MRT-RPA-LFB detection kit capable of simultaneously detecting three viral pathogens of bovine BVDV, BEFV and BRSV and detection method of mRT-RPA-LFB detection kit

Abstract

The invention discloses an mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens of bovine BVDV, BEFV and BRSV and a detection method of the mRT-RPA-LFB detection kit, and relates to the field of agricultural gene biology. The detection kit comprises three groups of RT-RPA primer probes and a lateral flow biosensor LFB, the BVDV-1 primer group is designed according to a 5 'UTR region GenBank: M31182.1, the BEFV primer group is designed according to a G gene GenBank: AF058321.2, and the BRSV primer group is designed according to an N gene GenBank: NC001989.1; the mRT-RPA-LFB detection method disclosed by the invention is rapid, sensitive and portable in detection, and has accuracy and stability.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biology, in particular to a virus detection kit and a detection method thereof. Background Art

[0002] The sharp increase in cattle breeding has led to an expansion in scale and quantity, resulting in an increase in cattle breeding density, more frequent transportation, and an increased risk of disease outbreaks. To effectively reduce risks, it is crucial to conduct comprehensive and timely cattle disease monitoring. Bovine viral diarrhea virus (BVDV), bovine ephemeral fever virus (BEFV), and bovine respiratory syncytial virus (BRSV) seriously affect the health of cattle and pose a huge challenge to the global livestock industry.

[0003] BVDV belongs to the genus Pestivirus of the family Flaviviridae and is a positive-strand RNA virus. BEFV belongs to the genus Ephemerovirus of the family Rhabdoviridae and is a negative-strand RNA virus. BRSV belongs to the genus Orthopneumovirus of the family Pneumoviridae and is also a negative-strand RNA virus. BRSV is usually transmitted through aerosols in cattle herds, and this virus is considered a pathogen with significant economic impact in global cattle agriculture. BVDV and BRSV are key viruses of bovine respiratory disease syndrome. They lay the foundation for opportunistic secondary bacterial infections by damaging the nasal mucosa, and then may cause cattle to suffer from severe respiratory diseases. It has been documented in the literature that BVDV and BRSV can co-infect cattle, resulting in mixed infections. Although the symptoms caused by BEFV infection in cattle are mild and the course of the disease is short, there may be significant delayed effects after the disease, such as the need to cull diseased animals, etc. The symptoms caused by the infection of the three viruses in cattle are similar, and it is very difficult for farmers to distinguish the virus infection situation only by observing the symptoms. Given the significant impact and wide prevalence of bovine viral diarrhea virus type 1 (BVDV-1), it is selected as the representative strain for BVDV detection. Importantly, by effectively diagnosing BVDV-1, BEFV, and BRSV, the health of cattle can be ensured, and thus their economic interests can be protected.

[0004] Serological and virus isolation methods have been used to identify BVDV, BEFV, and BRSV, respectively. However, these two methods not only require continuous cold-chain storage of samples but are also labor-intensive and time-consuming. Polymerase chain reaction (PCR) is the main method used for nucleic acid amplification. Although PCR-based methods are considered reliable, they require expensive thermal cyclers and professional laboratories and are time-consuming. The limitations of PCR-based detection have prompted the development of various isothermal amplification methods. Recombinase polymerase amplification (RPA) is an innovative isothermal nucleic acid amplification method that can be carried out with minimal heating equipment, using body temperature to facilitate the reaction. Loop-mediated isothermal amplification (LAMP) is a commonly used isothermal nucleic acid amplification method. LAMP and RPA aim to meet the criteria of cost-effectiveness, sensitivity, specificity, simplicity, user-friendliness, stability, and speed required for point-of-care diagnosis. Different from LAMP, which requires four to six primers, RPA can achieve amplification with only two primers. In addition, RPA can operate effectively at lower ambient temperatures (37 - 42°C), while LAMP requires higher isothermal conditions (above 55°C), indicating that RPA is superior to LAMP in multiple aspects.

[0005] RPA is based on the activities of three key proteins: recombinase, single-stranded DNA-binding protein (SSB), and polymerase. During the RPA process, the nucleoprotein formed by the binding of recombinase to each primer attaches to a specific sequence of the target DNA. SSB ensures its stability by adhering to the separated DNA strand and prevents the strands from reannealing. Subsequently, the polymerase replicates the target DNA sequence. In addition, reverse transcriptase has been integrated into the RPA system, enabling the detection of various RNAs. Currently, RPA technology has become a valuable tool for detecting viral DNA and RNA. Agarose gel electrophoresis can be used to visualize RPA products; however, purification is required before electrophoresis, which increases the overall detection time. Detection of RPA products using a lateral flow biosensor (LFB) can eliminate the need for purification. The combination of RPA and LFB has shown its effectiveness and suitability for point-of-care testing.

[0006] Considering the complex viral infections, there is a need in the market for a multiplex detection method, namely multiple reverse transcription recombinase polymerase amplification (mRT-RPA) combined with a lateral flow biosensor (LFB), which can detect these three pathogens simultaneously. Summary of the Invention

[0007] To address the deficiencies of the prior art, the objective of the present invention is to provide an mRT-RPA-LFB detection kit and its detection method for simultaneously detecting three viral pathogens, namely bovine BVDV, BEFV, and BRSV. The present invention combines RPA and LFB. mRT-RPA-LFB detection features rapid detection speed, simple operation, high specificity, and strong sensitivity.

[0008] To achieve the above objective, the present invention adopts the following technical solutions:

[0009] An mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens, namely bovine BVDV, BEFV, and BRSV, comprising: three groups of RT-RPA primer-probes and a lateral flow biosensor LFB;

[0010] The lateral flow biosensor LFB includes: a sample pad, a conjugate pad, an absorption pad, a backplane, a T1 test line coated with digoxin monoclonal antibody, a T2 test line coated with ROX monoclonal antibody, a T3 test line coated with anti-FAM antibody, an LFB test line provided on the sample pad, an NC membrane, a C line coated with goat anti-mouse IgG, and biotin-labeled colloidal gold particles;

[0011] The RT-RPA primer-probes include: a primer group for BVDV-1 virus designed for the 5'UTR region of BVDV-1 virus (GenBank: M31182.1), a primer group for BEFV virus designed for the G gene of BEFV virus (GenBank: AF058321.2), and a primer group for BRSV virus designed for the N gene of BRSV virus (GenBank: NC_001989.1). The upstream primer of the BVDV-1 virus primer group is labeled with biotin, and the downstream primer is labeled with digoxin Dig; the upstream primer of the BEFV virus primer group is labeled with biotin, and the downstream primer is labeled with ROX; the upstream primer of the BRSV virus primer group is labeled with biotin, and the downstream primer is labeled with FAM.

[0012] For the aforementioned mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens, namely bovine BVDV, BEFV, and BRSV, the RT-RPA primer-probes are as shown in SEQ ID No: 01 - SEQ ID No: 06 in Table 1.

[0013] For the aforementioned mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens, namely bovine BVDV, BEFV, and BRSV, the C line is coated with goat anti-mouse IgG at a concentration of 2 mg / mL, the T1 test line is coated with digoxin monoclonal antibody at a concentration of 0.75 mg / mL, the T2 test line is coated with ROX monoclonal antibody at a concentration of 0.6 mg / mL, and the T3 test line contains FAM monoclonal antibody at a concentration of 0.5 mg / mL.

[0014] The aforementioned mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens, namely bovine BVDV, BEFV, and BRSV. For mRT-RPA, the TwistAmp Basic RPA kit is used. In addition to the substances provided in the RPA lyophilized tube, the following components are sequentially added: 25 μL of buffer, 30 μM of RT-RPA primer-probe mixture, 1 μL of reverse transcriptase, 3 μL of template, 280 mM of magnesium acetate, with the concentration of Mg2+ being 2.8 to 16.8 nM. The total volume is adjusted to 50 μL with ddH2O. The amplification time is 5 - 25 minutes, the final concentration of the primer set is 150 - 500 nM, and the reaction temperature is 38 - 43°C.

[0015] For the aforementioned mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens, namely bovine BVDV, BEFV, and BRSV, the final concentration of the BVDV-1 and BRSV primer sets is 450 nM, and the final concentration of the BEFV primer set is 250 nM.

[0016] For the aforementioned mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens, namely bovine BVDV, BEFV, and BRSV, the reaction temperature is 42°C.

[0017] For the aforementioned mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens, namely bovine BVDV, BEFV, and BRSV, the amplification time is 20 minutes.

[0018] For the aforementioned mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens, namely bovine BVDV, BEFV, and BRSV, the concentration of Mg2+ is 16.8 nM.

[0019] The detection method of the aforementioned mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens, namely bovine BVDV, BEFV, and BRSV, includes the following steps:

[0020] a) Viral RNA extraction

[0021] Extract bovine viral diarrhea virus type 1 (BVDV-1), bovine ephemeral fever virus (BEFV), bovine respiratory syncytial virus (BRSV), peste des petits ruminants virus (PPRV), bovine rotavirus (BRV), bluetongue virus (BTV), bovine parainfluenza virus type 3 (BPIV3), bovine enterovirus (BEV), and bovine torovirus (BToV) using the kit.

[0022] b) Pseudovirus preparation and RNA quantification

[0023] Extract pseudoviruses BVDV-1, BEFV, and BRSV. After extracting the RNA of the pseudoviruses using a kit, quantify and reverse transcribe it, and then use a Nano-100 differential spectrophotometer to evaluate the concentration and purity of the cDNA;

[0024] c) Primer design

[0025] Design three sets of RT-RPA primers for the 5'UTR region of BVDV-1 (GenBank: M31182.1), the G gene of BEFV (GenBank: AF058321.2), and the N gene of BRSV (GenBank: NC_001989.1);

[0026] Preparation of LFB

[0027] The lateral flow biosensor LFB consists of five main parts: sample pad, conjugate pad, NC membrane, absorbent pad, and backplate; the conjugate pad is uniformly coated with colloidal gold and conjugated with monoclonal antibodies specific to the target analyte; the C line is coated with goat anti-mouse IgG at a concentration of 2 mg / mL, the T1 test line uses digoxin monoclonal antibody at a concentration of 0.75 mg / mL, the T2 test line is sprayed with ROX monoclonal antibody at a concentration of 0.6 mg / mL, and the T3 test line contains FAM monoclonal antibody at a concentration of 0.5 mg / mL;

[0028] e) mRT-RPA-LFB reaction.

[0029] The advantages of the present invention are as follows:

[0030] The mRT-RPA-LFB detection of the present invention can be completed within 20 minutes at 41°C;

[0031] The limits of detection (LODs) of BVDV, BEFV, and BRSV of the present invention are 2.62×10 1 、2.42×10 1 and 2.56×10 1 copies / μL;

[0032] The method of the present invention has no cross-reaction with six other bovine viruses, shows satisfactory within-run and between-run precision, and the average coefficient of variation ranges from 2.92% to 3.99%. The diagnostic sensitivity and specificity are 98.11% and 100% respectively, which are highly consistent with the RT-qPCR detection results, and the Kappa value is 0.988 (95% confidence interval);

[0033] The mRT-RPA-LFB detection of the present invention has the advantages of fast detection speed, simple operation, high specificity, and strong sensitivity, and has the potential to become a powerful tool for rapid screening of bovine diseases. Description of the drawings

[0034] Figure 1 This is the detection principle of mRT-RPA-LFB of the present invention. (a) Workflow of mRT-RPA-LFB detection. The mRT-RPA-LFB diagnostic test requires four steps: sample collection (10 seconds), rapid RNA extraction (7 minutes), mRT-RPA reaction (20 minutes), and result reporting (<2 minutes). The LFB detection results can be visualized and quantified using a test strip reader. The whole process can be completed within half an hour. (b) LFB principle of the mRT-RPA-LFB product. (b-i) Positive results for BVDV-1, BEFV, and BRSV are shown as the appearance of test line 1 (T1), test line 2 (T2), test line 3 (T3), and control line on the test strip. Negative results (only the control line is visible on the LFB). (b-ii) Goat anti-mouse IgG is sprayed at the control line position to verify the effectiveness of the LFB. Anti-Dig monoclonal antibody, anti-ROX monoclonal antibody, and FAM monoclonal antibody are sprayed at T1, T2, and T3 respectively to detect the amplification products of BVDV-1, BEFV, and BRSV. (c) Primer design. The primers for BVDV-1 target the 5'UTR gene, the primers for BEFV target the G gene, and the primers for BRSV target the N gene. For BVDV-1, BEFV, and BRSV, the upstream primers are all labeled with biotin, while the downstream primers are labeled with Dig, ROX, and FAM respectively. (d) Mechanism description of the mRT-RPA-LFB test;

[0035] Figure 2 . This is the optimization result of mRT-RPA-LFB of the present invention; the T / C value graph of the LFB reaction is drawn with the color intensity representing the reading intensity of the test strip reader, (a) Optimization of primer ratio, (b) Optimization of temperature, (c) Optimization of time, (d) Optimization of magnesium ion concentration;

[0036] Figure 3 This is the specificity analysis of mRT-RPA-LFB observed on the TAE gel of the present invention; Note: In the figure, "+" indicates spiked, "-" indicates unspiked; "M", marker;

[0037] Figure 4 This is the sensitivity analysis of mRT-RPA-LFB observed on the TAE gel of the present invention; Note: "M", Marker;

[0038] Figure 5Method performance evaluation of the present invention; (a) Specificity analysis of mRT-RPA-LFB. The bar chart reflects the results of using different viruses as templates in the mRT-RPA-LFB detection. The T / C values are provided by the test strip reader. "+" indicates that the corresponding virus was added during the detection, while "-" indicates that the corresponding virus was not added; (b) Sensitivity analysis of mRT-RPA-LFB. The mRT-RPA-LFB detection uses three mixtures of pseudovirus RNA with the same concentration and volume as templates, and the negative control (NC) uses ddH2O as a template. Positive reactions can be produced when the concentration of pseudovirus RNA ranges from 10 7 to 10 1 copies / μL. These pseudovirus RNAs include BVDV-1 (2.62×10 7 to 2.62×10 0 copies / μL), BEFV (2.42×10 7 to 2.42×10 0 copies / μL), and BRSV (2.56×10 7 to 2.56×10 0 copies / μL);

[0039] Figure 6 Repeatability evaluation of mRT-RPA-LFB of the present invention; (a) Intra-batch experimental procedure: Five LFBs are randomly selected from the same test batch, and each strip is read three times (T / C values), and the average value and CV are calculated. (b) Inter-batch experimental procedure: Five LFBs are randomly selected from different test batches, and each strip is read three times (T / C values); the average value is calculated and the CV is determined;

[0040] Figure 7 Performance of mRT-RPA-LFB of the present invention in actual samples; (a) Detection results of mRT-RPA-LFB and RT-qPCR for 292 actual samples. The mRT-RPA-LFB detection method is used to identify viral RNA in 292 actual samples. The T / C values are substituted into the standard curve of mRT-RPA-LFB to calculate the concentration of viral RNA in the samples. Each cell represents a test result, and the color ranges from white to red, reflecting the concentration from low to high; all other colored areas indicate negative results; the green area represents the results obtained using RT-qPCR as the detection method, and its concentration indication mechanism is similar to the above mechanism; (b) Consistency between the proposed mRT-RPA-LFB and RT-qPCR in the detection of 292 field samples; "+" indicates a positive result; "-" indicates a negative result; (c) Evaluation of the consistency of the detection of 292 field samples by comparing mRT-RPA-LFB with RT-qPCR; TP: True positive; FP: False positive; TN: True negative; FN: False negative. Detailed implementation mode

[0041] The present invention will be specifically introduced below in conjunction with the accompanying drawings and specific embodiments.

[0042] Method principle:

[0043] The mRT-RPA-LFB detection method relies on the principle that nucleic acid amplification products with unique labels specifically bind to the corresponding antibodies coated on the NC membrane (see Figure 1 ). The primer sets of the three viruses carry unique probe markers: the upstream primer of BVDV-1 is labeled with biotin, and the downstream primer is labeled with digoxin (Dig); the upstream primer of BEFV is labeled with biotin, and the downstream primer is labeled with ROX; the upstream primer of BRSV is labeled with biotin, and the downstream primer is labeled with FAM (see Figure 1 c). Specific capture antibodies are sprayed on different detection lines of the LFB NC membrane: detection line 1 (T1) is coated with anti-Dig antibody, detection line 2 (T2) is coated with anti-ROX antibody, and detection line 3 (T3) is coated with anti-FAM antibody. After RT-RPA amplification, the labeled amplification products were diluted 50-fold, and then 40 μL of the diluted solution was dropped onto the sample pad of the LFB (see Figure 1 b-ii). The sample then migrates along the NC membrane by capillary action (see Figure 1 b-ii). The specific antibody on the designated detection line binds to the labeled amplification product, resulting in the appearance of a red line. A positive signal on the test strip reader on any detection line indicates a positive result, while the absence of the target analyte results in no color change on the corresponding detection line, indicating a negative result. The C line is coated with goat anti-mouse IgG to capture excess colloidal gold particles labeled with biotin. Whether the sample contains the analyte or not, the C line must show color for the LFB to be considered valid (see Figure 1 b-i). When the target nucleic acid is present in the sample, the primers of BVDV-1, BEFV, and BRSV, which are primers with specific labels, produce amplification products with specific labels through RT-RPA. These products are captured by the LFB detection line, resulting in a positive test result shown by the LFB (see Figure 1 d).

[0044] Specific kit and detection method:

[0045] 1. Materials and methods

[0046] 1.1 Virus RNA extraction

[0047] Bovine viral diarrhea virus type 1 (BVDV-1), bovine ephemeral fever virus (BEFV), bovine respiratory syncytial virus (BRSV), peste des petits ruminants virus (PPRV), bovine rotavirus (BRV), bluetongue virus (BTV), bovine parainfluenza virus type 3 (BPIV 3), bovine enterovirus (BEV) and bovine torovirus (BToV) were provided by the Zhejiang Academy of Science and Technology Inspection and Quarantine (Hangzhou, China). The RNA of all viruses was extracted using the FastPure Viral DNA / RNA Mini Kit produced by Vazyme Biotech Co., Ltd. (Nanjing, China).

[0048] 1.2 Pseudovirus preparation and RNA quantification

[0049] Pseudoviruses BVDV-1, BEFV and BRSV were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). After extraction of the pseudoviruses, the RNA of the pseudoviruses was extracted and quantified using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver. 5.0 (Takara, Kyoto, Japan). Reverse transcription was performed using the PrimeScript TM RT Kit from Takara Co., Ltd. (Kyoto, Japan). The concentration and purity of the cDNA were evaluated using the Nano-100 Micro-spectrophotometer from Quesheng Instruments Co., Ltd. (Hangzhou, Zhejiang). A series of 10-fold dilutions of the reverse-transcribed cDNA were prepared, ranging from 107 to 10 1 copies / μL, for the quantification of the RNA of the BVDV-1, BEFV and BRSV pseudoviruses. These dilutions were used as templates for a single RT-qPCR assay for these three virus species. Next, the RNA copy numbers of the pseudoviruses were determined using a standard curve. Finally, the quantified pseudovirus RNA was stored frozen at -80 °C for later use.

[0050] 1.3 Primer design

[0051] Three sets of RT-RPA primers were designed for the 5'UTR region of BVDV-1 (GenBank: M31182.1), the G gene of BEFV (GenBank: AF058321.2) and the N gene of BRSV (GenBank: NC_001989.1), using the BeaconDesigner 7.0 software (Premier Biosoft, Palo Alto, CA, USA) (see Figure 1 c). Gene homology analysis between the three target genes was performed using NCBI BLAST (http: / / www.ncbi.nlm.nih.gov). More details of the primer design, location, sequence and modifications are shown in Figure 1 c.

[0052] The RT-RPA primers and probes are shown in Table 1:

[0053] Table 1 RPA primer sequences of BVDV 1, BEFV, and BRSV

[0054]

[0055] 1.4 Preparation of the lateral flow biosensor (LFB)

[0056] The lateral flow biosensor (LFB) consists of five main parts: a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, an absorbent pad, and a backing plate. All of the above components are from Zhejiang Dean Biotechnology Co., Ltd. (Hangzhou, Zhejiang, China). Three test lines are sprayed with corresponding concentrations of monoclonal antibodies and have been verified to obtain good test results. The conjugate pad is evenly coated with colloidal gold and conjugated with a monoclonal antibody specific to the target analyte. On the NC membrane of the LFB, the control line (Line C) is coated with goat anti-mouse IgG at a concentration of 2 mg / mL. The T1 test line uses a digoxin monoclonal antibody at a concentration of 0.75 mg / mL for the detection of BVDV-1. The T2 test line is sprayed with a ROX monoclonal antibody at a concentration of 0.6 mg / mL and is designed for the detection of BEFV, while the T3 test line contains a FAM monoclonal antibody at a concentration of 0.5 mg / mL for the detection of BRSV. The goat anti-mouse IgG and these three monoclonal antibodies are all from Hangzhou KuaiGe Biotechnology Co., Ltd. (Hangzhou, Zhejiang, China). The LFBs are cut to a uniform width (2.5 mm) and stored under dry conditions for subsequent applications.

[0057] 1.5 mRT-RPA-LFB reaction

[0058] mRT-RPA uses the TwistAmp Basic RPA kit (TwistDx, Cambridge, UK). In addition to the substances provided in the RPA lyophilized tube, the following components are added sequentially: 25 μL of buffer, a mixture of three target gene primers (each at 10 μM), 1 μL of reverse transcriptase (TaKaRa, Japan), 3 μL of template, and 280 mM magnesium acetate. Finally, the total volume is adjusted to 50 μL with ddH2O.

[0059] A lateral flow biosensor (LFB) and a test strip reader (Helmen, China) are used to verify the RT-RPA reaction. The device converts the light of the test (T) and control (C) lines into digital form and assigns values to the T and C lines respectively. The result is defined as the ratio of the T value to the C value (T / C value), which reflects the amplification intensity. Based on this T / C ratio, the samples are quantitatively evaluated.

[0060] 1.6 Optimization of mRT-RPA-LFB

[0061] By improving the mRT-RPA-LFB detection method, the detection efficiency was ensured, which included optimizing the final concentrations of three primer sets, adjusting the time and temperature of the amplification reaction, and fine-tuning the magnesium ion concentration. The improvement of mRT-RPA-LFB detection was carried out under 106 copies / μL of pseudovirus RNA. The initial concentrations of the upstream and downstream primers for BVDV-1, BEFV, and BRSV were 10 μM and could be used after mixing in equal volumes. The final concentrations of the three primer pairs were tested in the range of 150, 200, 250, 300, 350, 400, and 500 nM to determine the most effective primer ratio. Eight different combinations of final primer concentrations were used. The optimized final concentrations of the three primer sets are listed in Table 1. There were multiple options for the reaction time: 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, and 25 minutes. The optimal reaction temperature was determined from six groups: 38, 39, 40, 41, 42, and 43 °C. In addition, six levels of magnesium ion concentration were tested: 2.8, 5.6, 8.4, 11.2, 14, and 16.8 mM. All experiments were performed in triplicate.

[0062] Table 1 Optimization of the final concentrations of primer sets

[0063]

[0064] Optimization of mRT-RPA-LFB

[0065] By optimizing the basic elements, the performance of mRT-RPA-LFB detection was improved. When the final concentration of the BVDV 1 and BRSV primer sets was 450 nM and the final concentration of the BEFV primer set was 250 nM, a consistent detection line could be obtained, and the data read on the strip reader had very little variation ( Figure 2 a). Compared with the detection carried out at 42 °C, the detection carried out at 41 °C could not only amplify all three detection lines simultaneously but also had lower energy consumption ( Figure 2 b). In addition, within the time optimization range (5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, and 25 minutes), an amplification time of 20 minutes was the most effective amplification state ( Figure 2 c). Among the tested Mg2+ concentrations of 2.8 to 16.8 nM, 16.8 nM was determined to be the optimal concentration ( Figure 2 d).

[0066] 2. Experimental verification:

[0067] 2.1 Evaluation of the specificity and sensitivity of mRT-RPA-LFB

[0068] The specificity of the mRT-RPA-LFB assay was evaluated by detecting other templates such as PPRV, BRV, BTV, BPIV3, BEV, and BToV. In the specificity experiment, the concentration of all virus extracts was 106 copies / μL.

[0069] The sensitivity of mRT-RPA-LFB was evaluated based on the limit of detection (LOD). The same concentration and volume of BVDV-1, BEFV, and BRSV pseudovirus RNA were used as templates for the mRT-RPA-LFB assay, with concentrations ranging from 107 to 100 copies / μL. A negative control group was set up using ddH2O. Each LFB was scanned three times.

[0070] Specificity and sensitivity of mRT-RPA-LFB

[0071] The specificity of the mRT-RPA-LFB assay was 100%, and all positive controls, including BVDV 1, BEFV, and BRSV, showed positive results. Accordingly, all samples lacking the three target templates (the other six viruses) gave negative results ( Figure 5 a). Color development was observed on the test line only when the specific template was present. This indicates that there was no cross-reaction in the mRT-RPA-LFB assay. The detection products were detected on a 1% agarose gel, and the results were consistent with those of the LFB ( Figure 3 ).

[0072] To evaluate the sensitivity of the mRT-RPA-LFB assay, serial dilutions of template pseudoviruses (BVDV-1, BEFV, and BRSV) were used. As Figure 5 shown in 1 b, the LoD values of BVDV-1, BEFV, and BRSV determined by the mRT-RPA-LFB method were 2.62×10 1 copies / μL, 2.42×10 1 copies / μL, and 2.42×10 Figure 5 copies / μL, respectively. A logarithmic curve of RNA concentration was plotted on the x-axis, and the corresponding T / C value of the test line was plotted on the y-axis to construct a standard curve. The R-squared values of the standard curve (0.9806 for BVDV-1, 0.9819 for BEFV, and 0.987 for BRSV) verified the reliability of the detection method ( Figure 5 b). As Figure 4 shown in

[0073] b, the assay detected and identified three target genes in a single-tube reaction. The results of the mRT-RPA-LFB assay were consistent with those on a 1% agarose gel ( Figure 4 ). However, traditional visual monitoring methods (such as agarose gel electrophoresis) could not distinguish the target genes in multiplex assays. )

[0073] 2.2 Repeatability evaluation of mRT-RPA-LFB

[0074] Repeatability is a key factor in evaluating the performance of sensor data. In within-batch experiments, five sets of test strips from the same production batch were selected. The pseudovirus RNA template (10 6 copies / μL) was used as the standardized template for the amplification experiment. In between-batch experiments, test strips from five different batches were used, and the same template was used under the same conditions as in the within-batch experiment. Each sample was tested three times, and the coefficient of variation (CV) was calculated to evaluate repeatability.

[0075] Repeatability of mRT-RPA-LFB

[0076] The CV values of mRT-RPA-LFB for within-assay and between-assay detections were less than 3.99% and 3.44%, respectively. These results indicate the stability and reproducibility of the mRT-RPA-LFB detection method ( Figure 6 ).

[0077] Real-time RT-PCR analysis based on fluorescent probes:

[0078] RT-qPCR for detecting three viruses (BVDV-1, BEFV, and BRSV) was performed using three commercial kits purchased from Shanghai Xuanke Biotechnology Co., Ltd. According to the manufacturer's instructions, 5 μL of the sample was used for amplification in a reaction mixture with a total volume of 25 μL. RT-qPCR was performed on a QuantStudio TM 5 Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA). The RT-qPCR cycling parameters were set as follows: 50 °C for 10 minutes, 95 °C for 2 minutes, followed by 45 cycles, each cycle consisting of 95 °C for 15 seconds and 60 °C for 30 seconds.

[0079] Field sample detection:

[0080] During the period from July 2022 to June 2023, 292 field samples were collected. These data sets included 277 bovine nasal swab samples collected in cooperation with a farm in Hebei Province. In addition, 14 nasal swab samples positive for BEFV provided by the Zhejiang Academy of Science and Technology Inspection and Quarantine, and a nasal swab sample with a mixed infection of BVDV-1, BEFV, and BRSV. The RNA of all samples was extracted using the FastPure Viral DNA / RNA Mini Kit produced by Novoprotein Scientific Inc. (Nanjing, China). To evaluate the performance of mRT-RPA-LFB detection, these samples were analyzed and the results were compared with the RT-qPCR results.

[0081] Data processing

[0082] Microsoft Excel 2021 and PowerPoint 2021 (Microsoft Corporation, Redmond, WA, USA) were used for data analysis and the production of schematic diagrams. SPSS statistical software (IBM SPSS Inc., Chicago, USA) and Origin 2021 (Origin Lab, Northampton, USA) were used to analyze and process experimental data.

[0083] The reliability of mRT-RPA-LFB was evaluated using 292 actual samples for the detection of actual samples (Table 2).

[0084] Table 2 Information on 292 clinical samples

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091] The results showed good consistency between the two detection methods ( Figure 7 a). One sample showed a false negative result in the BVDV-1 detection ( Figure 7 b). There were 52 true positives (TP), 239 true negatives (TN), 0 false positives (FP), and 1 false negative (FN) ( Figure 7 c). The sensitivity of mRT-RPA-LFB was 98.11%, the specificity and positive predictive value (PPV) were both 100%, the negative predictive value (NPV) was 99.58%, and the kappa value was 0.988 (Table 3). Linear correlation analysis was performed on samples positive for BVDV-1, BEFV, and BRSV detection. The results showed a very high consistency between mRT-RPA-LFB and RT-qPCR, with R 2 being 0.9822 ( Figure 7 c).

[0092] Table 3 Comparison between mRT-RPA-LFB detection method and RT-qPCR detection method

[0093]

[0094] Note: "Kappa" represents the Kappa coefficient in statistical analysis. "+" indicates positive; "-" indicates negative. "PPV" represents the positive predictive value; "NPV" represents the negative predictive value.

[0095] The mRT-RPA-LFB detection method developed in this study is expected to become an effective and accurate tool for rapid on-site diagnosis. Its operation procedure is simple and clear, and even people without laboratory expertise can quickly perform BVDV-1, BEFV, and BRSV detections, thus promoting rapid and synchronous on-site diagnosis. This detection method combines RPA, reverse transcription, and multiplex detection, uses a nanoparticle-based biosensor, and can diagnose three bovine viral diseases with just one-step single-tube reaction. Compared with the original single-tube detection method, the mRT-RPA-LFB detection method requires at least two-thirds less reagent, while doubling the detection targets. The method constructed in this invention undergoes reaction amplification for 20 minutes under isothermal conditions at 41 °C, and results can be obtained within 2 minutes using LFB. The mRT-RPA-LFB detection method designed in this invention can simultaneously identify three virus targets, and its sensitivity thresholds are 2.62x 10 1 copies / μL, 2.42x 10 1 copies / μL, and 2.56x 10 1 copies / μL. The sensitivity of mRT-RPA-LFB is similar to the research results of previously published studies using RPA to detect RNA. Compared with the previously developed single RPA-LFB and dual RPA-LFB detection methods, the ability of this detection method has been improved, and it can simultaneously detect three pathogens (Table 4).

[0096] Table 4 Comparison of published BVDV, BEFV, and BRSV detection methods.

[0097]

[0098]

[0099] Compared with the previously developed single RPA-LFB and dual RPA-LFB methods for detecting BVDV, BEFV, and BRSV, RPA-mediated identification still relies on the initial RNA amplification step through reverse transcription. RNA is easily degraded by ubiquitous RNases, which may lead to false-negative results in on-site detections. In contrast, mRT-RPA-LFB can directly amplify and detect RNA, thereby reducing the possibility of sample contamination and the risk of virus transmission. The RPA-lateral flow assay can simultaneously detect three Plasmodium anasum, and the detection sensitivity ranges from 8.99×10 3 to 4.58×10 3The number of copies / μl varies, and the detection time is 60-70 minutes. In contrast, mRT-RPA-LFB is more sensitive and faster, with the entire process from sampling to displaying results taking only 30 minutes. In addition, when used with a test strip reader, the mRT-RPA-LFB assay can digitize visual signals, thereby reducing potential subjective errors. Traditional PCR and its derivatives have been used to detect BVDV, BRSV, and BEFV, as well as the combined detection of BVDV and BRSV. Although these detection techniques are highly sensitive, with a minimum detection limit as low as 1 copy, and can identify more pathogens than the three pathogens targeted by this assay (up to 19 pathogens can be detected simultaneously), PCR technology relies on thermal cycling or advanced precision equipment (Table 2). The mRT-RPA-LFB assay can be operated using a simple thermostat, such as the MiNiT-100H (ALSHENG, Hangzhou, China), which weighs only 0.9 kg and is easy to carry. Detection methods combined with a single LAMP have been used to detect viruses in animals, such as BVDV and BEFV, with sensitivities of 2.97×101 copies / μL and 20 copies / test tube, respectively (Table 2). In addition, a LAMP detection method can simultaneously identify PEDV, PoRV, and PBoV, with detection limits of 2.40×101, 2.89×10 1 and 2.62×10 1 However, due to the complex primer system of LAMP technology, the need for high temperature and the high probability of false positives, it may not be the best choice for regular testing in places with limited resources. The entire mRT-RPA-LFB process, from sample collection to the precise identification of species-specific viruses after collection, can be completed within 30 minutes ( Figure 1 ). In summary, the mRT-RPA-LFB test is rapid, sensitive, and portable. It provides quantitative detection and can distinguish BVDV-1, BEFV, and BRSV without the need for specialized personnel, making it ideal for use in field settings.

[0100] Comparison of mRT-RPA-LFB and RT-qPCR in actual samples is crucial for evaluating the constructed detection method. This study conducted a retrospective analysis of field samples. Among 292 samples, the mRT-RPA-LFB detection method detected one sample co-infected with all three viruses, two samples co-infected with BVDV-1 and BRSV, and 49 samples infected with one virus (BVDV-1, BEFV, or BRSV). However, there was a difference in the detection results of one sample: the mRT-RPA-LFB detection result was negative, while the RT-qPCR detection result was positive (Table 1). The result of detecting BVDV-1 using the mRT-RPA-LFB method was not found in sample No. 97. This might be due to uneven sample distribution, sample selection bias, or nucleic acid degradation. The fact that the two detection methods were not applied simultaneously to all samples might have affected this result. The collection of virus samples presented challenges, hindering the feasibility of repeating experiments on all samples. However, the kappa value of the detection results of 292 samples was 0.988, and the specificity of mRT-RPA-LFB and RT-qPCR was 100%, which proved a strong correlation between the two methods. Therefore, mRT-RPA-LFB was considered reliable (Table 1). The repeatability assessment demonstrated the accuracy and stability of mRT-RPA-LFB.

[0101] The mRT-RPA-LFB detection method has been successfully developed for detecting BVDV-1, BEFV, and BRSV infections. This detection method can quickly perform visual quantification and convert the signals on the LFB into quantitative data using a test strip reader to report the virus concentrations of BVDV-1, BEFV, and BRSV. The LODs of BVDV-1, BEFV, and BRSV obtained by the test strip reader were 2.62×10 1 、2.42×10 1 and 2.56×10 1 copies / μL, respectively. The analysis of sensitivity and specificity data indicated that the mRT-RPA-LFB detection method was a sensitive, reliable, and feasible technique for diagnosing BVDV-1, BEFV, and BRSV infections. This detection only requires a simple instrument to maintain a constant temperature (41°C) for 20 minutes. Given its high efficiency, simplicity, and reliability, the mRT-RPA-LFB platform has the potential to become a point-of-care diagnostic tool in animal transfer stations, breeding farms, and various field environments.

[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. Any technical solutions obtained by using equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens of bovine BVDV, BEFV, and BRSV, characterized in that, Including: Three sets of RT-RPA primer-probes and a lateral flow biosensor LFB; The lateral flow biosensor LFB includes: a sample pad, a conjugate pad, an absorbent pad, a back plate, a T1 test line coated with digoxin monoclonal antibody, a T2 test line coated with ROX monoclonal antibody, a T3 test line coated with anti-FAM antibody, an LFB test line disposed on the sample pad, an NC membrane, a C line coated with goat anti-mouse IgG, and biotin-labeled colloidal gold particles; The RT-RPA primer-probes include: a BVDV-1 virus primer set designed for the 5'UTR region of the BVDV-1 virus (GenBank: M31182.1), a BEFV virus primer set designed for the G gene of the BEFV virus (GenBank: AF058321.2), and a BRSV virus primer set designed for the N gene of the BRSV virus (GenBank: NC_001989.1). The upstream primer of the BVDV-1 virus primer set is labeled with biotin, and the downstream primer is labeled with digoxin (Dig); the upstream primer of the BEFV virus primer set is labeled with biotin, and the downstream primer is labeled with ROX; the upstream primer of the BRSV virus primer set is labeled with biotin, and the downstream primer is labeled with FAM.

2. The mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens of bovine BVDV, BEFV, and BRSV according to claim 1, wherein The C line is coated with goat anti-mouse IgG at a concentration of 2 mg / mL, the T1 test line is coated with digoxin monoclonal antibody at a concentration of 0.75 mg / mL, the T2 test line is coated with ROX monoclonal antibody at a concentration of 0.6 mg / mL, and the T3 test line contains FAM monoclonal antibody at a concentration of 0.5 mg / mL.

3. The mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens of bovine BVDV, BEFV, and BRSV according to claim 1, characterized in that, The RT-RPA primer-probes are as shown in SEQ ID No: 01 - SEQ ID No: 06 in Table 1.

4. The mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens of bovine BVDV, BEFV, and BRSV according to claim 1, characterized in that, mRT-RPA uses the TwistAmp Basic RPA kit. In addition to the substances provided in the RPA lyophilized tube, the following components are added sequentially: 25 μL of buffer, 30 μM of RT-RPA primer-probe mixture, 1 μL of reverse transcriptase, 3 μL of template, 280 mM of magnesium acetate, and the concentration of Mg 2+ is 2.8 to 16.8 nM. The total volume is adjusted to 50 μL with ddH2O. The amplification time is 5 - 25 minutes. The final concentration of the primer set is 150 - 500 nM, and the reaction temperature is 38 - 43 °C.

5. The mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens of bovine BVDV, BEFV, and BRSV according to claim 4, wherein, The final concentrations of the BVDV-1 and BRSV primer sets are 450 nM, and the final concentration of the BEFV primer set is 250 nM.

6. The mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens of bovine BVDV, BEFV, and BRSV according to claim 4, wherein, The reaction temperature is 42 °C.

7. The mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens of bovine BVDV, BEFV, and BRSV according to claim 4, wherein The amplification time is 20 minutes.

8. The mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens of bovine BVDV, BEFV, and BRSV according to claim 4, wherein Mg 2+ The concentration of 9. The detection method of the mRT-RPA-LFB detection kit for simultaneously detecting three viral pathogens of bovine BVDV, BEFV, and BRSV as described in claim 1, wherein Including the following steps: a) Viral RNA extraction Using a kit to extract bovine viral diarrhea virus type 1 (BVDV-1), bovine ephemeral fever virus (BEFV), bovine respiratory syncytial virus (BRSV), peste des petits ruminants virus (PPRV), bovine rotavirus (BRV), bluetongue virus (BTV), bovine parainfluenza virus type 3 (BPIV 3), bovine enterovirus (BEV), and bovine torovirus (BToV); b) Pseudovirus preparation and RNA quantification Extracting pseudoviruses BVDV-1, BEFV, and BRSV, using a kit to extract, quantify, and reverse-transcribe the pseudovirus RNA, and then using a Nano-100 differential spectrophotometer to evaluate the concentration and purity of the cDNA; c) Primer design Designing three sets of RT-RPA primers for the 5'UTR region of BVDV-1 (GenBank: M31182.1), the G gene of BEFV (GenBank: AF058321.2), and the N gene of BRSV (GenBank: NC_001989.1); c) Preparation of LFB The lateral flow biosensor LFB consists of five main parts Composition: sample pad, conjugate pad, NC membrane, absorbent pad and backplate; the conjugate pad is uniformly coated with colloidal gold and conjugated with monoclonal antibodies specific to the target analyte; the C line is coated with goat anti-mouse IgG at a concentration of 2 mg / mL, the T1 test line uses digoxin monoclonal antibody at a concentration of 0.75 mg / mL, the T2 test line is sprayed with ROX monoclonal antibody at a concentration of 0.6 mg / mL, and the T3 test line contains FAM monoclonal antibody at a concentration of 0.5 mg / mL; d) mRT-RPA-LFB reaction.

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