification reaction, during which the temperature of the device was kept stable.

[0056] Table 2 Cafl gene detection reaction system

[0057] Table 3 YPO0392 gene detection reaction system

[0058] Table 4 YPO2088 gene detection reaction system

[0059] 3. Chromatographic test strip detection (1) Sample loading and detection: After RPA amplification, use a pipette to accurately aspirate 8.5 μL of amplification product and add it to a centrifuge tube containing 100 μL of product diluent. After tightening the cap, invert or vortex to mix. Then take 100 μL of the mixture and add it to the sample loading well of the chromatography strip. (2) Result Interpretation: After loading the sample, place the chromatography strip flat at room temperature and observe the test results within 5-15 minutes. The interpretation conditions are shown below, and the schematic diagram for interpreting the test results is shown below. Figure 2 As shown: Positive: Clear red bands appear at both the T and C positions on the test strip.

[0060] Negative: A clear red band appears only at the C-band location.

[0061] Invalid: No red band at the C band position. Regardless of whether a red band appears at the T band position, the test is invalid. It is recommended to repeat the test once.

[0062] Experimental Example 1: Optimization of Reaction Temperature In this experimental example, a positive recombinant plasmid was used as the test sample. The reaction temperature in Example 3 was screened and optimized. The specific method is as follows: 1. Synthesis of positive recombinant plasmids: The Caf1 gene, YPO0392 gene, and YPO2088 gene were inserted into the LacZ site of the pUC57 plasmid to obtain the Caf1 gene plasmid, YPO0392 gene plasmid, and YPO2088 gene plasmid (the Caf1 gene plasmid, YPO0392 gene plasmid, and YPO2088 gene plasmid were synthesized by Shenzhen BGI Genomics Co., Ltd.; the accession number corresponding to the Caf1 gene is NC_017169.1, and the accession numbers corresponding to the YPO0392 gene and YPO2088 gene are NC_003143.1).

[0063] 2. Based on Example 3, the reaction temperature of the RPA amplification step was adjusted to 39℃, 40℃, and 41℃ respectively, while other reaction conditions remained unchanged. After RPA amplification and chromatographic detection using test strips, the results are as follows: Figure 3 As shown in Table 5 (since this is a plasmid sample, nucleic acid extraction is not required; RPA amplification and chromatography strip detection are performed directly).

[0064] Experimental results: In the reaction system targeting the Caf1 gene, a positive detection result was only obtained when the reaction temperature was set to 41℃. Figure 3 Therefore, when performing Caf1 gene detection, the reaction temperature for RPA amplification was chosen to be 41℃; in the reaction system targeting the YPO0392 gene, the positive T bandwidth in the 40℃ and 41℃ groups was wider than that in the 39℃ group (A). Figure 3 Therefore, when performing YPO0392 gene detection, the optimal reaction temperature for RPA amplification is 40-41℃; in the reaction system targeting the YPO2088 gene, the 39℃, 40℃, and 41℃ groups were all positive, but the positive band in the 41℃ group was wider (B). Figure 3 Therefore, the reaction temperature for RPA amplification during YPO2088 gene detection is 41℃. In summary, 41℃ isothermal RPA amplification is uniformly selected.

[0065] Table 5 Comparison of Reaction Temperature Optimization Results

[0066] Experimental Example 2: Optimization of Reaction Time In this experimental example, the positive recombinant plasmid was used as the test sample (same as in Experiment 1). The reaction time in Example 3 was screened and optimized. The specific method is as follows: Based on Example 3, the reaction time of the RPA amplification step was adjusted to 10 min, 15 min, 20 min, 25 min, and 30 min, respectively, while other reaction conditions remained unchanged. After RPA amplification and chromatographic detection using test strips, the results are as follows: Figure 4 As shown in Table 6 (since this is a plasmid sample, nucleic acid extraction is not required; RPA amplification and chromatography strip detection are performed directly).

[0067] Experimental results: In the reaction system targeting the Caf1 gene, the positive T bandwidth at a reaction time of 30 min was wider than that in other experimental groups ( Figure 4 Therefore, when performing Caf1 gene detection, the optimal reaction time for RPA amplification is 30 min; in the reaction system targeting the YPO0392 gene, no positive band was observed at a reaction time of 10 min, while the positive T band at a reaction time of 30 min was wider than in other experimental groups (15 min, 20 min, and 25 min groups). Figure 4 Therefore, when performing YPO0392 gene detection, the optimal reaction time for RPA amplification is 30 min; in the reaction system targeting the YPO2088 gene, a weak positive band was observed at a reaction time of 10 min, and the positive T band at a reaction time of 30 min was wider than that of other experimental groups (15 min group, 20 min group, and 25 min group). Figure 4 Therefore, when performing YPO2088 gene detection, the optimal reaction time for RPA amplification is 30 minutes. Thus, a uniform 30-minute isothermal RPA amplification time is selected.

[0068] Table 6 Comparison of Reaction Time Optimization Results

[0069] Experiment Example 3: Sensitivity Detection This experimental example uses a positive recombinant plasmid as the test sample (same as in Example 1) to verify the detection sensitivity of the RPA primer-probe set described in Example 1. The specific experimental steps are as follows: (1) The Caf1 gene plasmid was serially diluted 10-fold using enzyme-free water to obtain concentrations of 1 copies / μL to 10 copies / μL. 4 A series of templates with copies / μL were prepared; the YPO0392 gene plasmid was serially diluted 10-fold to obtain concentrations ranging from 1 copy / μL to 10 copies / μL. 4 A series of templates at copies / μL were prepared; the YPO2088 gene plasmid was serially diluted 10-fold to obtain concentrations ranging from 1 copies / μL to 10 copies / μL. 4 A series of templates with copies / μL; (2) The diluted template was tested according to the method described in Example 3 to verify the detection sensitivity of the RPA primer and probe set described in Example 1. During the detection process, the reaction conditions of the RPA amplification step were controlled as follows: constant temperature 41℃ reaction for 30 min, and wait for 5 min after sample loading and chromatography (since it is a plasmid sample, there is no need to extract nucleic acid, and RPA amplification and chromatography strip detection are performed directly).

[0070] Experimental results: The results are as follows Figure 5 As shown, Figure 5 The results from the A-test showed that the detection sensitivity of the Caf1 plasmid was 10 copies / μL; Figure 5 The results from the B test showed that the detection sensitivity of plasmid YPO0392 was 10 copies / μL; Figure 5 The results showed that the detection sensitivity of the YPO2088 plasmid was 10 copies / μL.

[0071] Experiment Example 4: Accuracy Testing This experiment used 20 Yersinia pestis samples as test samples to verify the detection accuracy of the RPA primer-probe set described in Example 1. The specific experimental steps are as follows: (1) Extraction of nucleic acid from samples: 20 Yersinia pestis samples were provided by the Qinghai Provincial Institute for Endemic Disease Prevention and Control. The nucleic acid extraction method was the same as in Example 3. (2) The test was performed according to the method described in Example 3.

[0072] Experimental results: The results are as follows Figure 6 As shown in the figure (the sample numbers are shown in the figure), all 20 positive samples tested positive for RPA-LFS, with a positive concordance rate of 100%.

[0073] Experimental Example 5: Methodological Comparative Analysis This experimental example provides a comparative analysis of low-concentration nucleic acid methodologies for 10 Yersinia pestis samples. The detection rates of qPCR and RPA-LFS at low nucleic acid concentrations were evaluated to compare the sensitivity of the two methods for detecting Yersinia pestis. The specific methods are as follows: (1) Ten Yersinia pestis samples were provided by the Qinghai Provincial Institute for Endemic Disease Prevention and Control. The ten Yersinia pestis samples were numbered, and nucleic acids were extracted from the ten samples numbered 1, 3 to 11 and the concentration of nucleic acid in the samples was determined. The nucleic acid extraction method was the same as in Example 3. (2) Dilute each sample to a concentration of 10 with enzyme-free water. -6 Times (10 fg / μL~100 fg / μL) and 10 -7The concentrations (1 fg / μL~10 fg / μL) were analyzed using qPCR and RPA-LFS to detect three target genes: Caf1, YPO0392, and YPO2088. RPA-LFS: The specific method is the same as in Example 3. The reaction is carried out at a constant temperature of 41℃ for 30 min, and the sample is loaded and chromatographically analyzed, then waited for 5 min. qPCR detection procedure: Prepare the reaction system. A 25 μL reaction system contains 2.5 μL of 10×PCR buffer, 0.64 mM dNTPs, 3 mM MgCl2, 2 U Taq DNA polymerase, 0.2 μM upstream primer, 0.2 μM downstream primer, and 0.16 μM probe. Add 2 μL of the nucleic acid to be tested to the prepared reaction system and place it in a real-time quantitative PCR instrument for amplification. The amplification program is shown in Table 7, and the qPCR primer and probe sequences are shown in Table 8.

[0074] Table 7 qPCR reaction conditions

[0075] Table 8 Primer and probe sequences

[0076] (3) The original nucleic acid concentration and the nucleic acid concentration after dilution of the samples are shown in Table 9; Experimental results: The results are as follows Figures 7-14 As shown.

[0077] When the sample is diluted to 10 -6 When the sample was diluted to 10 times, the positive detection rate of qPCR for the three genes was 40%–90%, while the positive detection rate of RPA-LFS was 50%–100%, with RPA-LFS showing better results than qPCR. -7 When the ratio is doubled, the positive detection rate of qPCR for the three genes is 10-30%, while the positive detection rate of RPA-LFS is 30-60%, and the detection results of RPA-LFS are better than those of qPCR.

[0078] In summary, this demonstrates that at low concentrations of genomic nucleic acids (1–100 fg / μL), RPA-LFS is more sensitive than qPCR, and it is also faster, more portable, and allows for visual observation of the results.

[0079] Table 9 Sample Nucleic Acid Concentration

[0080] Experimental Example 6: Specificity Detection This experimental example provides a specific detection method for the primer-probe set shown in Example 1. The specific experimental steps are as follows: The primer and probe set of Example 1 was used to specifically validate the genomic nucleic acids of 10 pathogens: Bacillus anthraci, Brucella bovis, Bacillus cereus, Francisella tularensis, Salmonella typhimurium, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, and Listeria monocytogenes (the genomic nucleic acids of the 10 pathogens were provided and preserved by the Qinghai Provincial Institute for Endemic Disease Prevention and Control). The genomic nucleic acid of Yersinia pestis was used as a positive control. The experimental method was the same as that in Example 3 (since the samples were genomic nucleic acid, nucleic acid extraction was not required; RPA amplification and chromatographic strip detection were performed directly).

[0081] Experimental results: The results are as follows Figure 15 As shown, only the positive control, i.e., when using the genomic nucleic acid of Yersinia pestis as a template for RPA-LFS, showed two clear bands, and all three genes (Caf1 gene, YPO0392 gene and YPO2088 gene) were positive, indicating that the RPA primer and probe set provided in Example 1 has good specificity for Yersinia pestis.

[0082] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An RPA primer and probe set for the detection of Yersinia pestis, characterized in that, The RPA primer and probe set includes a first primer and probe set targeting the Caf1 gene of Yersinia pestis, a second primer and probe set targeting the YPO0392 gene of Yersinia pestis, and a third primer and probe set targeting the YPO2088 gene of Yersinia pestis.

2. The RPA primer-probe set according to claim 1, characterized in that, The first primer-probe set includes a first upstream primer, a first downstream primer, and a first RPA probe; the nucleotide sequence of the first upstream primer is shown in SEQ ID NO.1; the nucleotide sequence of the first downstream primer is shown in SEQ ID NO.2; and the nucleotide sequence of the first RPA probe is shown in SEQ ID NO.

3. Preferably, the 5' end of the first RPA probe is modified with a fluorescent group; a cleavage site of an endonuclease is inserted at the middle position of the first RPA probe; a blocking group is attached to the 3' end of the first RPA probe; and the 5' end of the first downstream primer is modified with biotin. More preferably, the middle position of the first RPA probe forms a cleavage site for a nuclease by replacing one base with tetrahydrofuran (THF); the fluorescent group modified at the 5' end of the first RPA probe includes at least one of FITC and FAM; and the blocking group attached to the 3' end of the first RPA probe includes a C3 spacer.

3. The RPA primer-probe set according to claim 1, characterized in that, The second primer-probe set includes a second upstream primer, a second downstream primer, and a second RPA probe; the nucleotide sequence of the second upstream primer is shown in SEQ ID NO. 4; the nucleotide sequence of the second downstream primer is shown in SEQ ID NO. 5; and the nucleotide sequence of the second RPA probe is shown in SEQ ID NO.

6. Preferably, the 5' end of the second RPA probe is modified with a fluorescent group; a cleavage site of an endonuclease is inserted at the middle position of the second RPA probe; a blocking group is attached to the 3' end of the second RPA probe; and the 5' end of the second downstream primer is modified with biotin. More preferably, the middle position of the second RPA probe forms a cleavage site for a nuclease by replacing one base with tetrahydrofuran (THF); the fluorescent group modified at the 5' end of the second RPA probe includes at least one of FITC and FAM; and the blocking group attached to the 3' end of the second RPA probe includes a C3 spacer.

4. The RPA primer-probe set according to claim 1, characterized in that, The third primer-probe set includes a third upstream primer, a third downstream primer, and a third RPA probe; the nucleotide sequence of the third upstream primer is shown in SEQ ID NO. 7; the nucleotide sequence of the third downstream primer is shown in SEQ ID NO. 8; and the nucleotide sequence of the third RPA probe is shown in SEQ ID NO.

9. Preferably, the 5' end of the third RPA probe is modified with a fluorescent group; a cleavage site of an endonuclease is inserted at the middle position of the third RPA probe; a blocking group is attached to the 3' end of the third RPA probe; and the 5' end of the third downstream primer is modified with biotin. More preferably, the middle position of the third RPA probe forms a cleavage site for a nuclease by replacing one base with tetrahydrofuran (THF); the fluorescent group modified at the 5' end of the third RPA probe includes at least one of FITC and FAM; and the blocking group attached to the 3' end of the third RPA probe includes a C3 spacer.

5. A kit for detecting Yersinia pestis, characterized in that, The kit comprises the RPA primer and probe set as described in any one of claims 1-4.

6. The reagent kit according to claim 5, characterized in that, The kit also includes RPA lyophilized microspheres, activator, reconstitution buffer, product diluent, chromatography strips, positive control and / or negative control.

7. The reagent kit according to claim 6, characterized in that, The RPA lyophilized microspheres comprise recombinase, recombinase coenzyme, single-strand binding protein, strand displacement DNA polymerase, and endonuclease IV; and / or, the activator comprises Mg 2+ ; and / or, the reconstitution buffer comprises polyethylene glycol 35000; and / or, the product diluent comprises PBS buffer, sodium chloride, and Tween 20.

8. A method for detecting Yersinia pestis, wherein the detection method is not for the purpose of disease diagnosis and treatment, characterized in that, The detection method includes: detecting the sample to be tested using the kit according to any one of claims 5-7; Preferably, the detection method includes the following steps: Step 1: Extract genomic DNA from the sample to be tested; Step 2: Mix the reconstitution buffer and the RPA primer and probe set according to any one of claims 1-4 to obtain a dissolution solution; use the dissolution solution to dissolve the RPA lyophilized microspheres to obtain an RPA reconstitution solution; add an activator to the RPA reconstitution solution to obtain an RPA reaction system; Step 3: Using the genomic DNA obtained in Step 1 as a template, the template is amplified at an isothermal temperature in the RPA reaction system using the RPA primer and probe set described in any one of claims 1-4 to obtain the amplification product; Step 4: Dilute the amplification product obtained in Step 3 with product diluent, then perform chromatography on a chromatographic test strip and develop the color; determine whether Yersinia pestis is present in the sample based on the color development results.

9. The method according to claim 8, characterized in that, In step 3, the temperature of the isothermal amplification is 39~41℃; the time of the isothermal amplification is 10~30 min; and / or, in step 4, the time for chromatography and color development on the chromatographic test strip is 5~15 min.

10. The application of the RPA primer and probe set according to any one of claims 1-4, the kit according to any one of claims 5-7, or the method according to claim 8 or 9 in the detection of Yersinia pestis, wherein the application is not for the purpose of disease diagnosis and treatment.

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