LAMP (loop-mediated isothermal amplification) primer composition for nucleic acid chromatography, IBV (infectious bursal virus) detection test strip, kit, application of LAMP primer composition, IBV detection test strip and kit, and IBV detection method
By designing antibody/biotin-independent LAMP primer compositions and IBV test strips, the problem of IBV detection relying on professional personnel and expensive instruments has been solved, achieving highly sensitive, low-cost, and convenient IBV detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HEFEI SHANBEN BIOTECHNOLOGY CO LTD
- Filing Date
- 2025-12-19
- Publication Date
- 2026-05-12
Smart Images
Figure CN122012807A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of virology and molecular biology, specifically to a LAMP primer composition for nucleic acid chromatography, an IBV test strip, a kit, and their applications and methods for detecting IBV. Background Technology
[0002] Infectious bronchitis (IB) in chickens is a highly contagious acute upper respiratory tract disease caused by IBV, which has a significant impact on the global poultry industry. On the one hand, it causes a sharp decline in egg production from laying hens and a deterioration in egg quality (soft-shelled eggs and deformed eggs); on the other hand, it stunts the growth of broilers, reduces feed conversion ratio, and directly leads to severe economic losses. Therefore, achieving rapid and accurate diagnosis of IBV is a key step in controlling the spread of the disease and reducing losses in poultry farming.
[0003] Currently, nucleic acid detection methods for IBV mainly rely on technologies such as RT-PCR and real-time fluorescence RT-PCR. Although these methods have certain specificity and sensitivity, they have obvious limitations: First, they require high professional skills from operators, who need systematic training to ensure standardized operation and accurate results; second, they rely on expensive instruments such as PCR instruments and fluorescence quantitative detectors, resulting in high equipment procurement and maintenance costs, making it difficult to popularize them in grassroots farms.
[0004] To address the aforementioned issues, isothermal amplification techniques (such as loop-mediated isothermal amplification, LAMP) have gradually become a research hotspot for on-site IBV detection. LAMP technology requires only simple isothermal equipment to achieve nucleic acid amplification, and its reagent costs are lower than RT-PCR. When combined with colloidal gold lateral chromatography, it can further reduce reliance on reading instruments. However, the traditional LAMP-colloidal gold detection method has a core drawback: it relies on the binding mechanism of biotin-streptavidin (SA) and antibody-Digoxin / FAM / FITC to capture the signal. This is not only affected by the sensitivity and stability of the antibody and biotin, but also by the difficulty in controlling antibody quality during production, which can easily lead to fluctuations in detection results.
[0005] Therefore, developing an IBV LAMP-colloidal gold detection technology that is not dependent on antibodies / biotin, has higher sensitivity, and is easier to operate has become an urgent need in the current IBV detection field. Summary of the Invention
[0006] The purpose of this invention is to overcome the shortcomings of existing IBV detection methods, such as RT-PCR methods which rely on professional personnel and expensive instruments, and traditional LAMP-colloidal gold methods which rely on antibodies / biotin, resulting in unstable sensitivity, false negative risks, and high costs. This invention provides a LAMP primer composition for nucleic acid chromatography, IBV test strips, kits, and their applications and methods for detecting IBV. The method for detecting IBV in this invention improves the sensitivity of IBV detection, has high specificity, low cost, requires no professional skills training or expensive instruments, only basic temperature control equipment, and can complete the entire process from sample processing to result interpretation within one hour. It is suitable for on-site, real-time testing scenarios such as farms and grassroots veterinary stations.
[0007] To achieve the above objectives, in a first aspect, the present invention provides a LAMP primer composition for nucleic acid chromatography, the LAMP primer composition comprising an outer primer pair, an inner primer pair, and a loop primer pair; the outer primer pair comprising an outer forward primer F3 and an outer reverse primer B3; the inner primer pair comprising an inner forward primer FIP and an inner reverse primer BIP; and the loop primer pair comprising a loop forward primer LF and a loop reverse primer LB; wherein, The external forward primer F3 has the nucleotide sequence shown in SEQ ID NO: 1; The external reverse primer B3 has a nucleotide sequence as shown in SEQ ID NO: 2; The internal forward primer FIP has a nucleotide sequence as shown in SEQ ID NO: 3; The internal reverse primer BIP has a nucleotide sequence as shown in SEQ ID NO: 4; The circular forward primer LF has the nucleotide sequence shown in SEQ ID NO: 5; The circular reverse primer LB has the nucleotide sequence shown in SEQ ID NO: 6; The 5' end of the FIP is modified with a C3 spacer and DNA1, and the 5' end of the LF is modified with a C3 spacer and DNA2; wherein, The DNA1 has the nucleotide sequence shown in SEQ ID NO: 7; The DNA2 has a nucleotide sequence as shown in SEQ ID NO: 8.
[0008] Preferably, the LAMP primer composition is designed with the conserved N gene region of chicken infectious bronchitis virus as the target sequence.
[0009] Secondly, the present invention provides an application of the LAMP primer composition described herein in the preparation of IBV detection products.
[0010] Thirdly, the present invention provides an IBV test strip, the IBV test strip comprising a nitrocellulose membrane, a conjugate pad, a sample pad, and absorbent paper; The nitrocellulose membrane is provided with T-lines and C-lines; The T-line is prepared from a T-line nucleic acid probe solution containing DNA3-Biotin. The DNA3 is complementary to the DNA1 of the present invention, and the DNA3 has a nucleotide sequence as shown in SEQ ID NO: 9; The C-line is prepared from a C-line quality control solution, which contains an anti-DNP monoclonal antibody. The bonding pad is coated with a hybrid probe, the hybrid probe comprising a detection probe and a reference probe; The volume ratio of the detection probe to the reference probe is 2:1; The detection probe is DNA4-AuNPs. The DNA4 is complementary to the DNA2 of the present invention, and the DNA4 has a nucleotide sequence as shown in SEQ ID NO: 10.
[0011] Fourthly, the present invention provides a kit comprising the LAMP primer composition and LAMP amplification reagent described in the present invention.
[0012] Preferably, the LAMP amplification reagent comprises betaine, urea, lysis buffer, and an enzyme for performing the LAMP reaction.
[0013] Preferably, the final concentration of betaine is 1~1000 mmol / L, more preferably 200~600 mmol / L.
[0014] Preferably, the final concentration of the urea is 1~100 mmol / L, more preferably 10~30 mmol / L.
[0015] Preferably, the final concentration of the LAMP primer composition is 1~100 μmol / L, wherein the molar ratio of the outer primer pair F3 / B3, the inner primer pair FIP / BIP, and the loop primer pair LF / LB is 1:3~8:1~8, preferably 1:5~8:2~6.
[0016] Fifthly, the present invention provides a method for detecting IBV, the method comprising the following steps: 1) Extract RNA from the sample to be tested; 2) Using the RNA of the sample to be tested as a template, perform LAMP amplification reaction using the LAMP primer composition or the detection kit described in this invention; 3) After mixing the amplification product with the diluent, add it dropwise to the IBV nucleic acid test strip described in this invention. Let it soak for 8-15 minutes for color development and observation. If both the C line and the T line are visible, the result is positive. If both the C line and the T line are visible, the result is negative. If the C line is not visible, the result is invalid.
[0017] Preferably, in step 1), the sample to be tested is one or more of the following: chicken throat swab, chicken nasal mucus, chicken excrement, chicken tissue homogenate, and chicken blood supernatant.
[0018] Preferably, the sample to be tested is a chicken throat swab.
[0019] Preferably, in step 2), the molar ratio of the outer primer pair F3 / B3, the inner primer pair FIP / BIP, and the loop primer pair LF / LB in the LAMP amplification reaction is 1:3~8:1~8, more preferably 1:5~8:2~6.
[0020] Preferably, in step 2), the conditions for the LAMP amplification reaction include: a temperature of 60~80℃ and a time of 20~40 min.
[0021] In the above technical solution, the LAMP primer composition for nucleic acid chromatography of the present invention introduces a DNA-C3 spacer at the 5' end of the inner forward primer FIP and the circular forward primer LF to prevent the formation of blunt DNA ends after amplification. This results in sticky ends with single-stranded DNA, which can be used for base pairing in subsequent nucleic acid chromatography processes. This method changes the traditional Biotin-SA and antibody-Digoxin / FAM / FITC binding methods to nucleic acid chain hybridization, thus improving the detection sensitivity by approximately 5 times compared to the traditional LAMP-colloidal gold method, with a detection limit as low as 40 copies of IBV. The LAMP primer composition of the present invention uses primers designed for the conserved N gene of IBV, and it shows negative results for eight common avian pathogens, including avian influenza virus and Mycoplasma synoviae, with no cross-reactivity.
[0022] Meanwhile, the method for detecting IBV in this invention is easy to operate, does not require high professional skills from the experimenters, and does not require complex skills training; it does not require expensive professional instruments, only basic constant temperature equipment, and the entire process from sample processing to result interpretation can be completed within 1 hour.
[0023] Furthermore, the reagent components involved in the method for detecting IBV of the present invention are simple, avoiding dependence on high-valent antibodies and biotin, and are suitable for large-scale application.
[0024] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description
[0025] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 This is a schematic diagram illustrating the detection principle of the method for detecting IBV according to the present invention. Figure 2 The results are for nucleic acid test strips prepared using mixed probes with different coating amounts in this invention. Figure 3 The results of detecting IBV using different primer ratios in this invention are shown. Figure 4 The results of detecting IBV using different amplification promoters in this invention are shown. Figure 5 The image shows the fluorescence detection results of IBV with and without urea in this invention. Figure 6 The image shows the colloidal gold detection results for IBV with and without urea in this invention. Figure 7 The results of detecting IBV using detection probes prepared with colloidal gold solutions of different absorbances in this invention are shown. Figure 8 The results of IBV detection after amplification using different primer amounts are presented in this invention. Figure 9 This is a sensitivity test comparison diagram between the method for detecting IBV of the present invention and the traditional colloidal gold testing method; Figure 10 This is a graph showing the specificity test results of the detection probe in this invention. Detailed Implementation
[0026] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.
[0027] The endpoints and any values of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.
[0028] In a first aspect, the present invention provides a LAMP primer composition for nucleic acid chromatography, the LAMP primer composition comprising an outer primer pair, an inner primer pair, and a loop primer pair; the outer primer pair comprising an outer forward primer F3 and an outer reverse primer B3; the inner primer pair comprising an inner forward primer FIP and an inner reverse primer BIP; and the loop primer pair comprising a loop forward primer LF and a loop reverse primer LB; wherein, The external forward primer F3 has the nucleotide sequence shown in SEQ ID NO: 1; The external reverse primer B3 has a nucleotide sequence as shown in SEQ ID NO: 2; The internal forward primer FIP has a nucleotide sequence as shown in SEQ ID NO: 3; The internal reverse primer BIP has a nucleotide sequence as shown in SEQ ID NO: 4; The circular forward primer LF has the nucleotide sequence shown in SEQ ID NO: 5; The circular reverse primer LB has a nucleotide sequence as shown in SEQ ID NO: 6.
[0029] The nucleotide sequences shown in SEQ ID NO: 1-6 are as follows: SEQ ID NO:1 5'-AACAAGAGTTGTTTCAGAAGT-3'; SEQ ID NO:2 5'-AATGATAGCCACAAGTAGCT-3'; SEQ ID NO:3 5'-TAGCTTATCAG(C3 Spacer) ACAATGTATCAAACACCTGTCATCACTTTAAGTATTGGGATCAGGAG-3'; 5'-SEQ ID NO:4 5'-ACTGATGTTGA(C3 Spacer)CTACAGTCACGGCAGTTAGGATG-3'; SEQ ID NO:5 5'-CTACAGTCACGGCAGTTAGGATG-3'; SEQ ID NO:6 5'-ACTTTTGGTAATTTGTGTAGAA-3'.
[0030] This invention provides a LAMP primer composition for nucleic acid chromatography. By introducing a DNA-C3 spacer at the 5' end of the inner forward primer FIP and the circular forward primer LF, a blunt DNA end is formed after amplification, resulting in sticky ends with single-stranded DNA that can be used for base pairing in subsequent nucleic acid chromatography. This method replaces the traditional Biotin-SA and antibody-Digoxin / FAM / FITC binding methods with nucleic acid strand hybridization, significantly improving detection sensitivity. The detection sensitivity is approximately 5 times higher than the traditional LAMP-colloidal gold method, with a detection limit as low as 40 copies of IBV. The LAMP primer composition of this invention is designed with primers targeting the conserved N gene of IBV, and it shows negative results for eight common avian pathogens, including avian influenza virus and Mycoplasma synoviae, with no cross-reactivity.
[0031] To accommodate subsequent colloidal gold detection, the 5' end of the FIP is modified with a C3 Spacer and DNA1, constructing FIP-C3 Spacer-DNA1; the 5' end of the LF is modified with a C3 Spacer and DNA2, constructing LF-C3 Spacer-DNA2. The C3 Spacer prevents the formation of blunt DNA ends after amplification, ensuring that DNA1 and DNA2 retain single-stranded sticky ends, providing binding sites for subsequent nucleic acid hybridization. The DNA1 has the nucleotide sequence shown in SEQ ID NO: 7; The DNA2 has a nucleotide sequence as shown in SEQ ID NO: 8.
[0032] The nucleotide sequences shown in SEQ ID NO: 7-8 are as follows: SEQ ID NO: 7 5'-TAGCTTATCAG-3'; SEQ ID NO: 8 5'-ACTGATGTTGA-3'.
[0033] In this invention, the LAMP primer composition is designed with the conserved N gene region of chicken infectious bronchitis virus as the target sequence.
[0034] Secondly, the present invention provides an application of the LAMP primer composition described herein in the preparation of IBV detection products.
[0035] The IBV testing products include, but are not limited to, IBV testing reagents and IBV testing equipment.
[0036] Thirdly, the present invention provides an IBV test strip, the IBV test strip comprising a nitrocellulose membrane, a conjugate pad, a sample pad, and absorbent paper; The nitrocellulose membrane is provided with T-lines and C-lines; The T-line is prepared from a T-line nucleic acid probe solution containing DNA3-Biotin. The DNA3 is complementary to the DNA1 of the present invention, and the DNA3 has a nucleotide sequence as shown in SEQ ID NO: 9; The C-line is prepared from a C-line quality control solution, which contains an anti-DNP monoclonal antibody. The bonding pad is coated with a hybrid probe, the hybrid probe comprising a detection probe and a reference probe; The volume ratio of the detection probe to the reference probe is 2:1; The detection probe is DNA4-AuNPs. The DNA4 is complementary to the DNA2 of the present invention, and the DNA4 has a nucleotide sequence as shown in SEQ ID NO: 10.
[0037] In this invention, the preparation method of the reference probe adopts the general preparation method in the art, which is essentially the binding between DNP-BSA and DNP antibody.
[0038] The nucleotide sequences shown in SEQ ID NO: 9-10 are as follows: SEQ ID NO: 9 5'-CTGATAAGCTACCCCC-3'; SEQ ID NO: 10 5'-SH-CCCCCTAGACACCGTGTTCAACATCAGT-3'.
[0039] In this invention, DNA1, DNA2, DNA3, and DNA4 are non-specific sequences, but they need to be modified according to LAMP primers to prevent primer dimers and non-specific pairing between primers. In this invention, the nucleotide sequences of DNA1, DNA2, DNA3, and DNA4 are shown in SEQ ID NO: 7-10, respectively.
[0040] Fourthly, the present invention provides a kit comprising the LAMP primer composition and LAMP amplification reagent described in the present invention, wherein the LAMP amplification reagent is a commonly used LAMP amplification reagent in the art, such as LAMP amplification reaction enzyme and enzyme-catalyzed reaction reagent, anti-contamination reagent, signal detection reagent and reaction optimization reagent.
[0041] In a preferred embodiment of the present invention, the LAMP amplification reagent comprises betaine for eliminating nonspecific amplification and reducing the false positive rate, urea for accelerating the amplification reaction and shortening the fluorescence signal emission time, as well as lysate for breaking IBV virus particles and releasing viral RNA, and enzymes for performing the LAMP reaction. Preferably, to simplify the operation, the enzymes for the LAMP reaction are provided in the form of LAMP lyophilized microspheres.
[0042] In this invention, the final concentration of betaine is 1~1000 mmol / L, for example, it can be 1 mmol / L, 200 mmol / L, 300 mmol / L, 400 mmol / L, 500 mmol / L, 600 mmol / L, 700 mmol / L, 800 mmol / L, 900 mmol / L or 1000 mmol / L, etc., preferably 200~600 mmol / L.
[0043] In this invention, the final concentration of urea is 1~100 mmol / L, for example, it can be 1 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L or 100 mmol / L, etc., preferably 10~30 mmol / L.
[0044] In this invention, the final concentration of the LAMP primer composition is 1~100 μmol / L, for example, it can be 1 μmol / L, 10 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 70 μmol / L, 80 μmol / L, 90 μmol / L or 100 μmol / L, etc.
[0045] The molar ratio of the outer primer pair F3 / B3, the inner primer pair FIP / BIP, and the loop primer pair LF / LB is 1:3~8:1~8, for example 1:3:1, 1:4:2, 1:5:4, 1:6:5, 1:7:7, or 1:8:8; preferably 1:5~8:2~6, more preferably 1:8:4.
[0046] Fifthly, the present invention provides a method for detecting IBV, the method comprising the following steps: 1) Extract RNA from the sample to be tested; 2) Using the RNA of the sample to be tested as a template, perform LAMP amplification reaction using the LAMP primer composition or the detection kit described in this invention; 3) After mixing the amplification product with the diluent, add it dropwise to the IBV nucleic acid test strip described in this invention. Let it soak for 8-15 minutes for color development and observation. If both the C line and the T line are visible, the result is positive. If both the C line and the T line are visible, the result is negative. If the C line is not visible, the result is invalid.
[0047] The method for detecting IBV in this invention is easy to operate, requires minimal professional skills from laboratory personnel, and necessitates no complex skills training. It requires no expensive specialized equipment, only basic temperature control equipment, and the entire process from sample processing to result interpretation can be completed within one hour. Furthermore, the reagents involved in the method for detecting IBV in this invention are simple in composition, avoiding dependence on high-cost antibodies and biotin, making it suitable for large-scale application.
[0048] In this invention, the method for extracting RNA from the sample to be tested is a conventional method used in the art for extracting RNA from secretions or excrement, as long as the extracted RNA can be guaranteed to be intact and pure.
[0049] In step 1), the sample to be tested is one or more of the following: chicken throat swab, chicken nasal mucus, chicken excrement, chicken tissue homogenate, and chicken blood supernatant.
[0050] In this invention, in order to facilitate sampling and obtain more accurate results, the sample to be tested is a chicken throat swab.
[0051] In this invention, in step 2), the molar ratio of the external primer pair F3 / B3, the internal primer pair FIP / BIP, and the loop primer pair LF / LB in the LAMP amplification reaction is 1:3~8:1~8, preferably 1:5~8:2~6. It is understood that the forward and reverse primers of the external primer pair, the internal primer pair, and the loop primer pair typically use the same or approximately the same molar amounts. For example, the molar ratio of the external primer pair F3 / B3, the internal primer pair FIP / BIP, and the loop primer pair LF / LB is 1:3:1, 1:8:8, 1:5:3, or 1:6:4, etc.
[0052] In this invention, in step 2), the conditions for the LAMP amplification reaction include: a temperature of 60~80℃, specifically 60℃, 64℃, 68℃, 76℃, 78℃, 80℃, or any value between the two above; and a time of 20~40min, specifically 20 min, 25 min, 30 min, 35 min, 40 min, or any value between the two above.
[0053] In this invention, the room temperature is 15-30°C.
[0054] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto.
[0055] (1) Methods and apparatus used in the embodiments: Unless otherwise specified, the experimental methods and equipment described in the following examples are all conventional methods and equipment, and the reagents and agents are all commercially available products.
[0056] (2) Solutions used in the examples: • Washing solution: 1×PBS, 1%BSA, 1%Tween20; • Resuspension buffer: 1×PBS, 5% BSA, 10% sucrose, 1% Tween 20, 0.01mol / L Na3PO4; • Streaking solution: 2% sucrose, 1×PBS, 1% BSA, 0.05% PC-300; • Conjunctival pad treatment solution: 0.02M PB, 2.5% sucrose, 0.5% PVP, 0.02% Tween 20, 0.5% BSA, 0.05% P300, pH 7.58; • Sample pad treatment solution: 0.5% Triton X-100, 1% BSA, 2% glucose, 2% PEG, 0.01mol / L PB, pH 7.4.
[0057] (3) The primer sequences used in the examples are shown in Table 1.
[0058] Table 1
[0059] Example 1 Primer design: Using the conserved region N gene of IBV (GenBank accession number: PQ738163.1) from the National Center for Biotechnology Information (NCBI) database as the target sequence, a LAMP primer composition including outer primer pairs, inner primer pairs, and loop primer pairs was designed. The external primer pair includes external forward primer F3 and external reverse primer B3, the internal primer pair includes internal forward primer FIP and internal reverse primer BIP, and the loop primers include loop forward primer LF and loop reverse primer LB; The 5' end of the FIP is modified with C3 Spacer and DNA1 to form FIP-C3 Spacer-DNA1; the 5' end of the LF is modified with C3 Spacer and DNA2 to form LF-C3 Spacer-DNA2. The forward primer F3 has the nucleotide sequence shown in SEQ ID NO:1; The reverse primer B3 has the nucleotide sequence shown in SEQ ID NO:2; The forward primer FIP has the nucleotide sequence shown in SEQ ID NO:3; The reverse primer BIP has a nucleotide sequence as shown in SEQ ID NO:4; The circular forward primer LF has a nucleotide sequence as shown in SEQ ID NO:5.
[0060] The circular reverse primer LB has a nucleotide sequence as shown in SEQ ID NO:6.
[0061] The DNA1 has the nucleotide sequence shown in SEQ ID NO:7; The DNA2 has a nucleotide sequence as shown in SEQ ID NO:8.
[0062] Example 2 Preparation of hybrid probes: 1. Preparation of detection probes (DNA4-AuNPs) Take 100 μL of colloidal gold solution with 20 OD 40 nm and place it in a 1.5 mL centrifuge tube. Add 4 μL of activated thiol probe, i.e. DNA4 with thiol modification at the 5' end (nucleotide sequence as shown in SEQ ID NO: 10). Vortex to mix well and add 600 μL of n-butanol. Vortex again and centrifuge to remove the supernatant organic phase. Washing and resuspending: Wash the precipitate twice with 100 μL of washing solution, and finally add 100 μL of resuspension buffer. Sonicate for 5 seconds to fully resuspend the precipitate, so that the colloidal gold solution has a final concentration of 40 OD. Store at 4°C for later use.
[0063] 2. Preparation of reference probe 1) Dilute 100 OD of 40 nm colloidal gold with water to make a 40 OD colloidal gold solution; 2) Take 500 μL of the colloidal gold solution and add 30 μL of 0.1 mol / L K2CO3 solution, then add 30 μL of 1 mg / mL DNP-BSA (Hangzhou Boyue Biotechnology, catalog number: DNP411) solution, and incubate at room temperature for 1 h; 3) Add 40 μL of 2.5% sodium caseinate solution and incubate at room temperature for 30 min, then add 120 μL of 10% BSA solution and incubate at room temperature for 30 min, then add 5% PEG 20000 and incubate at room temperature for 30 min. Centrifuge to remove the supernatant, resuspend in 250 μL of resuspension buffer, sonicate for 5 s and resuspend thoroughly to obtain the reference probe solution, and store at 4℃ for later use.
[0064] 3. Preparation of hybrid probes The detection probe and the reference probe are mixed at a volume ratio of 2:1 and then vortexed to obtain a mixed probe for further experimentation.
[0065] Example 3 Test strip preparation: 1. Preparation of T-line nucleic acid probe solution A solution of 100 μmol / L Cap-DNA (DNA3 with a 3' end modified with biotin, nucleotide sequence as shown in SEQ ID NO:11) was mixed with a solution of 1 mg / mL recombinant streptavidin (rSA, Xinfan Biotech, catalog number: B2340) at a volume ratio of 1:3 and incubated at 4°C for 2 h to obtain the T-line nucleic acid probe solution.
[0066] 2. Preparation of C-line probe solution The anti-DNP monoclonal antibody (Hangzhou Boyue Biotechnology, catalog number: DNP101) solution was prepared into a 2 mg / mL solution using streak buffer, which is the C-line control solution.
[0067] 3. Preparation of test strips 1) The T-line nucleic acid probe solution and C-line quality control solution were respectively streaked onto CN95 nitrocellulose membrane at a concentration of 1.0 μL / cm, dried at 37℃ for 4 h to obtain the treated nitrocellulose membrane, and then stored in a dry environment for later use. 2) The mixed probe prepared in Example 2 was sprayed onto glass fiber paper at a rate of 2 μL / cm and dried at 37°C for 4 h to obtain the treated conjugate pad.
[0068] The conjunctival pad was obtained by soaking 1 cm of glass fiber paper in a conjunctival pad treatment solution for 30 min and then drying it overnight at 37°C; the sample pad was obtained by drying 1.5 cm of glass fiber paper in a sample pad treatment solution at 37°C overnight.
[0069] 4. Test strip assembly After assembling the sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper in sequence, cut them into 3 cm test strips and then put them into the test strip holder.
[0070] Example 4 Sample testing 1. Detection of nucleic acid extract Mix 1 μL of 100 μmol / L LAMP primer composition, 4 μL of 2 mol / L betaine, 1 μL of 0.4 mol / L urea, 1 μL of nucleic acid extraction buffer, and 13 μL of lysis buffer with one LAMP lyophilized microsphere to make a final solution volume of 20 μL. Then, amplify the solution at 70℃ for 30 min in a constant temperature device. Take 2 μL of LAMP nucleic acid amplification solution and add it to 80 μL of 4×SSC. Mix well and add the entire solution to the sample well of the test strip. After 10 min, determine the detection result / take a picture.
[0071] 2. Detection of real samples 1) Sample preparation a. Take sputum from a chicken using a throat swab, add it to a diluent tube and vortex to mix. Add 2-3 drops to a lysis buffer tube, vortex to mix, and obtain the nucleic acid lysis buffer. b. Crush a small amount of chicken tissue sample by hand, take a small amount of the crushed sample with a throat swab, add it to the diluent tube and vortex to mix, then add 2-3 drops to the lysis buffer tube and vortex to mix to obtain nucleic acid lysis buffer; c. Take chicken blood and place it at room temperature. After the blood coagulates, centrifuge and collect the supernatant. Pour out the liquid in the diluent tube and add 2-3 drops to the lysis buffer tube. Vortex to mix and obtain the nucleic acid lysis buffer. 2) Sample testing Mix 1 μL of 100 μmol / L LAMP primer composition, 4 μL of 2 mol / L betaine, 1 μL of 0.4 mol / L urea, 1 drop of the nucleic acid lysis buffer prepared in step 1), and 1 LAMP reaction lyophilized microsphere. Add lysis buffer to make the final volume of the solution 20 μL. Amplify at 70℃ for 30 min in a constant temperature device. Take 2 μL of LAMP nucleic acid amplification solution and add it to 80 μL of 4×SSC. Mix well and add the whole solution to the sample well of the test strip. Detect the results / take pictures after 10 min.
[0072] The LAMP reaction lyophilized microspheres and lysis buffer were both sourced from the DryMicro RT-LAMP Master Mix product of Genuin Biotechnologies LLC (a subsidiary of Hefei Shanben Biotechnology Co., Ltd.) (website: [website address missing]). https: / / www.genuinbiotech.com / product-page / drymicro-rt-lamp-master-mix#product information), which contains LAMP reaction complex enzyme preparations.
[0073] Example 5 This example illustrates the optimization of IBV detection conditions. 1. Effect of mixed probe coating amount on the color development effect of IBV test strips Using the amount of the mixed probe sprayed onto the bonding pad as the sole variable, and referring to the IBV test strip preparation method of Example 3, only the amount of the mixed probe sprayed was changed to prepare 4 sets of test strips. The specific spraying amounts were set as follows: Group 1: The spraying amount of the mixed probe is 1 μL / cm; Group 2: The spraying amount of the mixed probe is 2 μL / cm; Group 3: The spraying amount of the mixed probe is 3 μL / cm; Group 4: The amount of mixed probe sprayed is 4 μL / cm.
[0074] The remaining preparation steps are the same as in Example 3.
[0075] Colloidal gold test strip detection: Take 2 μL each of positive LAMP amplification solution (containing IBV amplification product) and negative LAMP amplification solution (without IBV amplification product), mix them with 80 μL of 4×SSC diluent, and then add them to the sample wells of the four test strips respectively. After 10 minutes, observe and record the color intensity of the T line (detection line) and the color development of the C line (control line) of each test strip, and take photos to record the results. The results are as follows. Figure 2 As shown.
[0076] Depend on Figure 2 The results showed that when the mixed probe coating volume was 1 μL / cm, the T-line of the positive sample was relatively light. When the coating volume was increased to 2 μL / cm, the T-line of the positive sample was clearly visible, and the C-line was also normally visible, proving the effectiveness of the detection. Further increasing the coating volume to 3 μL / cm and 4 μL / cm did not further enhance the color intensity of the T-line of the positive sample, nor did it improve the detection specificity. In conclusion, 2 μL / cm was determined to be the optimal coating volume for the mixed probe.
[0077] 2. Effect of different primer molar ratios on IBV detection results Using the molar ratio of F3 / B3:FIP / BIP:LF / LB in the LAMP primer composition as the sole variable, four LAMP primer compositions were prepared with a final concentration of 100 μmol / L. The specific ratios are as follows: Group 1: F3 / B3:FIP / BIP:LF / LB=1:8:1; Group 2: F3 / B3:FIP / BIP:LF / LB=1:8:2; Group 3: F3 / B3:FIP / BIP:LF / LB=1:8:4; Group 4: F3 / B3:FIP / BIP:LF / LB=1:8:6.
[0078] LAMP amplification reaction: Take 1 μL of each of the above 4 groups of LAMP primer compositions and add them to the corresponding reaction system containing 1 μL of positive nucleic acid lysis buffer (containing IBV RNA) or 1 μL of negative nucleic acid lysis buffer (IBV-free healthy chicken nucleic acid), 4 μL of 2 mol / L betaine, 1 μL of 0.4 mol / L urea, and 13 μL of lysis buffer. Then mix with 1 LAMP reaction lyophilized microsphere (total system 20 μL). Place all reaction systems in a 70℃ constant temperature device and amplify isothermally for 30 min to obtain the LAMP nucleic acid amplification solution of each group.
[0079] Colloidal gold test strip detection: Take 2 μL of LAMP nucleic acid amplification buffer from each group, mix it with 80 μL of 4×SSC diluent, and then add the entire amount to the sample well of the IBV test strip; after 10 minutes, observe and record the color development of the T and C lines on the test strip, and calculate the detection concordance rate of each group for negative / positive samples. The results are as follows: Figure 3 As shown.
[0080] Depend on Figure 3 The results showed that when the molar ratio of F3 / B3:FIP / BIP:LF / LB in the LAMP primer composition was 1:8:4, the positive sample showed a clear T-line, while the negative sample showed no T-line. The positive and negative concordance rate obtained was the best, significantly better than other ratios (1:8:1, 1:8:2, 1:8:6). Therefore, this ratio was determined to be the optimal primer preparation condition for IBV LAMP amplification reaction and colloidal gold lateral chromatography detection.
[0081] 3. The effect of different false positive elimination reagents on IBV detection results Using the type of false positive elimination reagent as the sole variable, four experimental groups were set up, each supplemented with a different false positive elimination reagent and one blank control group. Negative samples (RNA from healthy chickens without IBV) were tested in each group to verify the false positive elimination effect, with each group repeated three times. Experimental group 1: Add betaine. The reaction system consisted of 1 μL 2 mol / L betaine, 1 μL 100 μmol / L LAMP amplification primers, 1 μL negative nucleic acid solution, 17 μL lysis buffer, and 1 LAMP lyophilized microsphere, for a total system volume of 20 μL. Experimental Group 2: DMSO was added. The reaction system was the same as that of Experimental Group 1 except that “1 μL 2 mol / L betaine” was replaced with “1 μL DMSO”. Experimental Group 3: 1% BSA was added. The reaction system was the same as that of Experimental Group 1 except that “1 μL 2 mol / L betaine” was replaced with “1 μL 1% BSA”. Experimental Group 4: Urea was added. The reaction system was the same as that of Experimental Group 1 except that “1 μL 2 mol / L betaine” was replaced with “1 μL 2 mol / L urea”. Blank control group: No false positive elimination reagent was added. Except for omitting the false positive elimination reagent and adding 1 μL of enzyme-free water, the other components and amounts of the reaction system were the same as those of experimental group 1.
[0082] LAMP amplification reaction: All 5 reaction systems were placed in a 70℃ constant temperature device and amplified isothermally for 30 min to obtain LAMP nucleic acid amplification solution.
[0083] Colloidal gold test strip detection: Take 2 μL of LAMP nucleic acid amplification buffer from each group, mix it with 80 μL of 4×SSC diluent, and then add the entire amount to the sample well of the IBV test strip; observe the color development of the T and C lines on the test strip after 10 minutes, take pictures to record the results, and compare the inhibition effects of different false positive elimination reagents on false positive signals of negative samples. The results are as follows: Figure 4 As shown.
[0084] Depend on Figure 4 The content indicates that when DMSO and betaine are used as false positive elimination reagents, false positives in amplification are eliminated. However, considering that DMSO is a liquid, betaine is the best choice.
[0085] 4. The impact of IBV LAMP amplification results on dual detection results under urea variable conditions Two groups were set up, with urea addition as the variable. Each group contained three types of samples: positive (known high concentration of IBV RNA), weakly positive (known low concentration of IBV RNA), and negative (RNA from healthy chickens without IBV). Each group was repeated three times. Experimental group: containing urea, the reaction system consisted of 1 μL 2 mol / L betaine, 1 μL 0.4 mol / L urea, 1 μL 100 μmol / L LAMP amplification primers, 1 μL nucleic acid solution to be tested, 16 μL lysis buffer, and 1 LAMP lyophilized microsphere, with a total system volume of 20 μL; Control group: without urea, the reaction system was modified by replacing "1 μL 0.4 mol / L urea" with "1 μL enzyme-free water", and the other components and amounts were the same as the experimental group, with a total system volume of 20 μL.
[0086] LAMP amplification reaction: Both reaction systems were placed in a 70℃ constant temperature device and amplified isothermally for 30 min to obtain LAMP nucleic acid amplification solution.
[0087] Dual detection operation: Fluorescence detection: Simultaneously, 5 μL of LAMP nucleic acid amplification buffer was taken, and LAMP fluorescence detection reagent (containing the fluorescent dye EvaGreen) was used. Fluorescence signal changes during the amplification process were monitored using a fluorescence detector. The fluorescence elude time and endpoint fluorescence intensity were recorded. The detection results are as follows: Figure 5 As shown; Colloidal gold test strip detection: Mix 2 μL of LAMP nucleic acid amplification buffer with 80 μL of 4×SSC diluent, and add the entire mixture to the sample well of the IBV test strip. Observe and photograph the T / C line color development after 10 minutes. The detection results are as follows: Figure 6 As shown.
[0088] Depend on Figure 5 and Figure 6 The results show that when the experimental group containing urea and the control group without urea were tested using colloidal gold test strips, there was no significant difference in the detection of weak positive samples. However, when the fluorescence method was used, the weak positive samples in the experimental group containing urea appeared earlier. Therefore, it is best to add urea during the LAMP amplification reaction.
[0089] 5. The effect of colloidal gold solution absorbance on the performance of IBV detection probes Using the initial absorbance (OD value) of the colloidal gold solution during probe preparation as the sole variable, four experimental groups were set up. All groups prepared the probes (DNA4-AuNPs) according to the method described in Example 2. The specific steps are as follows: Colloidal gold dilution: Take 100OD of 40nm colloidal gold stock solution and dilute it with ultrapure water to 20OD, 40OD, 60OD and 80OD respectively to obtain four colloidal gold solutions with different absorbance. Probe coupling: Take 100 μL of each of the above four colloidal gold solutions and place them in a 1.5 mL centrifuge tube. Add 4 μL of activated thiol probe, i.e. DNA4 with thiol modification at the 5' end (nucleotide sequence as shown in SEQ ID NO: 10), vortex to mix, add 600 μL of n-butanol, vortex again, centrifuge, and remove the supernatant organic phase. Washing and resuspending: Wash the precipitate twice with 100 μL of washing buffer containing 1×PBS, 1% BSA and 1% Tween 20. Finally, add 100 μL of resuspending buffer and sonicate for 5 seconds to fully resuspend the precipitate, so that all groups are eventually detection probe solutions with an OD of 40. Store at 4℃ for later use.
[0090] The above four sets of detection probes were assembled into a test strip using the method described in Example 3.
[0091] Colloidal gold test strip detection: Take 2 μL each of positive LAMP amplification buffer (containing IBV amplification product) and negative LAMP amplification buffer (without IBV amplification product), mix them with 80 μL of 4×SSC diluent, and then add them to the sample wells of the four test strips respectively. After 10 minutes, observe and record the color intensity of the T line and the color development of the C line of each test strip, take photos and record the results. The results are as follows. Figure 7 As shown.
[0092] Depend on Figure 7 The results showed that the detection probe prepared with colloidal gold solution at an initial absorbance of 20 OD exhibited the best detection performance, with the deepest T-line color development in positive samples, clearly distinguishable T-lines in weakly positive samples, and no false positives in negative samples, with only the C-line showing color development. In contrast, detection probes prepared with colloidal gold solutions at initial absorbances of 40 OD, 60 OD, and 80 OD either showed weakened positive signal intensity or decreased detection stability in weakly positive samples. Therefore, the optimal initial absorbance of the colloidal gold solution for preparing the detection probe was determined to be 20 OD.
[0093] 6. Effect of LAMP primer composition dosage on IBV detection results Four experimental groups were set up with the amount of LAMP primer composition added as the only variable. Each group used LAMP primer composition with a final concentration of 100 μmol / L and a molar ratio of F3 / B3:FIP / BIP:LF / LB of 1:8:4. The specific amounts are as follows: Group 1: LAMP primer composition added in an amount of 0.5 μL; Group 2: LAMP primer composition added in an amount of 1 μL; Group 3: LAMP primer composition added in an amount of 1.5 μL; Group 4: 2 μL of LAMP primer composition added.
[0094] Each group added the appropriate amount of primer to one LAMP lyophilized bulb, and then, according to the reaction system ratio of Example 4, added 4 μL of 2 mol / L betaine, 1 μL of 0.4 mol / L urea, and corresponding volumes of lysis buffer (ensuring a total system volume of 20 μL) containing either 1 μL of positive nucleic acid lysis buffer (containing IBV RNA), 1 μL of weakly positive nucleic acid lysis buffer (containing low concentration of IBV RNA), or 1 μL of negative nucleic acid lysis buffer (IBV-free healthy chicken nucleic acid). After mixing, the LAMP amplification system was constructed.
[0095] LAMP amplification reaction: All four LAMP amplification systems were placed in a 70℃ constant temperature device and amplified isothermally for 30 min to obtain LAMP nucleic acid amplification solution.
[0096] Colloidal gold test strip detection: Take 2 μL of LAMP nucleic acid amplification buffer from each group, mix it with 80 μL of 4×SSC diluent, and then add the entire amount to the sample well of the IBV test strip; observe the color development of the T and C lines on the test strip after 10 minutes, take photos and record the results. The results are as follows: Figure 8 As shown.
[0097] Depend on Figure 8 The results showed that the amplification effect was optimal when the LAMP primer composition was added at a volume of 1 μL. Positive samples exhibited clear T-line development, weakly positive samples were consistently detected, and negative samples only showed C-line development with no false positives, resulting in the highest accuracy in distinguishing between positive and negative results. However, when the added volume was 0.5 μL, insufficient primer volume led to decreased amplification efficiency and false negatives in weakly positive samples. At volumes of 1.5 μL and 2 μL, excessive primers easily formed primer dimers, leading to false positives in negative samples. Therefore, the optimal volume of LAMP primer composition was determined to be 1 μL.
[0098] Example 6 This embodiment aims to clarify the limit of detection (LoD) of the LAMP primer composition of the present invention (F3 / B3:FIP / BIP:LF / LB molar ratio 1:8:4, final concentration 100 μmol / L) for IBV and to verify its detection sensitivity.
[0099] A recombinant plasmid containing the conserved N gene of IBV (GenBank ID: PQ738163.1) was selected as a standard template. After concentration determination, the plasmid was serially diluted with enzyme-free water to obtain a series of template solutions with final concentrations of 1000 copies / μL, 500 copies / μL, 200 copies / μL, 100 copies / μL, 80 copies / μL, 60 copies / μL, and 40 copies / μL. At the same time, 0 copies / μL (enzyme-free water) was set up as a negative control.
[0100] 1) Construction of LAMP amplification system: Take 1 μL of each serially diluted template solution and add it to the reaction system containing 1 μL of LAMP primer composition, 4 μL of 2 mol / L betaine, 1 μL of 0.4 mol / L urea, 1 LAMP reaction lyophilized microsphere and 13 μL of lysis buffer, for a total system volume of 20 μL. Each group was repeated 3 times. 2) LAMP amplification reaction: All reaction systems were placed in a 70℃ constant temperature device and amplified isothermally for 30 min to obtain LAMP nucleic acid amplification solutions for each group; 3) Colloidal gold test strip detection: Take 2 μL of amplification solution from each group, mix it with 80 μL of 4×SSC diluent, and add it to the sample well of the IBV test strip. Observe the color development of the T line and C line after 10 minutes, take pictures and record the results. The results are as follows: Figure 9As shown above, the test strips correspond to the detection results of 1000 copies, 500 copies, 200 copies, 100 copies, 80 copies, 60 copies, 40 copies, and 0 copies from left to right.
[0101] Depend on Figure 9 The results show that when the plasmid template concentration is 40-1000 copies / μL, the test strips all show C-line color development and clear T-line color development, indicating effective detection and clear positive discrimination. When the template concentration is 0 copies / μL (negative control), only the C-line is colored, and the T-line is not colored, indicating no false positives. Further observation revealed that even at a template concentration as low as 40 copies / μL, the T-line still shows stable color development, and no false negatives occurred. In summary, the limit of detection (LoD) of the LAMP primer composition of this invention for IBV is determined to be 40 copies.
[0102] Example 7 This embodiment aims to verify the sensitivity advantage of the LAMP-colloidal gold detection method based on nucleic acid hybridization (hereinafter referred to as "this method") for IBV detection by comparing it with the traditional colloidal gold detection method, and to clarify the sensitivity difference between the two methods and the reasons therefor.
[0103] Experimental group (this method): The detection system of the present invention is used, including a LAMP primer composition modified with FIP-C3 Spacer-DNA1 and LF-C3 Spacer-DNA2 (F3 / B3:FIP / BIP:LF / LB molar ratio 1:8:4), and a colloidal gold test strip containing DNA3-Biotin (T line), DNA4-AuNPs (detection probe), and DNP-BSA (reference probe). The test strip is prepared according to Example 3.
[0104] Control group (traditional colloidal gold method): A traditional LAMP-colloidal gold detection system was constructed as follows: Primer modification: FIP primers are labeled with Digoxin (or FAM) at the 5' end, LF primers are labeled with Biotin at the 5' end, and the remaining primer sequences are consistent with the experimental group; Test strip preparation: Refer to the test strip preparation process of Example 3, except that the reference probe "DNP-BSA labeled colloidal gold" is replaced with "streptavidin (SA) labeled colloidal gold", and the detection probe and anti-DNP antibody labeled colloidal gold are mixed at a volume ratio of 2:1 to prepare a gold label solution. The remaining steps are the same as in Example 3, and the conventional method colloidal gold test strip is obtained.
[0105] The detection limit-related nucleic acid concentration gradient determined in Example 6 was used, namely, the recombinant plasmid dilution containing the IBV conserved N gene (concentrations of 1000 copies / μL, 500 copies / μL, 200 copies / μL, 100 copies / μL, 80 copies / μL, 60 copies / μL, and 40 copies / μL, with 0 copies / μL as a negative control), to ensure that the same concentration gradient of templates was used in both groups of detections, thus guaranteeing the effectiveness of the comparison; LAMP amplification reaction: The experimental group and the control group constructed LAMP amplification systems using plasmids of different concentrations as templates: The total system was 20 μL, containing 1 μL of the corresponding primer, 4 μL of 2 mol / L betaine, 1 μL of 0.4 mol / L urea and 1 LAMP lyophilized microsphere, with the remainder being lysis buffer. The system was amplified at 70℃ for 30 min. Colloidal gold test strip detection: For both groups, 2 μL of amplification product was mixed with 80 μL of 4×SSC and added to the corresponding wells of the test strip. After 10 minutes, the color development of the T and C lines was observed. The detection rates of different template concentrations were recorded for both groups, and the results were photographed and stored. The results are as follows: Figure 9 As shown below.
[0106] Depend on Figure 9 As can be seen below, the control group (traditional colloidal gold method) can only stably detect plasmid templates at concentrations of 100 copies / μL and above. When the concentration is lower than 100 copies / μL (such as 80 copies / μL or 60 copies / μL), the T-line is blurred or absent, resulting in false negatives. In contrast, the experimental group (this method) can stably detect plasmid templates at 60 copies / μL, and the T-line is clearly visible. It also shows a weak positive signal for templates at 40 copies / μL, and the sensitivity is significantly improved compared to the traditional method.
[0107] Traditional colloidal gold methods rely on the binding of antigen-antibody (such as Digoxin-anti-Digoxin antibody) and biotin-SA to capture signals. The affinity of such biomolecular interactions is low. Within a limited amplification time (30 min), low concentrations of amplified products are difficult to fully bind with antibodies / SA to form a stable sandwich structure, leading to false negatives. In contrast, this method uses complementary hybridization of nucleic acid chains (DNA1-DNA3, DNA2-DNA4) to replace biomolecular binding. The specificity and affinity of nucleic acid hybridization are significantly higher than those of antigen-antibody interactions. Even low concentrations of amplified products can bind efficiently and accumulate at the T line, thus achieving higher sensitivity detection.
[0108] In summary, the LAMP-colloidal gold detection method based on nucleic acid hybridization of this invention has significantly better detection sensitivity than the traditional colloidal gold method, can effectively detect lower concentrations of IBV nucleic acid, and is more suitable for the early detection of low-load IBV infected samples.
[0109] Example 8 This embodiment is used to illustrate the specificity of the LAMP primer composition (F3 / B3:FIP / BIP:LF / LB molar ratio 1:8:4, final concentration 100μmol / L) and the matching detection method of the present invention, to clarify whether other pathogenic microorganisms that may infect chicken flocks in clinical practice will interfere with the IBV detection results, and to eliminate the risk of false positives caused by cross-reaction. Interfering pathogen selection: Eight common avian pathogens that overlap with IBV infection scenarios in clinical settings and may interfere with detection were selected. All of them were prepared as high-concentration nucleic acid templates (20,000 copies / μL, simulating severe clinical infection). Specifically, they included: avian influenza virus, Mycoplasma synoviae, Infectious bursal virus, avian adenovirus, Mycoplasma gallisepticum, avian porcine circovirus, duck avian circovirus, and Marek's virus. Positive control: An IBV positive template group was set up, and IBV recombinant plasmid of 100 copies / μL was selected (the concentration is 2.5 times lower than the detection limit of 40 copies / μL determined in Example 6 to ensure that the positive signal can be stably identified). Experimental grouping: A total of 9 groups of samples were prepared, with 3 replicates for each group. Simultaneously, 0 copies / μL of enzyme-free water was set as a negative blank control. LAMP amplification system construction: Take 1 μL of the corresponding pathogen nucleic acid template (or IBV positive template, negative control) for each group and add it to the reaction system containing 1 μL of the LAMP primer composition of this invention, 4 μL of 2 mol / L betaine, 1 μL of 0.4 mol / L urea, 1 LAMP reaction lyophilized microsphere and 13 μL of lysis buffer, with a total system volume of 20 μL. Vortex mix well. LAMP amplification reaction: All reaction systems were placed in a 70℃ constant temperature device and amplified isothermally for 30 min to obtain LAMP nucleic acid amplification solutions for each group; Colloidal gold test strip detection: Take 2 μL of LAMP nucleic acid amplification solution from each group, mix thoroughly with 80 μL of 4×SSC diluent, and then add the entire mixture to the sample well of the IBV test strip of this invention (prepared according to Example 3). Observe and record the color development of the T and C lines after 10 minutes, take photos to preserve the experimental results, and the results are as follows. Figure 10 As shown, the test strips correspond from left to right to the avian influenza virus group, mycoplasma synoviae group, infectious bursal virus group, avian adenovirus group, mycoplasma gallisepticum group, avian circovirus group, duck choriovirus group, Marek's virus group, and IBV positive control group. Depend on Figure 10 It can be seen that the eight potential interfering pathogens and the negative blank control group all showed only C-line color development, proving that the detection process was effective and there was no test strip failure or operational error. The T-line did not develop and the test results were all negative. In contrast, the IBV positive control group (100 copies / μL) showed both C-line and T-line color development, and the positive signal was clearly distinguishable. The above results show that even in the presence of high concentrations of interfering pathogens (20,000 copies / μL), the LAMP primer composition of the present invention can only specifically bind to the IBV target gene and initiate amplification, without non-specific amplification or cross-reaction with the nucleic acids of other avian pathogens. The matching colloidal gold detection system also only recognizes IBV amplification products and is not affected by other pathogens, further verifying the high specificity of the detection method of the present invention, which can meet the needs of accurate IBV detection in complex clinical infection scenarios. The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
[0110] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.
[0111] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.
Claims
1. A LAMP primer composition for nucleic acid chromatography, characterized in that, The LAMP primer composition includes an outer primer pair, an inner primer pair, and a loop primer pair. The outer primer pair includes an outer forward primer F3 and an outer reverse primer B3; the inner primer pair includes an inner forward primer FIP and an inner reverse primer BIP; and the loop primer pair includes a loop forward primer LF and a loop reverse primer LB. The external forward primer F3 has the nucleotide sequence shown in SEQ ID NO: 1; The external reverse primer B3 has a nucleotide sequence as shown in SEQ ID NO: 2; The internal forward primer FIP has a nucleotide sequence as shown in SEQ ID NO: 3; The internal reverse primer BIP has a nucleotide sequence as shown in SEQ ID NO: 4; The circular forward primer LF has the nucleotide sequence shown in SEQ ID NO: 5; The circular reverse primer LB has the nucleotide sequence shown in SEQ ID NO: 6; The 5' end of the FIP is modified with a C3 spacer and DNA1, and the 5' end of the LF is modified with a C3 spacer and DNA2; wherein, The DNA1 has the nucleotide sequence shown in SEQ ID NO: 7; The DNA2 has a nucleotide sequence as shown in SEQ ID NO:
8.
2. The LAMP primer composition according to claim 1, characterized in that, The LAMP primer composition was designed with the conserved N gene region of chicken infectious bronchitis virus as the target sequence.
3. The use of a LAMP primer composition as described in claim 1 or 2 in the preparation of IBV detection products.
4. An IBV test strip, characterized in that, The IBV test strip includes a nitrocellulose membrane, a conjugate pad, a sample pad, and absorbent paper; The nitrocellulose membrane is provided with T-lines and C-lines; The T-line is prepared from a T-line nucleic acid probe solution containing DNA3-Biotin. The DNA3 is complementary to the DNA1 of claim 1, and the DNA3 has a nucleotide sequence as shown in SEQ ID NO: 9; The C-line is prepared from a C-line quality control solution, which contains an anti-DNP monoclonal antibody. The bonding pad is coated with a hybrid probe, the hybrid probe comprising a detection probe and a reference probe; The volume ratio of the detection probe to the reference probe is 2:1; The detection probe is DNA4-AuNPs. The DNA4 is complementary to the DNA2 of claim 1, and the DNA4 has the nucleotide sequence shown in SEQ ID NO:
10.
5. A reagent kit, characterized in that, The kit includes the LAMP primer composition and LAMP amplification reagent as described in claim 1 or 2.
6. The reagent kit according to claim 5, characterized in that, The LAMP amplification reagent contains betaine, urea, lysis buffer, and an enzyme for performing the LAMP reaction; The final concentration of betaine is 1~1000 mmol / L, preferably 200~600 mmol / L; The final concentration of the urea is 1~100 mmol / L, preferably 10~30 mmol / L.
7. The kit according to claim 5 or 6, characterized in that, The final concentration of the LAMP primer composition is 1~100 μmol / L, wherein the molar ratio of the outer primer pair F3 / B3, the inner primer pair FIP / BIP, and the loop primer pair LF / LB is 1:3~8:1~8, preferably 1:5~8:2~6.
8. A method for detecting IBV, characterized in that, The method includes the following steps: 1) Extract RNA from the sample to be tested; 2) Using the RNA of the sample to be tested as a template, perform LAMP amplification reaction using the LAMP primer composition of claim 1 or 2 or the kit of any one of claims 5-7; 3) After mixing the amplification product with the diluent, add it dropwise to the IBV nucleic acid test strip as described in claim 4. Soak for 8-15 minutes for color development and observation. If both the C line and the T line are colored, the result is positive. If both the C line and the T line are colored, the result is negative. If the C line is not colored, the result is invalid.
9. The method according to claim 8, characterized in that, In step 1), the sample to be tested is one or more of the following: chicken throat swab, chicken nasal mucus, chicken excrement, chicken tissue homogenate, and chicken blood supernatant; preferably, it is a chicken throat swab.
10. The method according to claim 8 or 9, characterized in that, In step 2), the molar ratio of the outer primer pair F3 / B3, the inner primer pair FIP / BIP, and the loop primer pair LF / LB in the LAMP amplification reaction is 1:3~8:1~8, preferably 1:5~8:2~6; The conditions for the LAMP amplification reaction include: a temperature of 60-80℃ and a time of 20-40 min.