Joint detection method for edwardsiella tarda and aeromonas hydrophila based on hyper-branched rolling circle amplification transverse flow test strip method

Abstract

The invention discloses a joint detection method for edwardsiella tarda and aeromonas hydrophila based on a hyper-branched rolling circle amplification transverse flow test strip method, and relates to biological detection. Extracting sample genome DNA (deoxyribonucleic acid); the method comprises the following steps: designing specific lock-type probes PLP-Et and PLP-Ah, a universal primer with a marker and a specific primer; the method comprises the following steps: constructing three double-index colored latex microsphere transverse flow test strips with a double-color double-detection line, a single-color double-detection line and a double-color single-detection line; the three test strips can be used for simultaneously detecting two pathogenic bacteria; dNA templates and lock-type probes are added in the denaturation and connection process, corresponding primers are selected according to the types of the test strips, amplification is performed at 63 DEG C for 60 min, three test strips are used for detection, and a joint detection system is established; and verifying the specificity and sensitivity of the test strip. The hyper-branched rolling circle amplification is combined with the transverse flow test strip, so that two pathogenic bacteria can be jointly detected in the same system, the operation is convenient and rapid, and the method is suitable for field detection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a combined detection method for Edwardsiella tarda and Aeromonas hydrophila based on a hyperbranched rolling circle amplification (HRCA) lateral flow test strip method, which can be applied to the rapid detection of relevant pathogenic bacteria in aquaculture. Background Art

[0002] Edwardsiella tarda and Aeromonas hydrophila are both important pathogens in aquaculture. Among them, Edwardsiella tarda can cause diseases such as sepsis and gastroenteritis in fish (Zou Wenzheng, Yu Jianming, Cai Hongjiao, et al. Construction of a model and pathological analysis of zebrafish infected with Edwardsiella tarda [J]. Acta Microbiologica Sinica, 2023, 63(12): 4686-4697), while gill rot and sepsis in aquatic animals are both related to Aeromonas hydrophila (Guo Songlin, Guan Ruizhang. Effects of Aeromonas hydrophila infection on the immune function of different blood cells in American eel [J], Journal of Huazhong Agricultural University, 2009, 28(4): 463-466). Molecular detection methods that only target one pathogen are inefficient and cannot judge mixed infections of multiple pathogens. If two pathogens can be detected simultaneously and quickly and easily on-site, the detection cost can be effectively reduced and play a role in preventing and controlling related diseases.

[0003] Multiplex fluorescence quantitative PCR can detect multiple pathogens simultaneously (Xu Enhong, Qi Mingpu, Xiang Zhijie, et al. Establishment of a multiplex qPCR method for the joint detection of seven pathogens of bovine respiratory disease syndrome [J]. Journal of Huazhong Agricultural University, 2023, 42(02): 38-47). However, commercial fluorescence quantitative PCR instruments usually include a circulating temperature control system and a fluorescence detection system, which are relatively expensive and will increase the cost of grassroots on-site testing. In addition, high-throughput PCR methods for detecting different pathogens require multiplex PCR, which is prone to false positives in multiplex PCR, and different primer pairs affect each other, resulting in low amplification efficiency. HRCA is a constant temperature nucleic acid amplification detection method. Its principle is to design a specific padlock probe (PLP) and a pair of primers. The two ends of the padlock probe are complementary to the target DNA to form a ring. The primer recognizes the circular padlock probe, thereby realizing HRCA amplification of the padlock probe and achieving the purpose of signal amplification (Zhang Shijia, Feng Jianjun, Guo Songlin, et al., Application of Rolling Circle Amplification Technology in Pathogenic Microorganism Detection [J]. Journal of Analysis and Testing, 2021, 40(10): 1538-1544). The HRCA reaction has extremely high specificity and does not require an expensive circulating temperature control system. A simple constant temperature water bath can be used for operation. And because the universal primers in HRCA can equivalently amplify multiple different padlock probes, the problem of mutual interference between multiple pairs of primers in multiplex PCR detection is overcome. The lateral flow strip method (LFD) has many advantages such as low cost, easy operation and visualization. When combined with the HRCA method, it will be particularly suitable for on-site detection (Kong Chengjiang, Shi Yuhong, Li Haoyan, et al. Detection of infectious spleen and kidney necrosis virus by hyperbranched rolling circle amplification [J]. Fisheries Science, 2011, 30(9): 575-579.). Currently, there are no reports on the simultaneous detection of Edwardsiella tarda and Aeromonas hydrophila using the HRCA-LFD method, and even the detection of only one of the bacteria has not been found. In addition, the advantage of changing the marker from the common colloidal gold to latex microspheres is that it is stable and has a variety of colors (Liu Yuan, Zhang Kaihui, Wang Yingying, et al. Research progress of multiple immunochromatographic detection technology in rapid food safety detection [J]. Food and Fermentation Industries, 2023, 49(1): 337-346,360.), and different pathogens can be detected separately by color changes alone. Summary of the Invention

[0004] The present invention aims to provide a low-cost, easy-to-use, and visually accessible method for the combined detection of Edwardsiella tarda and Aeromonas hydrophila using HRCA-LFD, suitable for field applications. This method allows for the combined detection of Edwardsiella tarda and Aeromonas hydrophila in the same system, combining the visualization provided by dual-indicator colored latex microspheres to achieve multi-label detection using HRCA-LFD.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0006] The present invention provides a combined detection method for Edwardsiella tarda and Aeromonas hydrophila based on HRCA-LFD, comprising the following steps:

[0007] 1) Extracting genomic DNA from samples: Take the bacterial solution or tissue sample to be tested and extract the genomic DNA template using the adsorption column method;

[0008] 2) Probe and Primer Design: Designed specific padlock probes for Edwardsiella tarda and Aeromonas hydrophila, respectively. A universal primer was labeled with fluorescein isothiocyanate (FITC-F) at the 5' end. For PLP-Et, a pair of primers (FITC-Et1 and Bio-Et2) were labeled with FITC and biotin at the 5' end, respectively. For PLP-Ah, a pair of primers (TAMRA-Ah1 and Dig-Ah) were labeled with 5-carboxytetramethylrhodamine and digoxigenin at the 5' end, respectively. Both the padlock probes (PLP-Et) and PLP-Ah contained recognition sequences and linker sequences. The universal primer FITC-F combined with the two padlock probes achieved joint amplification.

[0009] 3) Design of dual-index colored latex microsphere lateral flow test strip:

[0010] Dual-color, dual-detection line test strip: The front section of the chromatography pad is coated with blue latex microspheres-bound streptavidin and red latex microspheres-bound mouse anti-TAMRA antibody; detection line T1 is coated with FITC antibody, detection line T2 is coated with digoxigenin antibody, and quality control line C is coated with goat anti-mouse secondary antibody;

[0011] Single-color dual-detection line test strip: The front end of the chromatography pad is coated with red latex microspheres-bound mouse anti-FITC antibody; detection line T1 is coated with streptavidin, detection line T2 is coated with digoxigenin antibody, and quality control line C is coated with goat anti-mouse secondary antibody;

[0012] Dual-color single-line test strip: The front section of the chromatography pad is coated with red latex microspheres-bound mouse anti-digoxigenin antibody and blue latex microspheres-bound streptavidin; the test line T is coated with FITC antibody, and the quality control line C is coated with goat anti-mouse secondary antibody and biotin;

[0013] 4) Establish a combined detection system: During the denaturation and ligation process, add 4 μL of bacterial genomic DNA template and 1 μL of each of the two padlock probes. During the amplification process, add corresponding labeled primers according to the test strip type, and select 63°C amplification for 60 min as the reaction condition. For dual-line dual-detection line test strips, add primers FITC-Et1, Bio-Et2, TMARA-Ah1, and Dig-Ah2 for multiplex amplification. After dilution, the amplified products are detected using dual-color dual-detection line test strips. For single-color dual-detection line test strips and dual-color single-detection line test strips, add primers FITC-F, Bio-Et2, and Dig-Ah2 for combined amplification. After different dilutions, the products are detected using the corresponding test strips.

[0014] 5) Visual interpretation of results: For two-color, two-line strips, blue test line T1 indicates positive for Edwardsiella tarda, red test line T2 indicates positive for Aeromonas hydrophila, and a red control line C indicates a valid test. For single-color, two-line strips, red test line T1 indicates positive for Edwardsiella tarda, red test line T2 indicates positive for Aeromonas hydrophila, and a red control line C indicates a valid test. For two-color, single-line strips, blue test line at the same location indicates positive for Edwardsiella tarda, red test line indicates positive for Aeromonas hydrophila, and purple test line indicates positive for both bacteria. A mixed color control line C indicates a valid test.

[0015] In step 1), the specific steps of extracting the sample genomic DNA may be: reviving the bacteria stored at -80°C, adding bacterial solution and LB liquid culture medium at a ratio of 1:100, and culturing at 250 rpm for 12 hours in a constant temperature shaker according to the optimal growth temperature of the strain to achieve bacterial rejuvenation; after rejuvenation, streaking on a solid culture medium plate, culturing for 18 hours, picking a single colony for expansion; taking 1 mL of the expanded culture solution, centrifuging and discarding the supernatant, resuspending and washing with PBS, resuspending with RB and adding proteinase K and CB for digestion, balancing the adsorption column, adding isopropanol, transferring to the adsorption column, IR washing, WB washing, drying, and finally adding sterile double distilled water and centrifuging. The obtained genomic DNA sample is stored at -20°C for use.

[0016] In step 2), the sequence of the specific padlock probe PLP-Et is shown in the sequence listing as SEQ ID NO: 1, specifically:

[0017] 5'-P-TCGGAAGTGAGAGGTATATCGACAAagctttcccaataggtccagaatgctgaatccgttagccagcagc cgcctcgacgaatACAAGAAGAACCACA-3';

[0018] The sequence of the specific padlock probe PLP-Ah is shown in the sequence listing as SEQ ID NO: 2, specifically:

[0019] 5'-P-GACAGTCCCAGGTCCAACATACGTTttcccaataggtccagaatgtggaaccgaggaaatagagaaccc cagtccctagctAGTGGTACAAACTTT-3';

[0020] The universal primer FITC-F with a label is shown in SEQ ID NO: 3, specifically:

[0021] 5'-FITC-ATTCTGGACCTATTGGGAA-3';

[0022] The specific primer FITC-Et1 with a label is shown in SEQ ID NO: 4, specifically:

[0023] 5'-FITC-GATGGTGGTTCTTCTTGTATT-3';

[0024] The specific primer Bio-Et2 with a marker is shown in SEQ ID NO: 5, specifically:

[0025] 5'-Bio-GCTGAATCCGTTAGCCAGCA-3';

[0026] The specific primer TAMRA-Ah1 with a marker is shown in SEQ ID NO: 6, specifically:

[0027] 5'-TAMRA-ACCTGGGACTGTCAAAGTTTGTACC-3';

[0028] The specific primer Dig-Ah2 with a marker is shown in SEQ ID NO: 7, specifically:

[0029] 5'-Dig-GTGGAACCGAGGAAATAGAG-3'.

[0030] In step 4), the reaction system further comprises: 2.5 μL 10× ligation buffer, 1 U T4 DNA ligase, 1 U Hi29 DNA polymerase, and 0.4 mM dNTPs.

[0031] In step 4), when the dual-color dual-detection line test strip is used for detection, the detection line T1 is used to detect the nucleic acid amplification product labeled with biotin and FITC, and the detection line T2 is used to detect the nucleic acid amplification product labeled with digoxigenin and TAMRA.

[0032] When the single-color dual-detection line test strip is used for detection, the detection line T1 is used to detect the nucleic acid amplification product labeled with biotin and FITC, and the detection line T2 is used to detect the nucleic acid amplification product labeled with digoxigenin and FITC.

[0033] When the dual-color single-detection-line test strip is used for detection, the detection line T is used to detect nucleic acid amplification products labeled with biotin and FITC and nucleic acid amplification products labeled with digoxigenin and FITC simultaneously according to color differentiation.

[0034] In step 4), the detection limit for genomic DNA of Edwardsiella tarda and Aeromonas hydrophila was approximately 50 ng / mL.

[0035] The present invention provides a detection kit comprising:

[0036] Test strips: any of the following: dual-color dual-detection line test strips, single-color dual-detection line test strips, and dual-color single-detection line test strips;

[0037] Padlock probes: PLP-Et (SEQ ID NO: 1) and PLP-Ah (SEQ ID NO: 2);

[0038] Primer set: including universal primer FITC-F (SEQ ID NO: 3), FITC-Et1 primer (SEQ ID NO: 4), Bio-Et2 primer (SEQ ID NO: 5), TMARA-Ah1 primer (SEQ ID NO: 6), Dig-Ah2 primer (SEQ ID NO: 7);

[0039] HRCA premix: contains T4 DNA ligase, Phi29 DNA polymerase, dNTPs and ligation buffer;

[0040] Controls: Aeromonas hydrophila and Edwardsiella tarda mixed genomic DNA positive control, sterile water negative control.

[0041] Furthermore, in the HRCA premix, the concentration of the T4 DNA ligase is 0.5 to 2 U / reaction; the concentration of the Phi29 DNA polymerase is 0.5 to 2 U / reaction; and the concentration of the dNTPs is 0.2 to 0.6 mM.

[0042] Preferably, the concentration of the T4 DNA ligase is 1 U / reaction; the concentration of the Phi29 DNA polymerase is 1 U / reaction; and the concentration of the dNTPs is 0.4 mM.

[0043] The present invention provides application of the combined detection method of Edwardsiella tarda and Aeromonas hydrophila based on the hyperbranched rolling circle amplification lateral flow test strip method in diagnosis of mixed infection of aquatic animal pathogens.

[0044] The aquatic animal may be Japanese eel, and the test sample may be genomic DNA extracted from liver, intestine, muscle, metanephrodisiac and gill tissues.

[0045] Compared with the prior art, the outstanding technical effects and advantages of the present invention are:

[0046] 1. This invention, based on HRCA technology, utilizes universal primers and specific padlock probes to achieve the combined amplification of Edwardsiella tarda and Aeromonas hydrophila in a single reaction system. Compared to single-target detection methods, this significantly improves detection efficiency and enables simultaneous detection of both pathogens, eliminating the need for separate testing. Because the universal primer, FITC-F, in HRCA can effectively amplify two different padlock probes, it reduces the potential for interference between multiple primer pairs in multiplex PCR assays.

[0047] 2. This invention combines the visualization effect of dual-index colored latex microspheres with the lateral flow test strip method to design three different test strip formats, capable of distinguishing between single and mixed infections of two pathogens. Specificity and sensitivity validation experiments demonstrated that this detection method has good specificity and can accurately identify the target pathogens. The detection limit for genomic DNA of Edwardsiella tarda and Aeromonas hydrophila is approximately 50 ng / mL, ensuring the accuracy of the test results.

[0048] 3. The present invention uses colored latex microsphere immunochromatographic test strips, which are easy to operate and have fast detection and reading. No complex instrumentation is required; test results can be obtained simply by visually observing the color change on the test strip, making it suitable for rapid on-site testing. Furthermore, the test strips are highly stable and can distinguish different test objects using different colors, reducing competition between latex microspheres of the same color. This allows for more intuitive determination of test results in high-throughput testing.

[0049] 4. The present invention simplifies the operation steps. There is no need to perform probe hybridization and re-detection after HRCA. Instead, the test strips can be used directly for detection after rolling circle amplification.

[0050] 5. This invention combines HRCA with a combined detection system and immunochromatographic test strips for the first time, designing various latex microsphere test strips, providing new ideas and methods for the combined detection of pathogens. This overcomes the limitations of traditional detection methods and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Preparation of three types of HRCA-LFD test strips: a is a dual-color dual-detection line test strip; b is a single-color dual-detection line test strip; and c is a dual-color single-detection line test strip.

[0052] Figure 2The working principles of three types of HRCA-LFD test strips. Type a is a two-color, two-detection-line test strip; type b is a single-color, two-detection-line test strip; and type c is a two-color, single-detection-line test strip.

[0053] Figure 3 Figure 1 shows the specificity and sensitivity of the dual-color, dual-line HRCA-LFD system for Edwardsiella tarda and Aeromonas hydrophila. Figure a shows the specificity of the two pathogens, with 1-9 representing the HRCA-LFD detection results for a mixture of the two pathogens, Edwardsiella tarda, Aeromonas hydrophila, Pseudomonas proteus, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Aeromonas enterocolitica, Aeromonas zooxanthellae, and Escherichia coli DNA. Figure 10 represents the negative control. Figure b shows the sensitivity of the two pathogens. The template concentrations for the genomic DNA mixture of the two pathogens in Figures 1-8 are: 5×10 4 ng / mL, 5×10 3 ng / mL, 5×10 2 ng / mL, 50ng / mL, 5ng / mL, 5×10 -1 ng / mL, 5×10 -2 ng / mL, 5×10 -3 ng / mL. 9 was the negative control.

[0054] Figure 4 Visualization results of actual samples tested using the HRCA-LFD method. Numbers 1-5 represent results obtained using genomic DNA from liver, intestine, muscle, metanephros, and gill tissues in the mixed pathogen challenge group. Numbers 6-10 represent results obtained using genomic DNA from five tissues in the negative control group.

[0055] Figure 5 Figure 1 shows the specificity and sensitivity of the single-color dual-detection line HRCA-LFD system for Edwardsiella tarda and Aeromonas hydrophila. Figure a shows the specificity of the two pathogens, with 1-9 representing the combined amplification system's detection of a mixture of the two pathogens, Aeromonas hydrophila, Edwardsiella tarda, Pseudomonas proteus, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Aeromonas enterocolitica, Aeromonas zooxanthellae, and genomic DNA from Escherichia coli. Figure 10 represents the negative control. Figure b shows the sensitivity of the two pathogens, with 1-8 representing the detection of the mixed genomic DNA of the two pathogens. The template concentrations for the detection are 5×10 4 ng / mL, 5×10 3 ng / mL, 5×10 2 ng / mL, 50ng / mL, 5ng / mL, 5×10 -1 ng / mL, 5×10 -2 ng / mL, 5×10 -3 ng / mL. 9 was the negative control.

[0056] Figure 6 Visualization results of actual samples tested using the HRCA-LFD method. Numbers 1-5 represent results obtained using genomic DNA from liver, intestine, muscle, metanephros, and gill tissues in the mixed pathogen challenge group. Numbers 6-10 represent results obtained using genomic DNA from five tissues in the negative control group.

[0057] Figure 7 Figure 1 shows the specificity and sensitivity of the dual-color single-line HRCA-LFD system for Edwardsiella tarda and Aeromonas hydrophila. (a) shows the specificity of the two pathogens, and 1-9 are the combined amplification system's detection results for a mixture of the two pathogens, Aeromonas hydrophila, Edwardsiella tarda, Pseudomonas proteus, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Aeromonas enterocolitica, Aeromonas zooxanthellae, and genomic DNA from Escherichia coli. (10) is the negative control. (b) shows the sensitivity of the two pathogens. 1 and 2 are positive controls for Edwardsiella tarda and Aeromonas hydrophila, respectively. 3-10 are the detection template concentrations for the mixed genomic DNA of the two pathogens, respectively, at 5×10 4 ng / mL, 5×10 3 ng / mL, 5×10 2 ng / mL, 50ng / mL, 5ng / mL, 5×10 -1 ng / mL, 5×10 -2 ng / mL, 5×10 -3 ng / mL. 11 was used as negative control.

[0058] Figure 8 The following are the visualization results of the actual samples tested by the HRCA-LFD method. 1-5 are the results obtained using genomic DNA from the liver, intestine, muscle, metanephros, and gill tissues of the mixed pathogen challenge group as templates. 6-10 are the results obtained using genomic DNA from the five tissues of the negative control group as templates. DETAILED DESCRIPTION

[0059] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the following embodiments will be further described in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention. On the contrary, the present invention encompasses any alternatives, modifications, equivalent methods, and solutions made within the spirit and scope of the present invention as defined by the claims. Methods, devices, and the like not described in detail may be implemented using conventional means.

[0060] This paper aims to establish a combined detection method for Edwardsiella tarda and Aeromonas hydrophila, combining the visualization effect of dual-index colored latex microspheres to perform multi-label detection of these two aquatic pathogens. The specificity and sensitivity of this method were verified. 1.1 Materials and Instruments

[0061] Low-temperature high-speed refrigerated centrifuge, ultrapure water system, electric constant temperature water bath, agarose gel electrophoresis apparatus; UV-visible spectrophotometer, single-person single-sided clean bench.

[0062] 1.2 Main solution

[0063] Ligation system reaction reagents, enzyme digestion system reaction reagents, HRCA reaction reagents; DL2501 DNA Marker; tissue DNA extraction kit, bacterial whole genome extraction kit; colored latex microspheres, anti-TRMRA antibody, digoxigenin antibody, goat anti-mouse secondary antibody, lateral flow test strips.

[0064] 1.2 Experimental methods

[0065] 1.2.1 Bacterial culture and nucleic acid extraction

[0066] The bacteria stored at -80°C were revived and expanded, and genomic DNA was extracted according to the instructions of the bacterial whole genome extraction kit. The obtained DNA samples were stored in a -20°C low-temperature refrigerator for subsequent experiments.

[0067] 1.2.2 Probe and primer design

[0068] Table 1 Padlock probes and primers

[0069]

[0070] The designed sequences of padlock probes and primers are shown in Table 1, wherein PLP-Et is a padlock probe specific for Edwardsiella tarda, and PLP-Ah is a padlock probe specific for Aeromonas hydrophila. The capital letters represent the recognition sequence, and the lowercase letters represent the connection sequence. Primer F corresponds to the position of the two padlock probes. represents primer Et1 and its corresponding position in the padlock probe sequence, represents primer Et2 and its corresponding position in the padlock probe sequence, Indicates primer Ah-1 and its corresponding position in the padlock probe sequence, Indicates primer Ah-2 and its corresponding position in the padlock probe sequence.

[0071] 1.2.3 Design of dual-index colored latex microsphere test strips

[0072] 1.2.3.1 Two-color, two-line test strips

[0073] A dual-color dual-detection line latex microsphere LFD was designed for dual-index detection. The front section of the chromatography pad was coated with blue latex microsphere-bound streptavidin and red latex microsphere-bound mouse anti-TAMRA antibody. The detection line T1 was coated with FITC antibody, the detection line T2 was coated with digoxigenin antibody, and the quality control line C was coated with goat anti-mouse secondary antibody ( Figure 1 a in the figure). Detection line T1 is used to detect nucleic acid amplification products labeled with biotin and FITC. When the product is positive, blue latex microspheres are enriched in T1, and a blue band is visible. Detection line T2 is used to detect nucleic acid amplification products labeled with digoxin and TAMRA. When the product is positive, red latex microspheres are enriched in T2, and a red band is observed. The test results of the positive experimental group should be as follows: when HRCA-LFD is performed on a single target sample of Edwardsiella tarda, the detection line T1 of the test strip shows a blue band, the detection line T2 does not show color, and the quality control line C shows color; when HRCA-LFD is performed on a single target sample of Aeromonas hydrophila, the detection line T1 of the test strip does not show color, the detection line T2 shows a red band, and the quality control line C shows color; when HRCA-LFD is performed on a mixed sample of two pathogens, the detection line T1 of the test strip shows a blue band, the detection line T2 shows a red band, and the quality control line C shows color ( Figure 2 In the negative control group, the test results showed that both the T1 and T2 test lines did not show color, and only the quality control line C showed color.

[0074] 1.2.3.2 Single-color dual-line test strips

[0075] According to the structure and principle of the chromatography test strip, a single-color double-detection line latex microsphere LFD for dual-index detection was designed. The front section of the chromatography pad was coated with red latex microsphere-bound mouse anti-FITC antibody, the detection line T1 was coated with streptavidin, the detection line T2 was coated with digoxigenin antibody, and the quality control line C was coated with goat anti-mouse secondary antibody ( Figure 1 b). Detection line T1 is used to detect nucleic acid amplification products labeled with biotin and FITC. When the product is positive, the latex microspheres are enriched in T1 and a band is visible. Detection line T2 is used to detect nucleic acid amplification products labeled with digoxigenin and FITC. When the product is positive, the latex microspheres are enriched in T2 and a band is visible. The test results of the positive experimental group should be as follows: when the single target sample of Edwardsiella tarda is tested by HRCA-LFD, the detection line T1 of the test strip shows a red band, the detection line T2 does not show color, and the quality control line C shows color; when the single target sample of Aeromonas hydrophila is tested by HRCA-LFD, the detection line T1 of the test strip does not show color, the detection line T2 shows a red band, and the quality control line C shows color; when the mixed sample of two pathogens is tested by HRCA, both the detection lines T1 and T2 of the test strip show red bands, and the quality control line C shows color ( Figure 2 b). The negative control group test result is that both test lines T1 and T2 do not show color, and only the quality control line C shows color. 1.2.3.3 Dual-color single test line test strips

[0076] According to the structure and principle of the chromatography test strip, a dual-color single-detection line latex microsphere LFD capable of dual-index detection was designed. The front section of the chromatography pad was coated with red latex microspheres combined with mouse anti-digoxigenin antibody and blue latex microspheres combined with streptavidin. The detection line T was coated with FITC antibody, and the quality control line C was coated with goat anti-mouse secondary antibody and biotin ( Figure 1 c in the figure). The detection line can be used to detect two products at the same time based on color differentiation: one is a nucleic acid amplification product labeled with biotin and FITC. When the product is positive, blue latex microspheres are enriched on the detection line; the other is a nucleic acid amplification product labeled with digoxigenin and FITC. When the product is positive, red latex microspheres are enriched on the detection line. The test results of the positive experimental group should be: when the single target sample of Edwardsiella tarda is tested by HRCA-LFD, the detection line T of the test strip is a blue strip, and the quality control line C is colored; when the single target sample of Aeromonas hydrophila is tested by HRCA-LFD, the detection line T of the test strip is red, and the quality control line C is colored; when the mixed sample of two pathogens is tested by HRCA, the detection line of the test strip is a purple mixed of red and blue, and the quality control line C is colored ( Figure 2 c). The negative control group test result is that the test line does not show color, and only the quality control line C shows color.

[0077] 1.2.4 Establishing a joint detection system for two pathogens

[0078] During the denaturation and ligation process, 4 μL of bacterial genomic DNA template and 1 μL of each of the two padlock probes were added. During the amplification process, corresponding primers were added according to the test strip type, and then amplified at 63°C for 60 minutes. The dual-line dual detection line test strip requires a total of four different markers. Primers FITC-Et1, Bio-Et2, TMARA-Ah1, and Dig-Ah2 are added to the HRCA multiple amplification reaction. Edwardsiella tarda and Aeromonas hydrophila are amplified separately in the same reaction tube. 10 μL of the product is added to 60 μL of sterile double-distilled water, and the strip is inserted into the dual-color dual detection line test strip. The test results are read after 15 minutes. The single-color dual detection line and dual-color single detection line test strips require three different markers. Primers FITC-F, Bio-Et2, and Dig-Ah2 are added to the HRCA combined amplification reaction. The two pathogens are jointly amplified using the universal primer FITC-F. 10 μL of the product is added to 60 μL of sterile double-distilled water, and the test results can be read using the single-color dual detection line test strip and the dual-color single detection line test strip.

[0079] 1.2.5 Quality Verification of Test Strip Test Results

[0080] Two detection templates were added to simulate the mixed infection of Edwardsiella tarda and Aeromonas hydrophila, and the specificity and sensitivity of the dual-index color latex microsphere test paper were verified respectively.

[0081] 1.2.6 Test strips for actual samples

[0082] To verify the detection effect of the present invention, Japanese eels weighing 20-30 g were starved for one week, and the experimental group was injected with 0.85% saline diluted to 1×10 8 A mixed bacterial solution of Edwardsiella tarda and Aeromonas hydrophila (CFU / mL) was administered to a control group, with a 0.85% saline solution injection rate of 0.1 mL / 10 g. Three days later, eels without obvious disease symptoms were selected. Liver, intestine, muscle, metanephros, and gill tissues were collected and preserved in 95% ethanol. Genomic DNA from these tissues was extracted and used as a template for HRCA combined amplification. The product was analyzed using test strips to verify the detection of the actual sample.

[0083] 1.3 Results

[0084] 1.3.1 Specificity, sensitivity, and actual sample detection results of dual-color dual-detection line test strips

[0085] The genomic DNA of Edwardsiella tarda and Aeromonas hydrophila were detected simultaneously using the HRCA-LFD multiplex amplification system. The results were observed using a two-color two-line test strip. The specificity and sensitivity results are shown in Figure 3 In the specificity experiment, the test results of adding genomic DNA of two pathogens were double positive. The test results of adding genomic DNA of Aeromonas hydrophila and Edwardsiella tarda were consistent with the individual detection system. The test results of other bacteria and negative controls were negative. The specificity results of the colored latex microsphere test strips can distinguish between the two pathogens alone or in mixed infection. The constructed HRCA-LFD multiple amplification system has good specificity. In the sensitivity experiment, the colored latex microsphere test strips can detect the genomic DNA of the two pathogens at a concentration of at least 50 ng / mL. Therefore, the detection limit of the constructed HRCA-LFD for the genomic DNA of Edwardsiella tarda and Aeromonas hydrophila is about 50 ng / mL.

[0086] The genomic DNA of liver, intestine, muscle, metanephros, and gill tissues of the mixed infection group of Edwardsiella tarda and Aeromonas hydrophila and the genomic DNA of the control tissue injected with normal saline were used as detection templates for HRCA-LFD double amplification detection. The test strip results are shown in the figure. Figure 4 All five tissues in the challenge group tested positive, while the control group tested negative. The established HRCA-LFD multiplex amplification method for two pathogens, coupled with a two-color, two-line strip-based visual detection method, can be used for actual sample testing.

[0087] 1.3.2 Specificity, sensitivity, and actual sample detection results of single-color dual-line test strips

[0088] The HRCA-LFD combined amplification system was used to simultaneously detect the genomic DNA of Edwardsiella tarda and Aeromonas hydrophila, and the results were observed using a single-color double-line test strip. The specificity and sensitivity results are shown in Figure 5 In the specificity experiment, the test results of adding genomic DNA of two pathogens were double positive; the test results of adding genomic DNA of Aeromonas hydrophila and Edwardsiella tarda were consistent with the individual detection system, and the test results of other bacteria and negative controls were negative. The specificity results of the single-color double-line test strip can distinguish between the two pathogens alone or in mixed infection. The constructed HRCA combined amplification system has good specificity. In the sensitivity experiment, the colored latex microsphere test strip can detect the genomic DNA of the two pathogens at a concentration of at least 50 ng / mL. Therefore, the detection limit of the constructed HRCA-LFD for the genomic DNA of Edwardsiella tarda and Aeromonas hydrophila is approximately 50 ng / mL. However, when the DNA concentration is high, the color of the detection line T1 is darker, and the color of the detection line T2 and the quality control line is not clear enough. It is speculated that this is because the capacity of the mouse anti-FITC antibody coated in the chromatography pad is limited, but it needs to bind to the HRCA products of the two bacteria, resulting in insufficient latex microspheres to display color at the detection line T2.

[0089] The genomic DNA of liver, intestine, muscle, metanephros, and gill tissues of the mixed infection group of Edwardsiella tarda and Aeromonas hydrophila and the genomic DNA of the control tissue injected with normal saline were used as detection templates for HRCA-LFD combined amplification detection. The test strip results are shown in the figure. Figure 6 All five tissues in the challenge group tested positive, while those in the control group tested negative. However, the test line T1 was often darker, while the test line T2 and the quality control line were unclear. The established HRCA-LFD combined amplification method for two pathogens and the visual detection method using single-color dual-line test strips can be used for actual sample testing.

[0090] 1.3.3 Specificity, sensitivity, and actual sample detection results of dual-color single-line test strips

[0091] The HRCA-LFD combined amplification system was used to simultaneously detect the genomic DNA of Edwardsiella tarda and Aeromonas hydrophila, and the results were observed using a two-color single-line test strip. The specificity and sensitivity results are shown in Figure 7In the specificity experiment, the test results of adding the genomic DNA of two pathogens showed a purple color mixed with red and blue. The test results of adding the genomic DNA of Aeromonas hydrophila and Edwardsiella tarda were red and blue respectively. The test results of other bacteria and negative controls were negative. The specificity results of the two-color single-line test strip can distinguish between the two pathogens alone or in mixed infection. The constructed HRCA combined amplification system has good specificity. In the sensitivity experiment, the colored latex microsphere test strip can detect the genomic DNA of the two pathogens at a concentration of at least 50 ng / mL. Therefore, the detection limit of the constructed HRCA-LFD for the genomic DNA of Edwardsiella tarda and Aeromonas hydrophila is about 50 ng / mL.

[0092] The genomic DNA from the liver, intestine, muscle, metanephros, and gill tissues of the mixed infection group of Edwardsiella tarda and Aeromonas hydrophila, as well as the genomic DNA from the control tissue injected with normal saline, were used as detection templates for HRCA-LFD combined amplification detection. The results of the two-color single-line test strips are shown in Figure 2. Figure 8 The test results of the five tissues in the challenge group were all positive, while those in the control group were negative. The established HRCA-LFD combined amplification method for the two pathogens and the visual detection method using single and double-color single-line test paper can be used for actual sample detection.

[0093] 1.4 Conclusion

[0094] This study designed specific padlock probes (PLP-Et and PLP-Ah) for two aquatic pathogens, Edwardsiella tarda and Aeromonas hydrophila. Combined with high-resolution amplification (HRCA) and colored latex microsphere LFD technology, this method enables rapid, visual detection of both pathogens. This is the first application of the HRCA multiplex amplification system combined with three different latex microsphere test strip designs to meet the needs of diverse application scenarios. The first dual-color, dual-detection line test strip requires a total of four markers in the positive test object. During amplification, the HRCA multiplex amplification method that combines two detection systems is adopted. Two specific padlock probes are added with a total of four primers during the amplification stage, which specifically bind to the two latex microsphere markers and the two detection line antibodies respectively. There is no competition between the latex microspheres, and the color development is good, which is suitable for high-precision detection. The second single-color dual-detection line test strip has all red latex microspheres labeled with mouse anti-FITC antibodies, and the two detection lines are coated with different recognition antibodies. Therefore, a total of three markers are required for the amplification product, which can be jointly amplified using universal primer F and specific downstream primers of the two padlock probes. Compared with the HRCA dual amplification system, one primer sequence can be saved, and the detection performance is the same, with low cost. The third dual-color, single-detection line test strip completes detection by using three different markers in the HRCA combined amplification product. The detection performance is consistent with the results of the first two test strip methods, but with good visual effects, the simplest operation, and the results can be judged only by color changes, making it suitable for rapid on-site diagnosis.

[0095] The above embodiments are only preferred embodiments of the present invention and should not be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent of the present invention.

Claims

1. A method for the combined detection of Edwardsiella tarda and Aeromonas hydrophila based on a hyperbranched rolling circle amplification lateral flow test strip method, characterized in that The following steps are involved: 1) Design of probes and primers: Design of a padlock probe PLP-Et (SEQ ID NO: 1) specific for Edwardsiella tarda and a padlock probe PLP-Ah (SEQ ID NO: 2) specific for Aeromonas hydrophila; as well as a labeled universal primer FITC-F (SEQ ID NO: 3) and labeled specific primers FITC-Et1 (SEQ ID NO: 4), Bio-Et2 (SEQ ID NO: 5), TAMRA-Ah1 (SEQ ID NO: 6), and Dig-Ah2 (SEQ ID NO: 7); 2) Design of lateral flow test strips, including: (1) Dual-color dual-detection line test strip: The front section of the chromatography pad is coated with blue latex microspheres conjugated to streptavidin and red latex microspheres conjugated to mouse anti-TAMRA antibody; detection line T1 is coated with FITC antibody, detection line T2 is coated with digoxigenin antibody, and quality control line C is coated with goat anti-mouse secondary antibody; (2) Single-color dual-detection line test strip: The front end of the chromatography pad is coated with red latex microspheres-conjugated mouse anti-FITC antibody; the detection line T1 is coated with streptavidin, the detection line T2 is coated with digoxigenin antibody, and the quality control line C is coated with goat anti-mouse secondary antibody; (3) Two-color single-detection line test strip: The front section of the chromatography pad is coated with red latex microspheres-bound mouse anti-digoxigenin antibody and blue latex microspheres-bound streptavidin; the detection line T is coated with FITC antibody, and the quality control line C is coated with goat anti-mouse secondary antibody and biotin. 3) Amplification: Extract genomic DNA from the sample to be tested, add the designed probe and primers to perform HRCA rolling circle amplification; 4) Interpretation of results: After dilution, the amplified product is visually detected using a lateral flow test strip. Single or mixed infection is determined based on the color of the test line.

2. The method for the combined detection of Edwardsiella tarda and Aeromonas hydrophila based on the hyperbranched rolling circle amplification lateral flow test strip method according to claim 1, characterized in that In step 3), the HRCA rolling circle amplification reaction system further includes: 2.5 μL 10× ligation buffer, 1 μL UT4 DNA ligase, 1 μL UpHi29 DNA polymerase, and 0.4 mM dNTPs.

3. The method for combined detection of Edwardsiella tarda and Aeromonas hydrophila based on the hyperbranched rolling circle amplification lateral flow test strip method according to claim 1, characterized in that In step 3), when a dual-color dual-detection line test strip is used, four primers are added to the HRCA rolling circle amplification: FITC-Et1, Bio-Et2, TAMRA-Ah1, and Dig-Ah2.

4. The method for combined detection of Edwardsiella tarda and Aeromonas hydrophila based on the hyperbranched rolling circle amplification lateral flow test strip method according to claim 1, characterized in that In step 3), when a single-color dual-detection line test strip or a dual-color single-detection line test strip is used, three primers are added to the HRCA rolling circle amplification: FITC-F, Bio-Et2, and Dig-Ah2.

5. The method for combined detection of Edwardsiella tarda and Aeromonas hydrophila based on the hyperbranched rolling circle amplification lateral flow test strip method according to claim 1, characterized in that In step 4), the specific criteria for determining single or mixed infection based on the color development of the test line are: Two-color dual-detection line test strips: detection line T1 is blue for Edwardsiella tarda positive, detection line T2 is red for Aeromonas hydrophila positive, and quality control line C is red, indicating that the test is effective; Single-color double-detection line test strips: red detection line T1 corresponds to positive Edwardsiella tarda, red detection line T2 corresponds to positive Aeromonas hydrophila, and red control line C indicates that the test is effective; For the dual-color single-detection line test strip, if the detection line T at the same position is blue, it is positive for Edwardsiella tarda, if it is red, it is positive for Aeromonas hydrophila, and if it is purple, it is positive for both bacteria. If the quality control line C shows mixed colors, it means the test is valid.

6. The method for the combined detection of Edwardsiella tarda and Aeromonas hydrophila based on the hyperbranched rolling circle amplification lateral flow test strip method according to claim 1, characterized in that In step 4), the detection limit of the assay was 50 ng / mL.

7. A detection kit, characterized in that Include: Lateral flow test strips: any of the following: dual-color dual-detection line test strips, single-color dual-detection line test strips, and dual-color single-detection line test strips; Padlock probes: PLP-Et (SEQ ID NO: 1) and PLP-Ah (SEQ ID NO: 2); Primer mixture: including a first primer mixture for a dual-color dual-detection line test strip or a second primer mixture for a single-color dual-detection line or dual-color single-detection line test strip; wherein the first primer mixture contains FITC-Et1 (SEQ ID NO: 4), Bio-Et2 (SEQ ID NO: 5), TAMRA-Ah1 (SEQ ID NO: 6), and Dig-Ah2 (SEQ ID NO: 7); the second primer mixture contains FITC-F (SEQ ID NO: 3), Bio-Et2 (SEQ ID NO: 5), and Dig-Ah2 (SEQ ID NO: 7); HRCA premix: contains T4 DNA ligase, Phi29 DNA polymerase, dNTPs and ligation buffer; Controls: Aeromonas hydrophila and Edwardsiella tarda mixed genomic DNA positive control, sterile water negative control.

8. A detection kit according to claim 7, characterized in that: Dual-color, dual-detection line test strip: The front section of the chromatography pad is coated with blue latex microspheres conjugated to streptavidin and red latex microspheres conjugated to mouse anti-TAMRA antibody; detection line T1 is coated with FITC antibody, detection line T2 is coated with digoxigenin antibody, and quality control line C is coated with goat anti-mouse secondary antibody; Single-color dual-detection line test strip: The front end of the chromatography pad is coated with red latex microspheres-bound mouse anti-FITC antibody; detection line T1 is coated with streptavidin, detection line T2 is coated with digoxigenin antibody, and quality control line C is coated with goat anti-mouse secondary antibody; Dual-color single-detection line test strip: The front section of the chromatography pad is coated with red latex microspheres-bound mouse anti-digoxigenin antibody and blue latex microspheres-bound streptavidin; the detection line T is coated with FITC antibody, and the quality control line C is coated with goat anti-mouse secondary antibody and biotin.

9. A detection kit as claimed in claim 7, characterized in that In the HRCA premix, the concentration of the T4 DNA ligase is 0.5 to 2 U / reaction; the concentration of the Phi29 DNA polymerase is 0.5 to 2 U / reaction; and the concentration of the dNTPs is 0.2 to 0.6 mM.

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