Visualized multiplex rt-lamp detection method and primer for foot-and-mouth disease, vesicular stomatitis and blue tongue disease

Abstract

The application discloses a visual multiple RT-LAMP detection method and primers for foot-and-mouth disease, vesicular stomatitis and blue tongue disease. A plurality of primers are designed, and specific primer combinations are optimized and screened. A fluorescent-quenching composite probe is added in the LAMP method for the first time, different fluorescent groups are marked at the 3' end of the probe, and finally, the multiple RT-LAMP detection method for differentially diagnosing FMDV, VSV and BTV in the same reaction tube is successfully established. The method has good specificity, high sensitivity, and can detect 1 copies / reaction viral RNA at the minimum, and the reaction time is short, and the detection can be completed in only 65 minutes. The result can be directly read by observing the color of the reaction product after the reaction, and the method can be used for clinical differential diagnosis and epidemiological investigation, and can be used for on-site quarantine in primary areas with poor conditions.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a visualization method and primers for multiplex RT-LAMP detection of foot-and-mouth disease, vesicular stomatitis and bluetongue. Background Technology

[0002] Foot-and-mouth disease (FMD), vesicular stomatitis (VSD), and bluetongue disease are three common, highly acute viral infectious diseases in cattle, prevalent globally. Outbreaks of these diseases cause significant economic losses to the cattle industry and are listed as notifiable animal diseases by the World Organisation for Animal Health (OIE). They are also important quarantine targets in my country's international trade in animals and animal products. FMD is an acute, highly contagious infectious disease of cloven-hoofed animals caused by the foot-and-mouth disease virus (FMDV). It is characterized by its wide spread, rapid onset, and high severity, primarily infecting over 70 species of domestic and wild mammals belonging to 20 families, including cattle, pigs, sheep, and camels. Vesicular stomatitis (VSV) is a highly contagious infectious disease of various mammals caused by the vesicular stomatitis virus (VSV). It can naturally infect cattle, pigs, horses, and other mammals. Insects and plants are also potential reservoirs of VSV, and arthropod vectors can transmit VSV. Bluetongue disease is a non-contact infectious disease of ruminants caused by the bluetongue virus (BTV). It can be transmitted to hosts through insects such as Culicoides midges, and mainly infects sheep, cattle and wild ruminants.

[0003] Animals infected with all three viruses exhibit symptoms such as fever, salivation, and blisters and erosions on the oral mucosa, teat skin, and hoof coronary skin. These infections are frequently mixed and difficult to distinguish. Once an FMDV outbreak occurs, the only recourse is slaughter and mass incineration. VSV is a mandatory imported disease in my country, while BTV is generally a latent infection in cattle and is widespread in many provinces, constantly threatening the healthy development of the livestock industry.

[0004] As my country's cattle industry gradually shifts towards large-scale and intensive operations, the trade in live animals and frozen semen both domestically and internationally is becoming increasingly frequent, making the prevention and control of various diseases increasingly difficult. In recent years, foot-and-mouth disease (FMD) has occurred repeatedly in my country, with complex and diverse outbreaks. The cross-border transmission of FMD strains from neighboring countries further complicates and increases the difficulty of FMD prevention and control in my country. Bluetongue disease has been reported to be prevalent in several provinces of my country. Although vesicular stomatitis, as an introduced disease, has not yet been found in my country, it continues to threaten the development of my country's livestock industry. Therefore, it is necessary to establish detection methods that can simultaneously and rapidly identify and diagnose these three diseases, conduct early screening of infected animals, and prevent the outbreak of animal diseases.

[0005] Currently, many researchers have established multiplex RT-PCR and fluorescent RT-PCR methods for different combinations of these three diseases, with varying sensitivities and specificities. However, the detection process still requires laboratory testing, which cannot meet the current demand for rapid field diagnosis of infectious diseases. Laboratory diagnosis of these three diseases currently mainly involves virus isolation, serological diagnosis, and biological methods. Serological diagnosis includes serum neutralization tests, complement fixation tests, agar diffusion tests, and ELISA. Biological methods mainly include RT-PCR and quantitative RT-PCR. However, both RT-PCR and fluorescent RT-PCR have their own limitations, such as lower sensitivity for RT-PCR and higher cost for fluorescent RT-PCR, requiring expensive equipment.

[0006] Loop-mediated isothermal amplification (LAMP), an emerging nucleic acid detection technology developed from PCR, has overcome the technical difficulties of isothermal amplification. It achieves high efficiency with six primers simultaneously, exhibiting high sensitivity and specificity, and has been applied to the detection of various diseases. However, due to limitations in its result interpretation methods (turbidity, color, dye addition), multiplex LAMP methods have not made significant progress. Although several researchers both domestically and internationally have established multiplex LAMP detection methods, these methods all have certain shortcomings: they can only detect whether a sample carries a pathogen, but cannot determine the specific pathogen causing the positive reaction, thus not providing true differential diagnosis. Summary of the Invention

[0007] One object of the present invention is to provide a complete set of reagents.

[0008] The complete set of reagents provided by this invention is as follows: 1) or 2):

[0009] The reagent shown in 1) consists of primer probe set I, primer probe set II, and primer probe set III;

[0010] The primer-probe set I consists of primer FMDV-F3, primer FMDV-B3, primer FMDV-FIP, primer FMDV-BIP, primer FMDV-Floop, primer FMDV-Bloop, probe FMDV-FD, and probe FMDV-BD;

[0011] The primer-probe set II consists of primer VSV-F3, primer VSV-B3, primer VSV-FIP, primer VSV-BIP, primer VSV-Floop, primer VSV-Bloop, probe VSV-FD, and probe VSV-BD.

[0012] The primer-probe set III consists of primer BTV-F3, primer BTV-B3, primer BTV-FIP, primer BTV-BIP, primer BTV-Floop, primer BTV-Bloop, probe BTV-FD, and probe BTV-BD.

[0013] 2) The reagent shown consists of primer probe set A, primer probe set B, and primer probe set C;

[0014] The primer-probe set A consists of primer FMDV-FIP, primer FMDV-BIP, primer FMDV-F3, primer FMDV-B3, complex FMDV FIP-FD, complex FMDV BIP-BD, primer FMDV-Floop, and primer FMDV-Bloop;

[0015] The primer-probe set B consists of primer VSV-FIP, primer VSV-BIP, primer VSV-F3, primer VSV-B3, complex VSV-FIP-FD, complex VSV-BIP-BD, VSV-Floop, and VSV-Bloop;

[0016] The primer-probe set C consists of primer BTV-FIP, primer BTV-BIP, primer BTV-F3, primer BTV-B3, complex BTV FIP-FD, complex BTV BIP-BD, primer BTV-Floop, and primer BTV-Bloop.

[0017] The complex FMDV FIP-FD was obtained by annealing the primer FMDV-FIP and the probe FMDV-FD, which is complementary to its F1C segment;

[0018] The complex FMDV BIP-BD was obtained by annealing the primer FMDV-BIP and the probe FMDV-BD, which is complementary to its B1C segment;

[0019] The complex VSV FIP-FD was obtained by annealing the primer VSV-FIP and the probe VSV-FD, which is complementary to its F1C segment;

[0020] The complex VSV BIP-BD was obtained by annealing the primer VSV-BIP and the probe VSV-BD, which is complementary to its B1C segment;

[0021] The complex BTV FIP-FD was obtained by annealing the primer BTV-FIP and the probe BTV-FD, which is complementary to its F1C segment;

[0022] The complex BTV BIP-BD was obtained by annealing the primer BTV-BIP and the probe BTV-BD, which is complementary to its B1C segment.

[0023] The nucleotide sequences of primers FMDV-F3, FMDV-B3, FMDV-FIP, FMDV-BIP, FMDV-Floop, FMDV-Bloop, probe FMDV-FD, probe FMDV-BD, primer VSV-F3, primer VSV-B3, primer VSV–FIP, primer VSV-BIP, VSV-Floop, VSV-Bloop, probe VSV-FD, probe VSV-BD, primer BTV-F3, primer BTV-B3, primer BTV–FIP, primer BTV-BIP, primer BTV-Floop, primer BTV-Bloop, probe BTV-FD, and probe BTV-BD are sequences 1 to 24, respectively, or sequences that are functionally identical to the original sequences obtained by substituting and / or deleting and / or adding one or more nucleotides from at least one of sequences 1 to 24.

[0024] The terminal quenching groups of the FMDV-FIP, FMDV-BIP, VSV-FIP, VSV-BIP, BTV-FIP, and BTV-BIP;

[0025] The FMDV-FD, FMDV-BD, VSV-FD, VSV-BD, BTV-FD, and BTV-BD are all labeled with fluorescent groups at their ends, and different pathogens are labeled with fluorescent groups of different colors. The FD probe and BD probe of the same pathogen are labeled with the same fluorescent group.

[0026] Each primer, probe, or complex in the above-mentioned reagent kit is individually packaged.

[0027] Another objective of this invention is to provide a loop-mediated isothermal amplification reagent.

[0028] The loop-mediated isothermal amplification reagent provided by the present invention contains the reagent shown in 2) above.

[0029] The above loop-mediated isothermal amplification reagents include the following complexes: FMDV FIP-FD, FMDV BIP-BD, VSV FIP-FD, VSV BIP-BD, BTV FIP-FD, BTV BIP-BD, primers FMDV-F3, FMDV-B3, VSV-F3, VSV-B3, BTV-F3, BTV-B3, FMDV-Floop, FMDV-Bloop, VSV-Floop, VSV-Bloop, BTV-Floop, BTV-Bloop, FMDVFIP, BTV FIP, VSV FIP, FMDV BIP, BTV BIP, and VSV. The molar ratio of BIP in the reagent is 8:8:8:8:8:8:1:1:1:1:1:1:1:2:2:2:2:2:2:8:8:8:8:8:8.

[0030] The above loop-mediated isothermal amplification reagents include the following complexes: FMDV FIP-FD, FMDV BIP-BD, VSV FIP-FD, VSV BIP-BD, BTV FIP-FD, BTV BIP-BD, primers FMDV-F3, FMDV-B3, VSV-F3, VSV-B3, BTV-F3, BTV-B3, FMDV-Floop, FMDV-Bloop, VSV-Floop, VSV-Bloop, BTV-Floop, BTV-Bloop, FMDVFIP, BTV FIP, VSV FIP, FMDV BIP, BTV BIP, and VSV. The concentrations of BIP in the reagent were 0.8 μM, 0.8 μM, 0.8 μM, 0.8 μM, 0.8 μM, 0.8 μM, 0.1 μM, 0.1 μM, 0.1 μM, 0.1 μM, 0.1 μM, 0.1 μM, 0.2 μM, 0.2 μM, 0.2 μM, 0.2 μM, 0.2 μM, 0.2 μM, 0.8 μM, 0.8 μM, 0.8 μM, 0.8 μM, 0.8 μM, 0.8 μM, and 0.8 μM, respectively.

[0031] The reagents mentioned above also include dUTP, thermosensitive UDG, a visual pH indicator, DNA polymerase, and reverse transcriptase.

[0032] The application of the above-mentioned complete set of reagents or the above-mentioned loop-mediated isothermal amplification reagents in the preparation of the kit is also within the scope of protection of this invention; the uses of the kit are as follows (d1) or (d2) or (d3):

[0033] (d1) To identify or assist in the identification of foot-and-mouth disease virus, vesicular stomatitis virus, or bluetongue virus;

[0034] (d2) Identify or assist in identifying whether the virus to be tested is foot-and-mouth disease virus, vesicular stomatitis virus and / or bluetongue virus;

[0035] (d3) Identify or assist in identifying whether the sample to be tested is infected with foot-and-mouth disease virus, vesicular stomatitis virus and / or bluetongue virus.

[0036] Kits containing the above-mentioned complete set of reagents or the above-mentioned loop-mediated isothermal amplification reagents are also within the scope of protection of this invention; the uses of the kits are as follows (d1) or (d2) or (d3):

[0037] (d1) To identify or assist in the identification of foot-and-mouth disease virus, vesicular stomatitis virus, or bluetongue virus;

[0038] (d2) Identify or assist in identifying whether the virus to be tested is foot-and-mouth disease virus, vesicular stomatitis virus and / or bluetongue virus;

[0039] (d3) Identify or assist in identifying whether the sample to be tested is infected with foot-and-mouth disease virus, vesicular stomatitis virus and / or bluetongue virus.

[0040] The preparation method of the above-mentioned reagent kit is also within the scope of protection of this invention. The method includes the step of individually packaging each substance in the above-mentioned kit.

[0041] Another objective of this invention is to provide a method for identifying or assisting in the identification of a virus to be tested as foot-and-mouth disease virus, vesicular stomatitis virus, or bluetongue virus.

[0042] This invention provides a method comprising the following steps:

[0043] (1) Extract total RNA from the virus to be tested;

[0044] (2) Using the total RNA obtained in step (1) as a template, loop-mediated isothermal amplification was performed using the above-mentioned loop-mediated isothermal amplification reagent, and the loop-mediated isothermal amplification products were detected.

[0045] If the fluorescent group labeled with FMDV-FD or FMDV-BD shows a color in the loop-mediated isothermal amplification product, then the virus to be tested is or is a candidate for foot-and-mouth disease virus; otherwise, it is not.

[0046] If the fluorescent group labeled with VSV-FD or VSV-BD shows a color in the loop-mediated isothermal amplification product, then the virus to be tested is or is a candidate for vesicular stomatitis virus; otherwise, it is not.

[0047] If the loop-mediated isothermal amplification product shows the color of the fluorescent group labeled BTV-FD or BTV-BD, then the virus to be tested is or is a candidate for bluetongue virus; otherwise, it is not.

[0048] If the loop-mediated isothermal amplification product shows a mixed color of any two combinations of fluorescent groups of the above probes, then the virus to be tested is or is a candidate for a mixture of viruses corresponding to the fluorescent group-labeled probes; otherwise, it is not.

[0049] If the loop-mediated isothermal amplification product shows a mixed color of the fluorescent groups of the three combinations of the above probes, then the virus to be tested is or is a candidate for a mixture of the three viruses; otherwise, it is not.

[0050] This invention provides a method for identifying or assisting in the identification of whether a sample is infected with foot-and-mouth disease virus, vesicular stomatitis virus, and / or bluetongue virus, comprising the following steps:

[0051] (1) Extract total RNA from the virus to be tested;

[0052] (2) Using the total RNA obtained in step (1) as a template, loop-mediated isothermal amplification was performed using the above-mentioned loop-mediated isothermal amplification reagent, and the loop-mediated isothermal amplification products were detected.

[0053] If the fluorescent group labeled with FMDV-FD or FMDV-BD shows a color in the loop-mediated isothermal amplification product, then the sample to be tested is infected or a candidate for infection with foot-and-mouth disease virus; otherwise, it is not.

[0054] If the fluorescent group labeled with VSV-FD or VSV-BD shows a color in the loop-mediated isothermal amplification product, then the sample to be tested is infected or a candidate for infection with foot-and-mouth disease virus (FMDV) or vesicular stomatitis virus; otherwise, it is not.

[0055] If the loop-mediated isothermal amplification product shows the color of the fluorescent group labeled BTV-FD or BTV-BD, then the sample to be tested is infected or a candidate for infection with foot-and-mouth disease virus or bluetongue virus; otherwise, it is not.

[0056] If the loop-mediated isothermal amplification product shows a mixed color of any two combinations of fluorescent groups of the above probes, then the sample to be tested is infected or the candidate infected with the virus corresponding to the fluorescent group labeled probe; otherwise, it is not.

[0057] If the loop-mediated isothermal amplification product shows a mixed color of the fluorescent groups of the three combinations of the above probes, then the sample to be tested is infected or a candidate for infection with a mixture of the three viruses; otherwise, it is not.

[0058] The application of the above-mentioned complete set of reagents, the above-mentioned loop-mediated isothermal amplification reagents, or the above-mentioned kits in any of the following is also within the scope of protection of this invention:

[0059] 1) To identify or assist in the identification of foot-and-mouth disease virus, vesicular stomatitis virus, or bluetongue virus;

[0060] 2) To identify or assist in identifying whether the virus to be tested is foot-and-mouth disease virus, vesicular stomatitis virus, or bluetongue virus;

[0061] 3) To identify or assist in identifying whether the sample to be tested is infected with foot-and-mouth disease virus, vesicular stomatitis virus and / or bluetongue virus.

[0062] This invention employs a novel technical approach: complementary probes FD and BD are added to both the inner primers FIP and BIP. The FD probe is complementary to the F1C fragment on the FIP. The 3' end of the FD probe is labeled with a fluorescent group (FD-F), and the 5' end of the FIP inner primer is labeled with a quencher group (FIP-Q). Before the reaction, the FIP and FD are annealed to form a fluorescence-quencher composite probe (FIP-Q / FD-F). Because the F1C positions of FD and FIP are complementary, the fluorescent group and the quencher group are close together, resulting in quenching of fluorescence. During amplification, the FIP retains its original inner primer function and continues amplification. During reverse amplification guided by BIP, the FD is separated from the FIP-Q / FD-F composite probe by the newly synthesized strand, thus releasing fluorescence. Similarly, the BD probe is complementary to the B1C fragment on the BIP. The 3' end of the BD probe is labeled with a fluorescent group (BD-F), and the 5' end of the inner primer BIP is labeled with a quencher group (BIP-Q). Before the reaction, the BIP and BD are annealed to form a fluorescence-quencher composite probe (BIP-Q / BD-F). Because the B1C positions of BD and BIP are complementary, the fluorescent group and the quencher group are close to each other, and the fluorescence is quenched and no light is emitted. During amplification, the BIP retains the function of the original inner primer and continues amplification. During reverse amplification guided by FIP, the BD is separated from the BIP-Q / BD-F composite probe by the newly synthesized strand, thereby releasing fluorescence. LAMP amplification efficiency is very high, and a large amount of DNA can be rapidly synthesized in a short time. Therefore, the reaction product contains a large amount of free FD-fluorescence and BD-fluorescence, which eventually reaches a level visible to the naked eye. The simultaneous dual fluorescent labeling of FIP and BIP results in a higher fluorescence increment after the reaction, which more effectively suppresses the generation of false positives and makes the distinction more obvious. Multiple identification detection can be performed based on the different fluorescent colors labeled by the probes. After the reaction is completed, the reaction results can be read directly by observing the color of the reaction tube. No electrophoresis or dye addition is required, which is convenient, fast, time-saving and labor-saving.

[0063] This method has the following advantages: 1. It inherits the advantages of LAMP: simplicity, speed, sensitivity, and low cost. 2. It does not require separate probe design. The probe sequence is complementary to the inner primer, and the 3' end is labeled with a fluorescent group. It is annealed with the inner primer before the reaction and directly added to the system for use. 3. Better specificity: Chain elongation during amplification leads to the release of the fluorescent group, which consumes reaction energy and can suppress the generation of false positives. Compared with ordinary PCR, LAMP primers are multiplied, with dozens of primers in the multiplex system. The primers are intertwined, which may lead to non-specific amplification. In this invention, non-specific amplification turbidity curves can occasionally be detected on a real-time turbidimeter, but after the reaction, the fluorescent group is not released under the multicolor fluorescence imaging analysis system, so it cannot emit light. This shows that the multiplex RT-LAMP method has good specificity. 4. It reduces nucleic acid contamination in the laboratory. The results can be directly interpreted by observing the color of the reaction tube after the reaction, without the need for electrophoresis or opening the cap to add dye, which greatly reduces subsequent aerosol contamination in LAMP. 5. Accurate Results: This invention uses three different fluorescent groups: ALEXA FLUOR 488, CY5, and CY3. These three fluorescent groups have different excitation and absorption wavelengths, thus exhibiting different colors: ALEXA FLUOR 488 absorbs at 520 nm, appearing yellow-green; CY5 absorbs at 664 nm, appearing bright red; and CY3 absorbs at 570 nm, appearing blue (CY3's original color is orange, but the imaging system imparts blue, making the results clearer). Furthermore, each fluorescent group can only be observed in specific channels; specifically, only ALEXA FLUOR 488 can be observed in channel 520, while CY5 cannot. Multiple positive results can be simultaneously observed in 2-3 corresponding channels, presenting a mixed color. Compared to precipitate observation, electrophoresis, and dye addition methods are more accurate, representing the first truly meaningful multiplex LAMP identification detection.

[0064] New England Biolabs' WarmStart 2× premix contains UDG and dye, as well as Bst 2.0 WarmStart DNA polymerase and WarmStart RT× reverse transcriptase. Both Bst 2.0 WarmStart DNA polymerase and WarmStart RT× reverse transcriptase are genetically engineered for higher amplification efficiency and performance in LAMP reactions and are resistant to room temperature. The dUTP and thermosensitive UDG in the premix effectively reduce the risk of residual contamination, eliminating substrate contamination caused by aerosols generated during previous amplification. The thermosensitive UDG is completely inactivated at temperatures above 50°C, thus having no impact on the reaction. The premix contains a visual pH indicator. During the LAMP reaction, the DNA polymerase polymerizes, changing the number of protons and thus altering the pH, turning the pink reaction solution yellow. The color change is clear and visible to the naked eye: positive results turn yellow, while negative results remain pink. This invention aims to identify and diagnose three acute infectious diseases in cattle. If clinical use only requires testing positive or unqualified samples and does not require differentiation of these three viruses, this premixed solution can be used for on-site quarantine. The results can be judged by visually observing the color change of the product after the reaction.

[0065] The method of this invention achieves a breakthrough in overcoming the greatest technical difficulties in the dual and triple layers, as detailed below:

[0066] 1. The search for a third type of fluorescence channel:

[0067] The multicolor fluorescence imaging analysis system, purchased from BIO-RAD (Universal HoodⅢ catalog number), has a detection wavelength of 495-750 nm, while the wavelength range of blue fluorescence is 422-455 nm. This means that existing instruments cannot detect blue fluorescence. The inventors compared four fluorescent groups (ROX, Alexa Flour 586, CY3, and Texas Red) and found that only CY3 fluorescence could be detected in channel 570, with no overlap with the other 520 nm and 670 nm channels. However, the original color of CY3 fluorescence is orange, not blue, and orange and green are difficult to distinguish with the naked eye. The inventors programmed CY3 to be displayed as blue in the imaging system, thus finding a third type of blue fluorescence that could be used for triplet reactions.

[0068] 2. The fluorescence-quenching composite probe inhibits the reaction; this inhibition can be eliminated by adding a certain concentration of internal primers.

[0069] The inventors discovered that when the fluorescence quenching composite probe FIP-FD (or BIP-BD) completely replaces the standard FIP (or BIP) primers, not only is the fluorescence background high, but it also has a certain relative inhibitory effect on amplification, or even no reaction. By combining FIP-FD (or BIP-BD) with a certain ratio of standard FIP (or BIP) primers, this inhibitory effect is significantly reduced.

[0070] This experiment optimized the ratio of the fluorescence quenching composite probe FIP-FD (or BIP-BD) to the standard inner primer in the triple fluorescence LAMP system. It was found that using an inner primer FIP (or BIP): fluorescence quenching composite probe FIP-FD (or BIP-BD) ratio of 1:1 can effectively reduce the inhibition effect and produce a clear fluorescence signal. At the same time, it can lower the fluorescence background value and ensure stable amplification, which is crucial for multiplex reaction detection.

[0071] 3. In a triple reaction system, the competition between the primer and probe leads to an imbalance in the entire reaction system, preventing the reaction from occurring.

[0072] Each reaction is enhanced by adding 5U Bst 2.0 WarmStart DNA polymerase and 5U WarmStart RT× reverse transcriptase, achieving a triple reaction equilibrium where each amplifies independently without interference. Without the enzymes, the entire reaction is inhibited and does not occur.

[0073] In summary, this invention designed multiple sets of primers, optimized and screened specific primer combinations, and for the first time incorporated a fluorescence-quenching composite probe into the LAMP method. The 3' end of the probe was labeled with fluorescent groups of different colors. Ultimately, a multiplex RT-LAMP detection method for differentiating and diagnosing FMDV, VSV, and BTV in the same reaction tube was successfully established. This method has good specificity and high sensitivity, detecting at least 1 copy of viral RNA per reaction. The reaction time is fast, and the entire detection process can be completed in just 65 minutes. After the reaction, the results can be directly read by observing the color of the reaction products. It can be used for clinical differential diagnosis and epidemiological investigation, and can be used for field quarantine in grassroots areas with poor conditions. Attached Figure Description

[0074] Figure 1 These are results specific to multiple RT-LAMP experiments.

[0075] Figure 2 This is the result of sensitivity to multiplex fluorescence RT-LAMP.

[0076] Figure 3 The results show interference from the multiple RT-LAMP detection method. Detailed Implementation

[0077] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0078] The following examples are provided to help better understand the present invention, but are not intended to limit the invention.

[0079] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0080] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0081] The following kits were purchased: WarmStart RT-LAMP nucleic acid amplification kit (Catalog No. #M1804S) from New England Biolab; Loopamp real-time turbidimeter (Catalog No. LA-320C) from Eiken Chemical (Japan); RNA / DNA extraction kit (Catalog No. ER201-01) from TransGen Biotech; plasmid mini-extraction kit (Catalog No. EM101-01) from TransGen Biotech; PCR SuperMix kit (Catalog No. AS122-11) from Beijing Quanshijin Biotech; pGM-T vector (Catalog No. VT202-01) from Tiangen Biotech; T7 in vitro transcription kit (Catalog No. BTN91106) from Beijing Bio-Rad Biotech; micro-nucleic acid analyzer (Catalog No. NanoDrop 2000) from Thermo Scientific (USA); and multicolor fluorescence imaging analysis system (Catalog No. Universal HoodⅢ) from Bio-Rad Biotech (USA).

[0082] The strains used in the examples are shown in Table 1 below:

[0083] Table 1 lists the various strains used in the examples.

[0084]

[0085]

[0086] Example 1: Primers and methods for LAMP detection of foot-and-mouth disease virus, vesicular stomatitis virus, and bluetongue virus.

[0087] I. Primer Design

[0088] Based on the conserved regions of the FMDV 3D gene, the VSV nucleoprotein N gene, and the BTV VP7 gene, sequence analysis was performed. Target sites were selected and primers were designed based on this sequence analysis, resulting in a primer library of thousands of primer pairs. Preliminary experiments were conducted on each primer pair in the library to test their sensitivity, specificity, and universality. The primers for LAMP detection were finally obtained, and a fluorescence-quenching composite probe was introduced into the inner primer FIP. During amplification, the composite probe separates and releases fluorescence. The 3' ends of probes FD and BD were labeled with the fluorophores ALEXA FLUOR 488, CY5, and CY3, respectively, while the 5' ends of the inner primers FIP and BIP were labeled with the corresponding BHQ series quenching groups. The detection results were interpreted based on the color of the reaction product.

[0089] Primer and probe sequences are shown in Table 2.

[0090] Table 2 shows the primer and probe sequences.

[0091]

[0092]

[0093] In the primers and probes mentioned above, the probe FD corresponding to each pathogen is complementary to F1C in the inner primer FIP, and the probe BD is complementary to B1C in the inner primer BIP.

[0094] The inner primers FIP for the above pathogens are named FIP-quencher (FIP-Q) after the 5' end of the inner primer is labeled with a quencher group, and the inner primer BIP is named BIP-quencher (BIP-Q) after the 5' end of the inner primer is labeled with a quencher group.

[0095] The probes FD for each of the above pathogens were labeled with fluorescent groups at their 3' ends and named probes FD-fluorescence (FD-F).

[0096] The probes BD of the above pathogens are labeled with fluorescent groups at their 3' ends and named probes BD-fluorescent (BD-F);

[0097] The primers and probes were diluted to the required concentrations with Takara Bio's RNase-free Water (product number 9012) to obtain the primer and probe solutions.

[0098] Before the reaction, the FIP-quenched (FIP-Q) inner primers of each pathogen and their corresponding FD-fluorescent (FD-F) probes were annealed to extinguish the fluorescence; the BIP-quenched (BIP-Q) inner primers of each pathogen and their corresponding FD-fluorescent (BD-F) probes were annealed to extinguish the fluorescence; the details are as follows:

[0099] Mix 50 μM FIP-Q solution and 50 μM FD-F solution, heat to 98 °C, and slowly cool the mixture to room temperature to complete the annealing process, thus obtaining the fluorescence-quenching composite probe FIP-FD for later use.

[0100] Mix 50 μM BIP-Q solution and 50 μM BD-F solution, heat to 98 °C, and slowly cool the mixture to room temperature to complete the annealing process, thus obtaining the fluorescence-quenching composite probe BIP-BD for later use.

[0101] II. Parameter Optimization of Multiple RT-LAMP Reaction Systems

[0102] The parameters for the LAMP reaction using the primers and probes shown in Table 2 above were optimized. The optimized parameters included both the reaction system parameters and the reaction procedure parameters. The reaction system parameters (20 μL) included: the concentrations of DNA polymerase, reverse transcriptase, fluorescence-quenching composite probe, F3, B3, Floop, and Bloop in the initial reaction system. The reaction procedure parameters included: the annealing extension temperature.

[0103] The fluorescence-quenching composite probe FMDV FIP-FD is prepared by mixing 50 μM FMDV-FIP solution and 50 μM FMDV-FD solution, heating to 98 °C, and then slowly cooling the mixture to room temperature to complete the annealing process.

[0104] The fluorescence-quenching composite probe VSV FIP-FD is prepared by mixing 50 μM VSV-FIP solution and 50 μM VSV-FD solution, heating to 98 °C, and then slowly cooling the mixture to room temperature to complete the annealing process.

[0105] The fluorescence-quenching composite probe BTV FIP-FD is prepared by mixing 50 μM BTV-FIP solution and 50 μM BTV-FD solution, heating to 98 °C, and then slowly cooling the mixture to room temperature to complete the annealing process.

[0106] The fluorescence-quenching composite probe FMDV BIP-BD was prepared by mixing 50 μM FMDV-BIP solution and 50 μM FMDV-BD solution, heating to 98 °C, and then slowly cooling the mixture to room temperature to complete the annealing process.

[0107] The fluorescence-quenching composite probe VSV BIP-BD was prepared by mixing 50 μM VSV-BIP solution and 50 μM VSV-BD solution, heating to 98 °C, and then slowly cooling the mixture to room temperature to complete the annealing process.

[0108] The fluorescence-quenching composite probe BTV BIP-BD was prepared by mixing 50 μM BTV-BIP solution and 50 μM BTV-BD solution, heating to 98 °C, and then slowly cooling the mixture to room temperature to complete the annealing process.

[0109] The recommended initial reaction system is:

[0110] The 20 μL standard LAMP reaction system is as follows: 1 μL template, 10 μL WarmStart 2× premix (containing UDG and dye, New England Biolabs, MA, USA), 0-25 U Bst 2.0 WarmStart DNA polymerase, 0-25 U WarmStart RT× reverse transcriptase, 0.8 μM FMDV FIP, 0.8 μM BTV FIP, 0.8 μM VSV FIP, 0.1-50 μM FMDV FIP-FD annealing probe (where the mass ratio of FMDV-FIP to FMDV-FD is 1:1), 0.1-50 μM BTV FIP-FD annealing probe (where the mass ratio of BTV-FIP to BTV-FD is 1:1), 0.1-50 μM VSV FIP-FD annealing probe (where the mass ratio of VSV-FIP to VSV-FD is 1:1), 0.8 μM FMDV BIP, 0.8 μM VSV BIP, 0.8μM BTV BIP, 0.1-50μM FMDV BIP-BD annealed composite probe (where the mass ratio of FMDV-BIP to FMDV-BD is 1:1), 0.1-50μM BTVBIP-BD annealed composite probe (where the mass ratio of BTV-BIP to BTV-BD is 1:1), 0.1-50μM VSV BIP-BD annealed composite probe (where the mass ratio of VSV-BIP to VSV-BD is 1:1), 0.01-20μM FMDV-F3, 0.01-20μM BTV-F3, 0.01-20μM VSV-F3, 0.01-20μM FMDV-B3, 0.1-20μM BTV-B3, 0.1-20μM VSV-B3, 0.02-20μM FMDV-Floop, 0.02-20μM BTV-Floop, 0.02-20μM VSV-Floop, 0.02-20μM FMDV-Bloop, 0.02-20μM MBTV-Bloop, 0.02-20μM VSV-Bloop, with the remainder being water.

[0111] The optimal reaction system is as follows:

[0112] The 20 μL standard LAMP reaction system is as follows: 1 μL template, 10 μL WarmStart 2× premix (containing UDG and dye, New England Biolabs, MA, USA), 5 U Bst 2.0 WarmStart DNA polymerase, 5 U WarmStart RT× reverse transcriptase, 0.8 μM FMDV FIP, 0.8 μM BTV FIP, 0.8 μM VSV FIP, 0.8 μM FMDV FIP-FD annealing probe (containing 0.4 μM FMDV-FIP and 0.4 μM FMDV-FD), 0.8 μM BTV FIP-FD annealing probe (containing 0.4 μM BTV-FIP and 0.4 μM BTV-FD), 0.8 μM VSV FIP-FD annealing probe (containing 0.4 μM VSV-FIP and 0.4 μM VSV-FD), 0.8 μM FMDV BIP, 0.8 μM VSV... BIP, 0.8μM BTV BIP, 0.8μM FMDV BIP-BD annealed composite probe (containing 0.4μM FMDV-BIP and 0.4μM FMDV-BD), 0.8μM BTV BIP-BD annealed composite probe (containing 0.4μM BTV-BIP and 0.4μM BTV-BD), 0.8μM VSV BIP-BD annealed composite probe (containing 0.4μM VSV-BIP and 0.4μM VSV-BD), 0.1μM FMDV-F3, 0.1μM BTV-F3, 0.1μM VSV-F3, 0.1μM FMDV-B3, 0.1μM BTV-B3, 0.1μM VSV-B3, 0.2μM FMDV-Floop, 0.2μM BTV-Floop, 0.2μM VSV-Floop, 0.2μM FMDV-Bloop, 0.2μM BTV-Bloop, 0.2μM VSV-Bloop, with the remainder being water.

[0113] Recommended reaction procedure: Amplify at 58-67℃ for 60 minutes, then inactivate the enzyme at 80℃ for 5 minutes to end the reaction;

[0114] The optimal reaction program is: amplification at 63℃ for 60 minutes, followed by enzyme inactivation at 80℃ for 5 minutes to end the reaction.

[0115] Therefore, the primers and probes required for amplification of each pathogen shown in Table 2 are packaged separately for use in preparing LAMP detection kits for foot-and-mouth disease virus, vesicular stomatitis virus, and bluetongue virus.

[0116] Alternatively, the FIP and FD of each pathogen in Table 2 can be replaced with the fluorescent-quenched composite probe FIP-FD, and the BIP and BD of each pathogen can be replaced with the fluorescent-quenched composite probe BIP-BD. These can be packaged separately with the inner primer, outer primer, and loop primer for the preparation of LAMP kits for the detection of foot-and-mouth disease virus, vesicular stomatitis virus, and bluetongue virus.

[0117] The kit may also include 2× premixed solution (such as WarmStart 2× premixed solution) containing UDG and a visual pH indicator, Bst 2.0 WarmStart DNA polymerase, and WarmStart RT× reverse transcriptase, etc.

[0118] III. Method Establishment

[0119] 1. Nucleic acid was extracted from the sample to be tested using an RNA / DNA co-extraction kit.

[0120] 2. Take the nucleic acid obtained in step 1 as a template and perform the following LAMP reaction.

[0121] The optimal reaction system is as follows: A 20 μL standard LAMP reaction system contains: 1 μL template, 10 μL WarmStart 2× premix (containing UDG and dye, New England Biolabs, MA, USA), 5 U Bst 2.0 WarmStart DNA polymerase, 5 U WarmStart RT× reverse transcriptase, 0.8 μM FMDV FIP, 0.8 μM BTV FIP, 0.8 μM VSV FIP, 0.8 μM FMDV FIP-FD annealing probe (containing 0.4 μM FMDV-FIP and 0.4 μM FMDV-FD), 0.8 μM BTV FIP-FD annealing probe (containing 0.4 μM BTV-FIP and 0.4 μM BTV-FD), 0.8 μM VSV FIP-FD annealing probe (containing 0.4 μM VSV-FIP and 0.4 μM VSV-FD), 0.8 μM FMDV BIP, 0.8 μM VSV... BIP, 0.8μM BTV BIP, 0.8μM FMDV BIP-BD annealed composite probe (containing 0.4μM FMDV-BIP and 0.4μM FMDV-BD), 0.8μM BTV BIP-BD annealed composite probe (containing 0.4μM BTV-BIP and 0.4μM BTV-BD), 0.8μM VSV BIP-BD annealed composite probe (containing 0.4μM VSV-BIP and 0.4μM VSV-BD), 0.1μM FMDV-F3, 0.1μM BTV-F3, 0.1μM VSV-F3, 0.1μM FMDV-B3, 0.1μM BTV-B3, 0.1μM VSV-B3, 0.2μM FMDV-Floop, 0.2μM BTV-Floop, 0.2μM VSV-Floop, 0.2μM MDF-Bloop, 0.2μM BTV-Bloop, 0.2μM VSV-Bloop, with the remainder being water.

[0122] Reaction procedure: Amplification at 63℃ for 60 minutes, enzyme inactivation at 80℃ for 5 minutes to end the reaction, using an LA-320 real-time turbidimeter or a water bath.

[0123] Reaction result reading: The reaction products were analyzed using a Bio-Rad image analyzer. The color of the fluorescent group in the reaction tube was observed and interpreted in the corresponding channel: FDMV positive (ALEXA FLUOR 488 labeled) appeared green in channel 520, VSV positive (CY5 labeled) appeared red in channel 670, and BTV positive (CY3 labeled) appeared blue in channel 570. The original color of CY3 is orange, but the image analyzer assigns it blue, which does not affect the interpretation of the results.

[0124] The results can also be read using a real-time turbidimeter. The horizontal axis represents the reaction time, and the vertical axis represents the turbidity intensity, i.e. the amount of white precipitate magnesium pyrophosphate, a byproduct of RT-LAMP. However, the turbidity curve can only detect positive samples and cannot be used for pathogen identification.

[0125] Example 2: Specificity test for LAMP detection of foot-and-mouth disease virus, vesicular stomatitis virus, and bluetongue virus.

[0126] The samples to be tested were: inactivated foot-and-mouth disease virus type A (FMDV A), type O (FMDV O), Asia type 1 (FMDV Asia 1), inactivated vesicular stomatitis virus type New Jersey (NJ) and Indian (IND), inactivated BTV type 4 (i.e., bluetongue virus type 4), inactivated BTV type 18 (i.e., bluetongue virus type 18), and equal amounts (same number of viruses) of each of the following mixed samples: FMDV (A) + VSV (NJ) + BTV (8).

[0127] Bluetongue disease (IBRV) reference strain, PPRV (small ruminant virus) inactivated virus, foot-and-mouth disease (MB) Guangxi isolate and swine vesicular disease (SVDV) inactivated virus were used as control pathogens.

[0128] The detection was performed according to the method described in Example 1, Section 3. IBRV, PPRV, MB, SVDV, and VEV were used as control pathogens.

[0129] The results are as follows Figure 1 As shown, 1: FMDV type A, 2: FMDV type O, 3: FMDV Asia type 1, 4: VSV IND type, 5: VSVNJ type, 6: BTV type 4, 7: BTV type 18, 8: FMDV (type A) + VSV (type NJ) + BTV (type 8) triplet, 9: PPRV, 10: SVDV, 11: IBRV, 12: MB, 13: negative control (RNA extracted from healthy bovine blood), 14 blank control (water); it can be seen that the multiplex RT-LAMP system can simultaneously amplify FMDV, VSV, BTV viruses and their mixed samples. FMDV positive tubes appear green (1-3), VSV positive tubes appear red (4-5), BTV positive tubes appear blue (6-7), and mixed sample positive tubes appear mixed color (8), which can be seen in the corresponding channels, but no amplification is observed for other control viruses, indicating good specificity.

[0130] Example 3: Sensitivity test of LAMP detection for foot-and-mouth disease virus, vesicular stomatitis virus, and bluetongue virus.

[0131] I. Preparation of Standards

[0132] Total RNA was extracted from inactivated FMDV type A virus and reverse transcribed into cDNA. A 226bp fragment (sequence 25) was amplified using FMDV outer primers (FMDV-B3, FMDV-F3) and PCR (premixTaq, Takara, Dalian).

[0133] Total RNA was extracted from inactivated VSV NJ virus and reverse transcribed into cDNA. A 214bp fragment (sequence 26) was amplified by PCR using VSV outer primers (VSV-F3, VSV-B3) (premixTaq, Takara, Dalian).

[0134] Total RNA was extracted from inactivated BTV type 1 (i.e., bluetongue virus type 1) and reverse transcribed into cDNA. A 223bp fragment (sequence 27) was amplified by PCR using BTV outer primers (BTV-F3, BTV-B3) (premixTaq, Takara, Dalian).

[0135] The PCR products were purified by agarose gel extraction and ligated into the pGM-T vector (Tiangen, Beijing). Positive recombinant bacteria containing specific fragments of the target virus were screened. Plasmids of the positive recombinant bacteria were extracted using a kit (MidiPlasmidkid, Tiangen, Beijing) to obtain various standards.

[0136] Plasmid FMDV is obtained by cloning the double-stranded DNA molecule shown in sequence 25 of the sequence listing into the pGM-T vector.

[0137] Plasmid VSV is obtained by cloning the double-stranded DNA molecule shown in sequence 26 of the sequence listing into the pGM-T vector.

[0138] Plasmid BTV is obtained by cloning the double-stranded DNA molecule shown in sequence 27 of the sequence listing into the pGM-T vector.

[0139] Following the instructions of the T7 in vitro transcription kit (Fermentas), plasmids FMDV, VSV, and BTV were linearized by enzyme digestion. DNA contamination was removed using DNase, and the RNA was transcribed in vitro to obtain high-purity RNA. The RNA concentration was measured using a NanoDrop 2000 nucleic acid analyzer, yielding in vitro transcribed RNA from plasmids FMDV, VSV, and BTV.

[0140] Concentrations were converted to copy numbers using Avogadro's constant, and the three in vitro prepared RNAs with calculated copy numbers were diluted to 3.33 × 10⁻⁶. 8~1 copies / μL, then mix equal volumes of RNA of the same concentration, and dilute 10-fold to prepare 10-fold serially diluted mixed sample RNA standards, where the concentration of each RNA standard can reach 1×10 8 ~1 copies / μL, store at -70℃ for later use.

[0141] II. LAMP Reaction

[0142] The prepared RNA standards were mixed after being serially diluted 10-fold at different concentrations (each in vitro transcribed RNA concentration was 1×10⁻⁶). 8 -1 copies / μL), 1 μL of each gradient standard was used as a template, and the detection was performed according to the method of Example 1, Section 3 to evaluate its sensitivity.

[0143] The results are as follows Figure 2 As shown, 1:1×10 8 1 copy / μL, 2: 1×10 7 1 copy / μL, 3: 1×10 6 1 copy / μL, 4: 1×10 5 1 copy / μL, 5: 1×10 4 1 copy / μL, 6: 1×10 3 1 copy / μL, 7: 1×10 2 1 copy / μL, 8: 1×10 1 1 copy / μL, 9: 1 copy / μL (mixed template standard of FMDV, VSV and BTV RNA), 10: negative control; the sensitivity of multiplex fluorescent RT-LAMP is 1 copy / reaction viral RNA, and this method has high sensitivity.

[0144] Example 4: Interference Experiment of LAMP Detection for Foot-and-Mouth Disease Virus, Vesicular Stoma Virus, and Bluetongue Virus

[0145] In multiplex reactions, multiple sets of primers compete for the reaction system within the same reaction tube. High-concentration templates may rapidly capture reaction components at the beginning of the reaction, inhibiting the amplification of low-concentration templates.

[0146] Three plasmids, FMDV in vitro transcribed RNA, VSV in vitro transcribed RNA, and BTV in vitro transcribed RNA, were combined at different concentrations to prepare simulated mixed infection samples of different concentrations: Sample 1 (10 2 FMDV+10 2 VSV+10 8 BTV), Sample 2 (10 2 FMDV+10 3VSV+10 8 BTV), Sample 3 (10 2 FMDV+10 4 VSV+10 7 BTV), Sample 4 (10 2 FMDV+10 5 VSV+10 6 BTV), Sample 5 (10 2 FMDV+10 6 VSV+10 5 BTV), Sample 6 (10 2 FMDV+10 7 VSV+10 4 BTV), Sample 7 (10 2 FMDV+10 8 VSV+10 3 BTV), Sample 8 (10 2 FMDV+10 8 VSV+10 2 BTV).

[0147] Sample 1 was obtained by mixing equal volumes of in vitro transcribed RNA from plasmid FMDV, plasmid VSV, and plasmid BTV, with each RNA having a concentration of 10. 2 copies / μL, 10 2 copies / μL, 10 8 copies / μL;

[0148] The other samples were similar, the only difference being the concentration.

[0149] Using the simulated mixed infection samples of different concentrations as templates, the method in Example 1, Part 3 was used for detection to evaluate whether there was interference between the templates when the concentrations of different templates differed significantly.

[0150] The results are as follows Figure 3 As shown, 1-8 correspond to samples 1-8 respectively. When one template has a high concentration and another has a low concentration, multiplex RT-LAMP can still detect three templates simultaneously, indicating that the concentration of each sample in the mixed sample does not affect the amplification results of each other and has little interference.

[0151] Example 5: Practical Application of the Method

[0152] The samples to be tested were 33 suspected bovine samples.

[0153] The method described in Example 1, Section 3, was used to test 33 suspected bovine samples. The results showed that 9 FMDV positive samples, 0 vesicular stomatitis positive samples, and 1 BTV positive sample were detected simultaneously. No mixed infection samples were detected.

[0154] Meanwhile, fluorescent RT-PCR was performed on 33 suspected bovine samples using OIE recommended primers (Table 3). The results were consistent with those of the method in Example 1 above. Nine FMDV positive samples, zero vesicular stomatitis positive samples, and one BTV positive sample were detected. No mixed infection samples were detected.

[0155] Therefore, the detection results of the method of the present invention are consistent with the OIE-recommended fluorescent RT-PCR detection method, with a 100% concordance rate and good clinical diagnostic effect.

[0156] Table 3 lists the primers recommended by OIE.

[0157] SEQUENCE LISTING <110> Guangxi Zhuang Autonomous Region Veterinary Research Institute <120> Visualized multiplex RT-LAMP detection method and primers for foot-and-mouth disease, vesicular stomatitis and bluetongue. <160> 27 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> DNA <213> Artificial sequence <400> 1 actgggtacg gcaaatgc 18 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <400> 2 caggaaggtc tgacaagcaa 20 <210> 3 <211> 41 <212> DNA <213> Artificial sequence <400> 3 gcagtatctg gttccatggc gtactgagca tctacgaggc a 41 <210> 4 <211> 41 <212> DNA <213> Artificial sequence <400> 4 ttgacttcga gaacggcacg gttctccatg agctctaggg c 41 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <400> 5 aagaccgtcg acgcctttga 20 <210> 6 <211> 16 <212> DNA <213> Artificial sequence <400> 6 tcggacccga ggtcgc 16 <210> 7 <211> 22 <212> DNA <213> Artificial sequence <400> 7 acgccatgga accagatact gc 22 <210> 8 <211> 21 <212> DNA <213> Artificial sequence <400> 8 ccgtgccgtt ctcgaagtca a 21 <210> 9 <211> 22 <212> DNA <213> Artificial sequence <400> 9 agaagacaaa tacacaacag aa 22 <210> 10 <211> 19 <212> DNA <213> Artificial sequence <400> 10 cttgtcaaac tctgccttg 19 <210> 11 <211> 44 <212> DNA <213> Artificial sequence <400> 11 ccaagccagt ccacgacatt tgatggtctg aagaaattag atgt 44 <210> 12 <211> 42 <212> DNA <213> Artificial sequence <400> 12 ggaaaaccca caccagatat gctgtctttt cacgaagtga ct 42 <210> 13 <211> 22 <212> DNA <213> Artificial sequence <400> 13 tcccaccaaa aggaagaaat gt 22 <210> 14 <211> 23 <212> DNA <213> Artificial sequence <400> 14 acttcgcaag aagagcagtc aac 23 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <400> 15 aatgtcgtgg actggcttgg 20 <210> 16 <211> 22 <212> DNA <213> Artificial sequence <400> 16 gcatatctgg tgtgggtttt cc 22 <210> 17 <211> 19 <212> DNA <213> Artificial sequence <400> 17 gtactagcga cgccagaga 19 <210> 18 <211> 20 <212> DNA <213> Artificial sequence <400> 18 ctccagcatt cagtgacact 20 <210> 19 <211> 41 <212> DNA <213> Artificial sequence <400> 19 ccacgtcgaa gtctccccag taccttttac aacggaagcg g 41 <210> 20 <211> 40 <212> DNA <213> Artificial sequence <400> 20 agagactttc caacccggga gatccggacc acacactacc 40 <210> 21 <211> 20 <212> DNA <213> Artificial sequence <400> 21 cacgcgagca atctcattcg 20 <210> 22 <211> 18 <212> DNA <213> Artificial sequence <400> 22 ttcatgcgtg ccgctcaa 18 <210> 23 <211> 21 <212> DNA <213> Artificial sequence <400> 23 actggggaga cttcgacgtg g 21 <210> 24 <211> 22 <212> DNA <213> Artificial sequence <400> 24 tctcccgggt tggaaagtct ct 22 <210> 25 <211> 226 <212> DNA <213> Artificial sequence <400> 25 actgggtacg gcaaatgccc cactgagcat ctacgaggca atcaaaggcg tcgacggtct 60 tgacgccatg gaaccagata ctgcgcctgg tctccccggg gccctccagg ggaagcgccg 120 cggcgcactg attgacttcg agaacggcac ggtcggaccc gaggtcgcgg ctgccctaga 180 gctcatggag aaaagagaat acaaatttgc ttgtcagacc ttcctg 226 <210> 26 <211> 214 <212> DNA <213> Artificial sequence <400> 26 agaagacaaa tacacaacag aaaaagatga tggtctgaag aaattagatg tcccaccaaa 60 aggaagaaat gtcgtggact ggcttggctg gtatgatgac aatgggggaa aacccacacc 120 agatatgctc aacttcgcaa gaagagcagt caactctctg cagtcacttc gtgaaaagac 180 aattggcaaa tatgccaagg cagagtttga caag 214 <210> 27 <211> 223 <212> DNA <213> Artificial sequence <400> 27 gtactagcga cgccagagat accttttaca acggaagcgg cgaatgagat tgctcgcgtg 60 actggggaga cttcgacgtg gggaccagcg cgtcagccct atggtttttt cttgaaact 120 gaagagactt tccaacccgg gagatggttc atgcgtgccg ctcaagcagt aaccgcggta 180 gtgtgtggtc cggatatgat tcaagtgtca ctgaatgctg gag 223

Claims

1. A kit of parts, consisting of primer probe set A, primer probe set B and primer probe set C; the primer probe set A consists of primer FMDV-FIP, primer FMDV-BIP, primer FMDV-F3, primer FMDV-B3, complex FMDV FIP-FD, complex FMDV BIP-BD, primer FMDV-Floop and primer FMDV-Bloop; the primer probe set B consists of primer VSV-FIP, primer VSV-BIP, primer VSV-F3, primer VSV-B3, complex VSV FIP-FD, complex VSV BIP-BD, primer VSV-Floop and primer VSV-Bloop; the primer probe set C consists of primer BTV-FIP, primer BTV-BIP, primer BTV-F3, primer BTV-B3, complex BTV FIP-FD, complex BTV BIP-BD, primer BTV-Floop and primer BTV-Bloop; the complex FMDV FIP-FD is obtained by annealing the primer FMDV-FIP and the probe FMDV-FD complementary to the F1C segment of FMDV-FIP; the complex FMDV BIP-BD is obtained by annealing the primer FMDV-BIP and the probe FMDV-BD complementary to the B1C segment of FMDV-BIP; the complex VSV FIP-FD is obtained by annealing the primer VSV-FIP and the probe VSV-FD complementary to the F1C segment of VSV-FIP; the complex VSV BIP-BD is obtained by annealing the primer VSV-BIP and the probe VSV-BD complementary to the B1C segment of VSV-BIP; the complex BTV FIP-FD is obtained by annealing the primer BTV-FIP and the probe BTV-FD complementary to the F1C segment of BTV-FIP; the complex BTV BIP-BD is obtained by annealing the primer BTV-BIP and the probe BTV-BD complementary to the B1C segment of BTV-BIP; the nucleotide sequences of the primer FMDV-F3, primer FMDV-B3, primer FMDV-FIP, primer FMDV-BIP, primer FMDV-Floop, primer FMDV-Bloop, probe FMDV-FD, probe FMDV-BD, primer VSV-F3, primer VSV-B3, primer VSV-FIP, primer VSV-BIP, primer VSV-Floop, primer VSV-Bloop, probe VSV-FD, probe VSV-BD, primer BTV-F3, primer BTV-B3, primer BTV-FIP, primer BTV-BIP, primer BTV-Floop, primer BTV-Bloop, probe BTV-FD and probe BTV-BD are respectively sequence 1 to sequence 24. The 5' end of the FMDV-FIP, the FMDV-BIP, the VSV-FIP, the VSV-BIP, the BTV-FIP, the BTV-BIP is labeled with a quenching group; The 3' end of the FMDV-FD, the FMDV-BD, the VSV-FD, the VSV-BD, the BTV-FD, the BTV-BD is labeled with a fluorescent group, and different pathogenic bacteria are labeled with different colors of fluorescent groups, and the FD probe and the BD probe of the same pathogenic bacteria are labeled with the same fluorescent group.

2. A loop-mediated isothermal amplification reagent characterized by: The reagent contains the kit of claim 1. 3.The loop-mediated isothermal amplification reagent according to claim 2, characterized in that: The molar ratio of the complex FMDV FIP-FD, the complex FMDV BIP-BD, the complex VSV FIP-FD, the complex VSV BIP-BD, the complex BTV FIP-FD, the complex BTV BIP-BD, the primer FMDV-F3, the primer FMDV-B3, the primer VSV-F3, the primer VSV-B3, the primer BTV-F3, the primer BTV-B3, the primer FMDV-Floop, the primer FMDV-Bloop, the primer VSV-Floop, the primer VSV-Bloop, the primer BTV-Floop, the primer BTV-Bloop, the primer FMDV-FIP, the primer BTV-FIP, the primer VSV-FIP, the primer FMDV-BIP, the primer BTV-BIP and the primer VSV-BIP in the reagent is 8:8:8:8:8:8:1:1:1:1:1:1:2:2:2:2:2:2:8:8:8:8:8:

8.

4. The loop-mediated isothermal amplification reagent according to claim 2 or 3, characterized in that: The concentrations of the complex FMDV FIP-FD, complex FMDV BIP-BD, complex VSV FIP-FD, complex VSV BIP-BD, complex BTV FIP-FD, complex BTV BIP-BD, primer FMDV-F3, primer FMDV-B3, primer VSV-F3, primer VSV-B3, primer BTV-F3, primer BTV-B3, primer FMDV-Floop, primer FMDV-Bloop, primer VSV-Floop, primer VSV-Bloop, primer BTV-Floop, primer BTV-Bloop, primer FMDV-FIP, primer BTV-FIP, primer VSV-FIP, primer FMDV-BIP, primer BTV-BIP and primer VSV-BIP in the reagent are 0.8 µM, 0.8 µM, 0.8 µM, 0.8 µM, 0.8 µM, 0.8 µM, 0.1 µM, 0.1 µM, 0.1 µM, 0.1 µM, 0.1 µM, 0.1 µM, 0.2 µM, 0.2 µM, 0.2 µM, 0.2 µM, 0.2 µM, 0.2 µM, 0.8 µM, 0.8 µM, 0.8 µM, 0.8 µM, 0.8 µM and 0.8 µM, respectively.

5. Use of the kit reagent of claim 1 or the loop-mediated isothermal amplification reagent of any one of claims 2-4 in the preparation of a kit; the use of the kit is as follows (d1) or (d2) or (d3): (d1) identifying or assisting in identifying foot-and-mouth disease virus, vesicular stomatitis virus or blue tongue virus; (d2) identifying or assisting in identifying whether the virus to be tested is foot-and-mouth disease virus, vesicular stomatitis virus and / or blue tongue virus; (d3) identifying or assisting in identifying whether the sample to be tested is infected with foot-and-mouth disease virus, vesicular stomatitis virus and / or blue tongue virus.

6. The kit containing the kit reagent of claim 1 or the loop-mediated isothermal amplification reagent of any one of claims 2-4; the use of the kit is as follows (d1) or (d2) or (d3): (d1) identifying or assisting in identifying foot-and-mouth disease virus, vesicular stomatitis virus or blue tongue virus; (d2) identifying or assisting in identifying whether the virus to be tested is foot-and-mouth disease virus, vesicular stomatitis virus and / or blue tongue virus; (d3) identifying or assisting in identifying whether the sample to be tested is infected with foot-and-mouth disease virus, vesicular stomatitis virus and / or blue tongue virus.

7. The preparation method of the kit of claim 6, comprising the step of individually packaging each substance in the kit reagent of claim 1.

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