Triple LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection kit for simultaneously detecting three bovine diarrhea syndrome viruses and detection method thereof

Abstract

The invention discloses a triple LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection kit for simultaneously detecting three bovine diarrhea syndrome viruses and a detection method thereof, and relates to the field of agricultural gene detection.The detection kit comprises a binding pad coated with a colloidal gold solution labeled by an antibiotin monoclonal antibody, a sample pad, an NC (nitrocellulose) membrane, an absorption pad and a quality control line C covered with a goat anti-mouse polyclonal antibody, the kit comprises a detection line T1 covered with an anti-digoxin antibody, a detection line T2 covered with an anti-ROX antibody, a detection line T3 covered with an anti-FAM monoclonal antibody, an LFB detection line, a primer designed aiming at M31182.1, a primer designed aiming at JN191349.1 and a primer designed aiming at JN790188.1; the detection method provided by the invention has the advantages of quick result, high cost benefit and higher accuracy.

Description

Technical Field

[0001] The invention relates to the field of agricultural gene detection, in particular to a triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses and a detection method thereof. Background Art

[0002] One of the biggest problems facing farm farming is infectious diseases in cattle. Bovine viral diarrhea is associated with significant disease and mortality in calves and can cause abortions in dairy cows, ultimately leading to a significant drop in milk and meat production. BVDV, BRV, and BPV mainly cause diarrhea, dehydration, and lethargy in ruminants, which have a significant impact on the health of ruminants and cause significant economic losses worldwide. BVDV belongs to the Flaviviridae family and is a 12.5kb long virus with a single-stranded RNA structure. The genetic material includes a long nucleotide sequence without a stop codon interruption, flanked by two regions that do not encode proteins. The large polyprotein encoded by the open reading frame (ORF) produces four structural proteins and eight non-structural proteins. Eleven double-stranded RNA segments make up the non-enveloped virus known as BRV, encoding six structural proteins (VP1, VP2, VP3, VP4, VP6, and VP7) and six non-structural proteins (NSP1-NSP6). The VP7 and VP4 proteins on the outer cap are the basis for dividing the G and P genotypes. BPV is a DNA virus containing a 5.5 kb linear single-stranded DNA genome surrounded by non-identical palindromic hairpins. Its genetic material contains ORF1, ORF2, and ORF3. NS1 is required for DNA replication and is encoded by ORF1. NP1, encoded by ORF2, is involved in the processing of viral RNA. ORF3 uses a special splicing method to generate two viral proteins (VP1 and VP2).

[0003] Researchers have developed detection methods that can detect various bovine diarrhea syndrome viruses, including virus isolation, serology, and molecular detection. Traditional virus isolation methods are not suitable for rapid detection, and immunological methods are also affected by environmental factors. With the development of molecular biology techniques, diagnostic methods based on nucleic acid detection have become an important tool for animal epidemiological detection. Derivative technologies based on polymerase chain reaction, including TaqMan real-time polymerase chain reaction, multiplex droplet digital polymerase chain reaction, and multiplex real-time fluorescence quantitative polymerase chain reaction, have been shown to be highly sensitive, specific, and effective in detecting cattle pathogens. Standard PCR usually lasts for 90 minutes and has high sensitivity. However, the addition of gold nanoparticles and combined with double-primer oligonucleotides during the PCR process can increase the sensitivity by 100 times, but the amplified products cannot be directly observed on the surface and usually need to be verified by agarose gel electrophoresis after the reaction. The market needs a technology that can be directly observed on the surface and can be detected without specific laboratory conditions and equipment. LAMP-LFD technology is expected to solve this problem and provide a useful and reliable tool for detecting viral infections in resource-limited areas. Summary of the invention

[0004] To address the deficiencies of the prior art, the present invention aims to provide a triple LAMP-LFD detection kit and a detection method thereof for simultaneously detecting three bovine diarrhea syndrome viruses; the present invention identifies BVDV, BRV and BPV by combining LAMP and LFD technology, and the detection method has the advantages of rapid results, high cost-effectiveness and higher accuracy.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses, comprising: three sets of LAMP primer probes, a lateral flow biosensor LFB;

[0007] The lateral flow biosensor LFB comprises: a binding pad coated with a colloidal gold solution labeled with an anti-biotin monoclonal antibody, a sample pad, a nitrocellulose NC membrane, an absorption pad, a quality control line C covered with a goat anti-mouse polyclonal antibody, a detection line T1 covered with an anti-digoxin antibody, a detection line T2 covered with an anti-ROX antibody, a detection line T3 covered with an anti-FAM monoclonal antibody, and an LFB detection line arranged on the sample pad;

[0008] The LAMP primer probes include: a BVDV virus primer set designed for the GenBank accession number of BVDV virus: M31182.1, a BPV virus primer set designed for the GenBank accession number of BPV virus: JN191349.1, and a BRV virus primer set designed for the GenBank accession number of BRV virus: JN790188.1, wherein the BVDV virus primer set uses a circular primer labeled with biotin and ROX; the BRV virus primer set uses a circular primer labeled with biotin and FAM; and the BPV virus primer set uses a circular primer labeled with biotin and digoxigenin;

[0009] The aforementioned triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses, the LAMP primer probes are shown in SEQ ID No: 01 to SEQ ID No: 18 in Table 1.

[0010] The aforementioned triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses, the LAMP-LFD system of three sets of LAMP primer probes is: F3 / B3 0.2μM, FIP / BIP 1.6μM, LF / LB 0.8μM, (NH4)2SO410mM, MgSO4 8mM, KCl 10mM, Tris-HCl 20mM, 5% Tween-20 0.5μL, mixed dNTPs 1.0mM, Bst DNA polymerase 8U with a concentration of 0.16-0.80U / μL, virus standard plasmid 2.5μL, reaction temperature: 61℃-69℃, reaction time: 5-40min.

[0011] In the aforementioned triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses, the ratio of the BVDV virus primer set, the BRV virus primer set and the BPV virus primer set is 1:1:1.6.

[0012] The aforementioned triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses has a BstDNA polymerase concentration of 0.16U / μL.

[0013] The aforementioned triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses has a dNTPs concentration of 1.4 mM.

[0014] The aforementioned triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses has a reaction temperature of 65°C.

[0015] The aforementioned triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses has a reaction time of 30 minutes.

[0016] The aforementioned triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses comprises the following steps:

[0017] a) Viral RNA extraction:

[0018] Kits were used to extract BVDV, BRV, BPV, bovine enterovirus BEV, bovine coronavirus BCoV, bluetongue virus BTV, bovine epidemic fever virus BEFV, and bovine respiratory syncytial virus BRSV;

[0019] b) Preparation of standard plasmid:

[0020] Prepare a recombinant plasmid containing the gene fragments of 5'-UTR accession number of BVDV gene: M31182.1, BRV-VP6 gene accession number: JN790188.1 and BPV-VP2 gene accession number: JN191349.1, and after transfection, culture, plasmid separation, measure with a microspectrophotometer, and store for future use;

[0021] c) LAMP primer design:

[0022] Primers were designed based on the conserved sequences of the nucleic acid sequences of BVDV GenBank accession number: M31182.1, BPV GenBank accession number: JN191349.1 and BRV GenBank accession number: JN790188.1 as the target sequences for RT-LAMP and qRT-PCR; BVDV used biotin and ROX labeled circular primers, BRV used biotin and FAM labeled circular primers, and BPV used biotin and digoxigenin labeled circular primers;

[0023] d) Preparation of immunochromatographic test strips:

[0024] The lateral flow biosensor LFB comprises: a binding pad coated with a colloidal gold solution labeled with an anti-biotin monoclonal antibody, a sample pad, a nitrocellulose NC membrane, an absorption pad, a quality control line C covered with a goat anti-mouse polyclonal antibody, a detection line T1 covered with an anti-digoxin antibody, a detection line T2 covered with an anti-ROX antibody, a detection line T3 covered with an anti-FAM monoclonal antibody, and an LFB detection line arranged on the sample pad; after the assembly is completed, the sample is dried and divided into small pieces;

[0025] e) Triple LAMP-LFD system:

[0026] The LAMP solution includes: F3 / B3 0.2μM, FIP / BIP 1.6μM, LF / LB 0.8μM, (NH4)2SO4 10mM, MgSO4 8mM, KCl 10mM, Tris-HCl 20mM, 5% Tween-20 0.5μL, mixed dNTPs 0.8-2.0mM, Bst DNA polymerase 4-20U at a concentration of 8000U / mL; 2.5μL of virus standard plasmid is added to 25μL of LAMP reaction mixture; after the reaction is completed, amplify at a constant temperature of 61℃-69℃ in a thermal cycler for 5-40min, and then incubate to inactivate the enzyme and terminate the reaction; after the reaction is completed, dilute the LAMP amplification product with buffer; then take the dilution and put it into the sample well of the test paper; after waiting, observe the color development of the test paper; when quantitative analysis, use a colloidal gold reader to read the color signal on the test paper.

[0027] The aforementioned triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses comprises the following steps:

[0028] a) Viral RNA extraction:

[0029] Kits were used to extract BVDV, BRV, BPV, bovine enterovirus BEV, bovine coronavirus BCoV, bluetongue virus BTV, bovine epidemic fever virus BEFV, and bovine respiratory syncytial virus BRSV;

[0030] b) Preparation of standard plasmid:

[0031] A recombinant plasmid containing the gene fragments of the 5'-UTR accession number of the BVDV gene: M31182.1, the BRV-VP6 gene accession number: JN790188.1 and the BPV-VP2 gene accession number: JN191349.1 was prepared, and the plasmid was transfected into E. coli DH5α, followed by culture; the plasmid was separated using a plasmid extraction kit and then measured using a microspectrophotometer; the determination formula of the DNA copy number is as follows:

[0032]

[0033] The concentration of DNA standard is 10 times higher than that of 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 and 10 0 copies / μL, and the DNA standards were stored at −20°C until use;

[0034] c) LAMP primer design:

[0035] Primers were designed based on the conserved sequences of the nucleic acid sequences of BVDV GenBank accession number: M31182.1, BPV GenBank accession number: JN191349.1 and BRV GenBank accession number: JN790188.1 as the target sequences for RT-LAMP and qRT-PCR; BVDV used biotin and ROX labeled circular primers, BRV used biotin and FAM labeled circular primers, and BPV used biotin and digoxigenin labeled circular primers;

[0036] d) Preparation of immunochromatographic test strips:

[0037] The lateral flow biosensor LFB includes: a binding pad coated with a colloidal gold solution labeled with an anti-biotin monoclonal antibody, a sample pad, a nitrocellulose NC membrane, an absorption pad, a quality control line C covered with 2 mg / mL goat anti-mouse polyclonal antibody, a detection line T1 covered with 0.75 mg / mL anti-digoxin antibody, a detection line T2 covered with 0.6 mg / mL anti-ROX antibody, a detection line T3 covered with 0.5 mg / mL anti-FAM monoclonal antibody, and an LFB detection line arranged on the sample pad; after the assembly is completed, the sample is dried and divided into small pieces of 2.5 mm;

[0038] e) Triple LAMP-LFD system:

[0039] LAMP solution includes: F3 / B3 0.2μM, FIP / BIP 1.6μM, LF / LB 0.8μM, (NH4)2SO4 10mM, MgSO4 8mM, KCl 10mM, Tris-HCl 20mM, 5% Tween-20 0.5μL, mixed dNTPs 1.0mM, BstWarmStart 2.0 DNA polymerase is 8U; 2.5μL of virus standard plasmid is added to 22.5μL of LAMP reaction mixture; after the reaction is completed, amplify at 65℃ in a thermal cycler for 40min, and then incubate at 95℃ for 1min to inactivate the enzyme and terminate the reaction; after the reaction is completed, dilute the LAMP amplification product 50 times with PBS (3% Tween-80); then take 40μL of the dilution solution and put it into the sample well of the test paper; wait for 5min and observe the color development of the test paper; for quantitative analysis, use a colloidal gold reader to read the color signal on the test paper.

[0040] The present invention is beneficial in that:

[0041] The triple LAMP-LFD assay can detect three target viruses simultaneously within a short time of 30 min. The detection limits of BVDV, BRV and BPV were 2.62×101 copies / μL, 2.43×10 1 copies / μL and 2.50×10 1 copies / μL, thus showing extremely high sensitivity;

[0042] The present invention does not require specialized equipment or controlled laboratory conditions and can efficiently detect BVDV-5'UTR, BRV-VP6 and BPV-VP2 within 30 minutes at a constant temperature of 65°C, making it a more practical option for testing in the wild or in areas with limited resources;

[0043] The triple LAMP-LFD method is ideal for timely detection of various diseases in the field due to its rapid results, high cost-effectiveness, and improved accuracy. It is expected that the method developed in this study will be widely used in clinical diagnosis for the detection of BDS virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of triple LAMP-LFD analysis of the present invention ((A) sample extraction, (B) triple LAMP-LFD principle; C: quality control line; T1: detection line 1; T2: detection line 2; T3: detection line 3);

[0045] Figure 2 It is a schematic diagram of the improved triple LAMP-LFD test of the present invention; ((A) primer ratio, (B) Bst DNA polymerase concentration, (C) dNTPs concentration, (D) reaction temperature, (E) reaction time. The left figure is the test line and control line values, and the right figure is the T / C value; T1: BPV; T2: BVDV; T3: BRV: BRV);

[0046] Figure 3 The sensitivity and specificity of the triple LAMP-LFD detection of the present invention; ((A) Sensitivity test; the unit in the figure is copies / μL, and the BVDV concentration is 2.62×10 7 -10 0 copies / μL, BRV concentration was 2.43×10 7 -10 0 copies / μL, BPV concentration was 2.50×10 7 -10 0 copies / μL; (B) Standard curve. With the logarithm of the viral plasmid concentration (copies / μL) as the horizontal axis, determine the linear equation between the logarithm of the viral plasmid concentration and the T / C value; (C) Specific detection, (D) T value of specific detection. T1: BPV; T2: BVDV; T3: BRV);

[0047] Figure 4 The triple LAMP-LFD and qRT-PCR results for sample detection of the present invention. ((A) Intra-batch experimental results. (B) Inter-batch experimental results, (C) BVDV, (D) BRV, (E) BPV, (F) Total, "+" indicates positive; "-" indicates negative, (G) The actual detection results of the three viruses, as shown in the color scale, the color from light to dark indicates the concentration from low to high. One color band represents one sample). DETAILED DESCRIPTION

[0048] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0049] 1. Detection principle of the present invention:

[0050] like Figure 1 As shown. The reaction system uses three sets of primers, each with a circular primer labeled with a specific fluorescent group. As amplification proceeds, amplification products with different labels accumulate. Specific antibodies are used to capture products with corresponding labels on different detection lines. On the control line, uncaptured gold particles are fixed by secondary antibodies. The red bands represent the experimental results. If there is a target, the corresponding test will have a red band. If there is no target, no red band will be displayed.

[0051] 2. Testing materials and methods:

[0052] 2.1. Viral DNA / RNA extraction:

[0053] BVDV, BRV, BPV, Bovine enterovirus (BEV), Bovine coronavirus (BCoV), Bluetongue virus (BTV), Bovine epihemeral fever virus (BEFV), and Bovine respiratory syncytial virus (BRSV) were provided by Zhejiang Institute of Inspection and Quarantine (Hangzhou, China). Viral DNA or RNA was extracted using a DNA / RNA isolation kit (TIANGEN, Beijing) and reverse transcribed using the TIANScript II RT kit (TIANGEN, Beijing). DNA / cDNA was stored at −80°C until use.

[0054] 2.2. Preparation of standard plasmid

[0055] The 5'-UTR of the BVDV gene (accession number: M31182.1), the BRV-VP6 gene (accession number: JN790188.1) and the BPV-VP2 gene (accession number: JN191349.1) were commissioned to Shanghai Bioengineering Co., Ltd. to prepare the recombinant plasmid containing the above three gene fragments, and the plasmid was transfected into E. coli DH5α, followed by culture. The plasmid was isolated using the EU-MN-P-250 plasmid extraction kit from Suzhou Yilandi Biotechnology Co., Ltd., China, and then measured using the Nano100 microspectrophotometer from Hangzhou Aosheng Instrument Co., Ltd., China. The formula for determining the DNA copy number is as follows:

[0056]

[0057] The concentration of DNA standard is 10 times higher than that of 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 and 10 0 copies / μL, and store the DNA standards at -20°C until use.

[0058] 2.3.LAMP Primer Design

[0059] The conserved sequences of the reported nucleic acid sequences of BVDV (GenBank accession number: M31182.1), BPV (GenBank accession number: JN191349.1), and BRV (GenBank accession number: JN790188.1) were selected as the target sequences for RT-LAMP and qRT-PCR. The specific primers for LAMP were designed using the online LAMP primer design program Primer Explorer V5 (https: / / primerexplorer.jp / lampv5 / index.html). In order to distinguish different amplification products, biotin and ROX-labeled circular primers were used for BVDV, biotin and FAM-labeled circular primers were used for BRV, and biotin and digoxigenin-labeled circular primers were used for BPV. All primers were produced by Shanghai Sangon Biotech Co., Ltd. (Table 1).

[0060] Table 1 Primer sequences of triple LAMP-LFD

[0061]

[0062]

[0063] 2.4. Preparation of immunochromatographic test strips

[0064] The LFD consists of four parts: a conjugate pad, a sample pad, a nitrocellulose (NC) membrane, and an absorbent pad. The conjugate pad is coated with a colloidal gold solution labeled with anti-biotin monoclonal antibodies. The control line (C) and test lines 1, 2, and 3 (T1, T2, and T3, respectively) are the four lines that make up the strip. Sheep anti-mouse polyclonal antibodies (2 mg / mL) are used to cover the C line, while 0.75 mg / mL of anti-digoxin antibodies, 0.6 mg / mL of anti-ROX antibodies, and 0.5 mg / mL of anti-FAM monoclonal antibodies are used to cover the T1, T2, and T3 lines. These antibodies can recognize BVDV, BRV, and BPV. After assembly, the samples were dried and cut into 2.5 mm pieces.

[0065] 2.5. Triple LAMP-LFD system

[0066] The LAMP solution contains the specified concentrations of LAMP primers: F3 / B3 is 0.2μM, FIP / BIP is 1.6μM, LF / LB is 0.8μM, (NH4)2SO4 is 10mM, MgSO4 is 8mM, KCl is 10mM, Tris-HCl is 20mM, 5% Tween-20 is 0.5μL, mixed dNTPs is 1.0mM, and BstWarmStart 2.0 DNA polymerase is 8U. Then, 2.5μL of the virus standard plasmid is added to the 22.5μL LAMP reaction mixture. After the reaction is completed, the amplification is carried out at 65℃ for 40min in a thermal cycler, and then incubated at 95℃ for 1min to inactivate the enzyme and terminate the reaction. After the reaction is completed, the LAMP amplification product is diluted 50 times with PBS (3% Tween-80). Then 40μL is taken and placed in the sample well of the test paper. After waiting for 5min, the color development of the test paper is observed. During quantitative analysis, a colloidal gold reader is used to read the color signal on the test paper.

[0067] 2.6. Optimization of triple LAMP-LFD system

[0068] By using 10 5The LAMP-LFD reaction was optimized with a standard plasmid of 1000 copies / μL. The primer ratios of BVDV, BRV, and BPV were 1:1:1, 1:1:1.2, 1:1:1.4, 1:1:1.6, 1:1:1.8, and 1:1:2.0, respectively. In addition, a gradient of 0.16U / μL was set for Bst enzyme concentrations from 0.16 to 0.8U / μL, and a gradient of 0.2mM was set for dNTP concentrations from 0.8 to 2.0mM. Amplification experiments were performed at different temperatures, including 59°C, 61°C, 63°C, 65°C, 67°C, and 69°C. In addition, the reaction time was from 5min to 40min, with a gradient set every 5min, and a colloidal gold reader was used for color signal acquisition and analysis. All experiments were repeated three times to ensure accuracy.

[0069] 2.7. Evaluation of triple LAMP-LFD detection specificity

[0070] To analyze the specificity of triple LAMP-LFD, BEV, BCoV, BTV, BEFV, and BRSV were used as references. Viral DNA or cDNA was then used as a template to verify the specificity of the assay. In addition, BVDV, BRV, and BPV were amplified simultaneously or separately to confirm that there was no cross-reaction between the three assays.

[0071] 2.8. Evaluation of triple LAMP-LFD detection sensitivity

[0072] Equal volumes and concentrations of three viral standard plasmids were mixed as templates, and sterile water was used as a negative control to evaluate the sensitivity of triple LAMP-LFD.

[0073] 2.9. Repeatability evaluation of triple LAMP-LFD assay

[0074] Ensuring consistency of results is an important aspect of test strip performance. To evaluate the reproducibility of triple LAMP-LFD, we performed intra-batch experiments on five test strips from the same batch and inter-batch experiments using five different batches of test strips at 10% viral DNA or cDNA concentration. 5 We recorded the T and C values ​​for all experiments and calculated the T / C values. To measure the repeatability of triple LAMP-LFD, we calculated the coefficient of variation (CV) between and within batches.

[0075] 2.10.qRT-PCR system

[0076] qRT-PCR primers and probes were created using Beacon Designer 7.9 software (PREMIER Biosoft, San Francisco, CA, USA) and then produced by Shanghai Sangon Biotechnology Co., Ltd. The qRT-PCR setup included 10 μL 2× Premix Ex Taq (Probe qPCR, TaKaRa), 0.2 μL RoxII, 0.8 μM primers, 0.1 μM probes, 2.5 μL template, and ddH2O. In qRT-PCR, the temperature was first adjusted to 95°C for 5 min. Then 40 cycles were performed, alternating between 95°C for 10 s and 60°C for 30 s.

[0077] 2.11. Actual sample testing

[0078] A total of 156 anal swab samples were collected from a cattle farm in Hebei Province and stored at -20°C after extraction until use. Nucleic acids in liquid samples were extracted using the FastPure Viral DNA / RNA Mini Kit from Vazyme Biotechnology Co., Ltd. (Nanjing, China). Viral RNA was then reverse transcribed using the TIANScript II RT Kit from Tianjian, Beijing, China. Thermo Fisher Scientific, Waltham, MA, USA, provided the QuantStudioTM 5 Real-Time PCR Instrument for evaluation of all samples using triple LAMP-LFD technology and qRT-PCR.

[0079] Data analysis

[0080] The qRT-PCR results with a CT value lower than 35 were positive, and the qRT-PCR results with a CT value higher than 35 were negative. The triple LAMP-LFD standard curve was plotted using the T / C ratio and the logarithm of the viral plasmid concentration (copy number per volume). The schematic diagram was drawn using Adobe Illustrator 2022. Origin 2021 software and Microsoft Excel 2021 and PowerPoint 2021 software were used to create charts.

[0081] 3. Triple LAMP-LFD system optimization

[0082] The quantitative analysis uses a colloidal gold reader. Based on the absorbance of the test line reading, the T / C value is used to measure the intensity of each test line. When the ratio of BVDV, BRV and BPV primers is 1:1:1.6, the colors of the three test lines are bright and uniform, and the T / C values ​​are equivalent ( Figure 2 A).

[0083] In the study, we investigated the effect of different concentrations of Bst DNA polymerase on amplification efficiency. The results showed that when the concentration of Bst DNA polymerase was 0.16U / μL, the amplification efficiency reached a peak, and there was no significant increase in the amount of amplified product ( Figure 2 B). Based on the data obtained by the colloidal gold reader, 0.16U / μL Bst DNA polymerase was determined to be the optimal enzyme concentration for preserving experimental reagents.

[0084] dNTPs are essential substrates for amplification reactions. Insufficient dNTPs will slow down the reaction and affect the amount of product, while excess will hinder the activity of the enzyme and lead to a decrease in product. In our study, we found that higher concentrations of dNTPs favored the amplification of BRV and BVDV, but caused a significant decrease in the amplification of BPV. However, when the concentration reached 1.4 mM, the T / C values ​​between the three targets did not differ much ( Figure 2 C).

[0085] The correct temperature is crucial for the amplification reaction. Studies have found that 65°C is the most effective and closest temperature for amplifying the three target fragments. Temperatures below 61°C or above 69°C are not suitable for the reaction ( Figure 2 D).

[0086] The last parameter to be optimized is the reaction time. As the amplification time increases, the amplification product gradually accumulates. At 30 minutes, all three detection lines are close to saturation, and the T / C values ​​are relatively close ( Figure 2 E), so the optimal reaction time is 30 min.

[0087] 4. Evaluation of Triple LAMP-LFD Detection Specificity

[0088] The triple LAMP-LFD technique showed high specificity in identifying BVDV, BRV, and BPV, with red bands appearing on the test line when using viral DNA or reverse transcribed cDNA. However, no red bands appeared on the test line when using DNA or cDNA templates of BEV, BCoV, BTV, BEFV, and BRSV, confirming the absence of cross-reactions between viruses ( Figure 3 ).

[0089] 5. Evaluation of Triple LAMP-LFD Assay Sensitivity

[0090] The sensitivity of the triple LAMP-LFD assay was evaluated using a mixture of three viral plasmids as templates. A standard curve was generated based on their T / C values. The detection limits of BVDV, BRV, and BPV were 2.62×10 1 copies / μL, 2.43×10 1 copies / μL and 2.50×101 copies / μL( Figure 3 ).

[0091] 6. Repeatability Evaluation of Triple LAMP-LFD Assay

[0092] The reproducibility of triple LAMP-LFD was verified by intra- and inter-batch experiments. A mixture of three viral standard plasmids was used as template for amplification, with a template concentration of 10 5 copies / μL. Results ( Figure 4 ) In the intra-batch study, the CV values ​​of T1 / C for BPV were 4.39%, T2 / C for BVDV were 2.74%, and T3 / C for BRV were 2.06%. Similarly, in the inter-batch test, the CV values ​​of T1 / C for BPV were 2.14%, T2 / C for BVDV were 2.45%, and T3 / C for BRV were 4.54%. The low CV values ​​indicate that the reproducibility of triple LAMP-LFD is high.

[0093] 7. Actual sample testing

[0094] 156 samples were analyzed using triple LAMP-LFD and qRT-PCR to compare their test results and confirm the accuracy of the triple LAMP-LFD method. The test results showed that the incidence of mixed viral infection was high, and infection with multiple viruses at the same time was common ( Figure 4 ). In addition, the qRT-PCR results were completely consistent with the actual results, and the agreement rate between the triple LAMP-LFD and qRT-PCR results exceeded 99%, thus proving the accuracy and reliability of the method in actual detection (Table 1). The positive predictive value (PPV) of both techniques was 97.14%, the negative predictive value (NPV) was 100%, and the Kappa coefficient was 98.14% (Table 2).

[0095] Table 1 Triple LAMP-LFD and qRT-PCR sample detection results

[0096]

[0097] “(+)”: positive; “(-)”: negative

[0098] Table 2 PPV, NPV and Kappa coefficient of triple LAMP-LFD assay and qRT-PCR assay

[0099]

[0100] The present invention adopts LAMP-LFD detection method to identify BVDV, BRV and BPV by combining LAMP and LFD technology. The detection method has extremely high sensitivity and specificity; the addition of UNG enzyme and dUTP in the detection system of the present invention can reduce false positive results to a minimum; the detection method of the present invention can simultaneously detect three target viruses in a short time of 30 minutes. The detection limits of BVDV, BRV and BPV are 2.62×10 1 copies / μL, 2.43×10 1 copies / μL and 2.50×10 1 copies / μL, thus showing extremely high sensitivity.

[0101] In conclusion, the triple LAMP-LFD method is ideal for timely detection of various diseases in the field due to its rapid results, high cost-effectiveness, and improved accuracy. The method developed in this study is expected to be widely used in clinical diagnosis for the detection of BDS virus.

[0102] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses, characterized in that: include: Three sets of LAMP primers and probes, lateral flow biosensor LFB; The lateral flow biosensor LFB comprises: a binding pad coated with a colloidal gold solution labeled with an anti-biotin monoclonal antibody, a sample pad, a nitrocellulose NC membrane, an absorption pad, a quality control line C covered with a goat anti-mouse polyclonal antibody, a detection line T1 covered with an anti-digoxin antibody, a detection line T2 covered with an anti-ROX antibody, a detection line T3 covered with an anti-FAM monoclonal antibody, and an LFB detection line arranged on the sample pad; The LAMP primer probe includes: a BVDV virus primer set designed for the GenBank accession number of BVDV virus: M31182.1, a BPV virus primer set designed for the GenBank accession number of BPV virus: JN191349.1, and a BRV virus primer set designed for the GenBank accession number of BRV virus: JN790188.

1. The BVDV virus primer set uses a circular primer labeled with biotin and ROX; the BRV virus primer set uses a circular primer labeled with biotin and FAM; and the BPV virus primer set uses a circular primer labeled with biotin and digoxigenin.

2. A triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses according to claim 1, characterized in that: The LAMP primer probe is shown in SEQ ID No: 01 to SEQ ID No: 18 in Table 1.

3. A triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses according to claim 1, characterized in that: The LAMP-LFD system of the three sets of LAMP primer probes is: F3 / B3 0.2μM, FIP / BIP 1.6μM, LF / LB 0.8μM, (NH4)2SO4 10mM, MgSO4 8mM, KCl 10mM, Tris-HCl 20mM, 5% Tween-20 0.5μL, mixed dNTPs 1.0mM, BstDNA polymerase 8U at a concentration of 0.16-0.80U / μL, virus standard plasmid 2.5μL, reaction temperature: 61°C-69°C, reaction time: 5-40min.

4. A triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses according to claim 3, characterized in that: The ratio of the BVDV virus primer set, the BRV virus primer set and the BPV virus primer set is 1:1:1.

6.

5. A triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses according to claim 3, characterized in that: The concentration of the Bst DNA polymerase is 0.16 U / μL.

6. A triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses according to claim 3, characterized in that: The dNTPs concentration is 1.4 mM.

7. A triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses according to claim 3, characterized in that: The reaction temperature was 65°C.

8. A triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses according to claim 3, characterized in that: The reaction time is 30 minutes.

9. A triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses as claimed in claim 1, characterized in that: The steps include: a) Viral RNA extraction: Kits were used to extract BVDV, BRV, BPV, bovine enterovirus BEV, bovine coronavirus BCoV, bluetongue virus BTV, bovine epidemic fever virus BEFV, and bovine respiratory syncytial virus BRSV; b) Preparation of standard plasmid: Prepare a recombinant plasmid containing the gene fragments of 5'-UTR accession number of BVDV gene: M31182.1, BRV-VP6 gene accession number: JN790188.1 and BPV-VP2 gene accession number: JN191349.1, and after transfection, culture, plasmid separation, measure with a microspectrophotometer, and store for future use; c) LAMP primer design: Primers were designed based on the conserved sequences of the nucleic acid sequences of BVDV GenBank accession number: M31182.1, BPV GenBank accession number: JN191349.1 and BRV GenBank accession number: JN790188.1 as the target sequences for RT-LAMP and qRT-PCR; BVDV used biotin and ROX labeled circular primers, BRV used biotin and FAM labeled circular primers, and BPV used biotin and digoxigenin labeled circular primers; d) Preparation of immunochromatographic test strips: The lateral flow biosensor LFB comprises: a binding pad coated with a colloidal gold solution labeled with an anti-biotin monoclonal antibody, a sample pad, a nitrocellulose NC membrane, an absorption pad, a quality control line C covered with a goat anti-mouse polyclonal antibody, a detection line T1 covered with an anti-digoxin antibody, a detection line T2 covered with an anti-ROX antibody, a detection line T3 covered with an anti-FAM monoclonal antibody, and an LFB detection line arranged on the sample pad; after the assembly is completed, the sample is dried and divided into small pieces; e) Triple LAMP-LFD system The LAMP solution includes: F3 / B3 0.2μM, FIP / BIP 1.6μM, LF / LB 0.8μM, (NH4)2SO4 10mM, MgSO48mM, KCl 10mM, Tris-HCl 20mM, 5% Tween-20 0.5μL, mixed dNTPs 0.8-2.0mM, and Bst DNA polymerase 4-20U at a concentration of 8000U / mL; 2.5μL of virus standard plasmid is added to 25μL of LAMP reaction mixture; after the reaction is completed, amplify at a constant temperature of 61℃-69℃ in a thermal cycler for 5-40min, and then incubate to inactivate the enzyme and terminate the reaction; after the reaction is completed, dilute the LAMP amplification product with a buffer; then take the dilution and put it into the sample well of the test paper; after waiting, observe the color development of the test paper; when quantitative analysis is performed, use a colloidal gold reader to read the color signal on the test paper.

10. A triple LAMP-LFD detection kit for simultaneously detecting three bovine diarrhea syndrome viruses as claimed in claim 9, characterized in that: The steps include: a) Viral RNA extraction: Kits were used to extract BVDV, BRV, BPV, bovine enterovirus BEV, bovine coronavirus BCoV, bluetongue virus BTV, bovine epidemic fever virus BEFV, and bovine respiratory syncytial virus BRSV; b) Preparation of standard plasmid: A recombinant plasmid containing the gene fragments of the 5'-UTR accession number of the BVDV gene: M31182.1, the BRV-VP6 gene accession number: JN790188.1 and the BPV-VP2 gene accession number: JN191349.1 was prepared, and the plasmid was transfected into E. coli DH5α, followed by culture; the plasmid was separated using a plasmid extraction kit and then measured using a microspectrophotometer; the determination formula of the DNA copy number is as follows: The concentration of DNA standard is 10 times higher. 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 and 10 0 copies / μL, and the DNA standards were stored at −20°C until use; c) LAMP primer design: Primers were designed based on the conserved sequences of the nucleic acid sequences of BVDV GenBank accession number: M31182.1, BPV GenBank accession number: JN191349.1 and BRV GenBank accession number: JN790188.1 as the target sequences for RT-LAMP and qRT-PCR; BVDV used biotin and ROX labeled circular primers, BRV used biotin and FAM labeled circular primers, and BPV used biotin and digoxigenin labeled circular primers; d) Preparation of immunochromatographic test strips: The lateral flow biosensor LFB includes: a binding pad coated with a colloidal gold solution labeled with an anti-biotin monoclonal antibody, a sample pad, a nitrocellulose NC membrane, an absorption pad, a quality control line C covered with 2 mg / mL goat anti-mouse polyclonal antibody, a detection line T1 covered with 0.75 mg / mL anti-digoxin antibody, a detection line T2 covered with 0.6 mg / mL anti-ROX antibody, a detection line T3 covered with 0.5 mg / mL anti-FAM monoclonal antibody, and an LFB detection line arranged on the sample pad; after the assembly is completed, the sample is dried and divided into small pieces of 2.5 mm; e) Triple LAMP-LFD system LAMP solution includes: F3 / B3 0.2μM, FIP / BIP 1.6μM, LF / LB 0.8μM, (NH4)2SO4 10mM, MgSO4 8mM, KCl 10mM, Tris-HCl 20mM, 5% Tween-20 0.5μL, mixed dNTPs 1.0mM, BstWarmStart 2.0 DNA polymerase is 8U; 2.5μL of virus standard plasmid is added to 22.5μL of LAMP reaction mixture; after the reaction is completed, amplify at 65℃ in a thermal cycler for 40min, and then incubate at 95℃ for 1min to inactivate the enzyme and terminate the reaction; after the reaction is completed, dilute the LAMP amplification product 50 times with PBS (3% Tween-80); then take 40μL of the dilution solution and put it into the sample well of the test paper; wait for 5min and observe the color development of the test paper; for quantitative analysis, use a colloidal gold reader to read the color signal on the test paper.

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