Loop-mediated isothermal amplification primer composition for combined detection of multiple equine animal viruses and application of loop-mediated isothermal amplification primer composition

Abstract

The invention provides a loop-mediated isothermal amplification primer composition for combined detection of multiple equine animal viruses, a disposable centrifugal micro-fluidic chip, an LAMP micro-fluidic chip kit and a nucleic acid detection method of the LAMP micro-fluidic chip kit. The loop-mediated isothermal amplification primer composition comprises nucleotide sequences as shown in SEQ ID: 1 to SEQ ID: 16. A fluorescent dye and a loop-mediated isothermal amplification primer composition are pre-embedded in the disposable centrifugal micro-fluidic chip. The LAMP micro-fluidic chip kit comprises a disposable centrifugal micro-fluidic chip and an LAMP amplification working solution. The problem of multiple detection of VSV, NiV, EEEV and WEEV in equine animal viruses is solved, and the detection method is high in specificity, short in reaction time, safe, simple and convenient to operate, high in sensitivity, low in cost and suitable for being used when a temporary virus detection laboratory is established.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to the use of a microfluidic chip combined with a loop-mediated isothermal amplification method to jointly detect nucleic acids of four equine animal viruses. Background Art

[0002] Horses were once important riding animals with a profound cultural impression around the world. With the development of the modern horse industry, the use of horses has gradually shifted from agriculture, military, and transportation to sports and entertainment activities such as horse racing and equestrianism. Due to the change in use, the bloodline, breeding conditions, and medical health of horses have become new industry demands. At present, my country has many horses with excellent bloodlines in stock, and horse import and export activities are frequent.

[0003] Vesicular Stomatitis Virus (VSV) belongs to the family Rhabdoviridae. Symptoms include lethargy, fever, and vesicular lesions. In horses, it causes lameness and weight loss. The wounds after the blisters rupture are easily infected and lead to secondary diseases. Its transmission route is not very clear, and it can infect humans. Nipah Virus (NiV) belongs to the family Paramyxoviridae. It is a zoonosis. There have been many large-scale outbreaks in history. It can cause encephalitis and is often confused with other encephalitis viruses, leading to misdiagnosis. Equine Encephalitis Virus (EEV) is divided into three types: Western Equine Encephalitis Virus (WEEV), Eastern Equine Encephalitis Virus (EEEV), and Venezuelan Equine Encephalitis Virus (VEEV). All belong to the genus Alphavirus of the family Togaviridae and are zoonotic. The three have the same symptoms and can cause equine encephalitis and other neurological diseases. Although most reported cases are concentrated in the Americas, there is also evidence that EEEV and WEEV may have been weaponized.

[0004] In recent years, nucleic acid detection methods based on PCR as the main method and serological detection methods such as enzyme-linked immunosorbent assay and colloidal gold immunochromatography have been established for infectious diseases caused by four equine viruses: VSV, NiV, EEEV, and WEEV. However, the PCR method is expensive and requires highly skilled professionals to perform detection and interpretation in a well-equipped laboratory, making it difficult to cope with the increasing demand for detection. Colloidal gold immunochromatography test strips require a relatively high viral load, which is not conducive to the early detection of the virus and the blocking of the spread of the epidemic.

[0005] Therefore, a rapid, accurate and simple virus detection method needs to be established urgently. Summary of the invention

[0006] In order to overcome the above technical defects, the first aspect of the present invention provides a loop-mediated isothermal amplification primer composition for jointly detecting Vesicular Stomatitis Virus (VSV), Eastern Equine Encephalitis Virus (EEEV), Western Equine Encephalitis Virus (WEEV) and Nipah Virus (NiV), wherein the multiple equine viruses include Vesicular Stomatitis Virus, Eastern Equine Encephalitis Virus, Western Equine Encephalitis Virus and Nipah Virus, and the primer composition comprises 16 inner and outer primers whose nucleotide sequences are shown in SEQ ID: 1 to SEQ ID: 16.

[0007] The present invention comprises a LAMP primer combination required for detecting four viruses, each virus comprising a pair of inner primers (FIP, BIP) and a pair of outer primers (F3, B3), a total of 16 primers (SEQ ID No: 1 to SEQ ID No: 16), which solves the problem of multiple detection of equine viruses. The nucleotide sequence is shown in the following table:

[0008]

[0009] The second aspect of the present invention provides a disposable centrifugal microfluidic chip for joint detection of multiple equine viruses, wherein the multiple equine viruses include vesicular stomatitis virus, eastern equine encephalomyelitis virus, western equine encephalomyelitis virus and Nipah virus, wherein the disposable centrifugal microfluidic chip is pre-embedded with a fluorescent dye and the above-mentioned loop-mediated isothermal amplification primer composition, thereby facilitating production and storage.

[0010] Furthermore, a disposable centrifugal microfluidic chip, a centrifugal microfluidic chip comprises 4 large sectors, each of which contains two adjacent small sectors. The small sector contains a liquid storage area with a loading hole, and the liquid storage area is connected to the reaction pool through a fine groove. The connection pool between the liquid storage area and the fine groove has a gear-mounted reserved area for evenly distributing the sample added to the liquid storage area through the loading hole. There is a ball valve in the middle of the fine groove to prevent the liquid from flowing back from the reaction pool to the liquid storage area after centrifugation. The microfluidic chip is sealed with a sticky bottom membrane to form a chamber, and the loading hole is sealed with a sticky aluminum foil membrane. The fluorescent dye and the loop-mediated isothermal amplification primer composition are pre-buried in the reaction pool by a vacuum drying method. Thereby facilitating production and storage.

[0011] The third aspect of the present invention provides a LAMP microfluidic chip kit for joint detection of multiple equine viruses, wherein the multiple equine viruses include vesicular stomatitis virus, eastern equine encephalomyelitis virus, western equine encephalomyelitis virus and Nipah virus; the LAMP microfluidic chip kit includes: the above-mentioned disposable centrifugal microfluidic chip and LAMP amplification working solution; the LAMP amplification working solution includes a buffer system, a fluorescent dye and / or a color developer, Bst DNA polymerase and dNTP, and the solvent is RNase Free ddH2O. The buffer system includes Tris-HCl, (NH4)2SO4, KCl, MgSO4, and Triton X-100.

[0012] Preferably, the color developer is neutral red dye (NeuRed).

[0013] The working solution can be supplemented with an appropriate concentration of MgSO4 solution to balance the effect of the additional fluorescent dye on the enzyme activity.

[0014] Preferably, the detection limit of the working solution is 100 copies / μL.

[0015] Furthermore, the LAMP amplification working solution includes the following components:

[0016] (1) Buffer system: 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100;

[0017] (2) 8 U / μL Bst DNA polymerase;

[0018] (3) 6 mM dNTP;

[0019] (4) Fluorescent dye: SYBR GreenⅠDNA Dye.

[0020] Furthermore, the fluorescent dye pre-embedded in the disposable centrifugal microfluidic chip is the same as the fluorescent dye in the LAMP amplification working solution, which is SYBR Green Ⅰ DNA Dye. Preferably, the fluorescent dye pre-embedded in the disposable centrifugal microfluidic chip can be the same as or different from that in the LAMP amplification working solution. More preferably, SYBR Green Ⅰ DNA Dye consistent with that in the LAMP amplification working solution is used. The LAMP amplification working solution can change the fluorescent dye to adapt to different instruments; more preferably, it is SYBR Green Ⅰ DNA Dye corresponding to the FAM channel.

[0021] The fourth aspect of the present invention provides a LAMP microfluidic chip kit nucleic acid detection method for jointly detecting multiple equine viruses, wherein the multiple equine viruses include vesicular stomatitis virus, eastern equine encephalitis virus, western equine encephalitis virus and Nipah virus; the detection method comprises the following steps:

[0022] Step S1: extracting and purifying nucleic acid samples using a fully automatic nucleic acid extractor, or using nucleic acid samples obtained by other extraction or reverse transcription;

[0023] Step S2: taking the nucleic acid in step S1 and adding it to the LAMP amplification working solution, vortexing and mixing, and performing a loop-mediated isothermal amplification reaction using the loop-mediated isothermal amplification primer composition as claimed in claim 1;

[0024] Step S3: injecting the mixed solution in step S2 into the loading well of the disposable microfluidic chip, wherein the loading well is pre-buried with the above-mentioned LAMP primer combination and SYBR Green I DNA Dye nucleic acid dye mixture;

[0025] Step S4: Use an aluminum foil membrane to seal the sample loading hole and place it in a centrifugal microfluidic nucleic acid amplifier to perform a loop-mediated isothermal amplification reaction;

[0026] Step S5: Set the fluorescence threshold and output the result report. The threshold is preferably 10 times the standard deviation of the fluorescence signal in the previous 10 minutes.

[0027] The present invention provides a microfluidic method for rapid detection of four equine viruses, namely VSV, NiV, EEEV and WEEV. The present invention has the characteristics of strong specificity, short reaction time, safe and simple operation, high sensitivity and low cost, and is suitable for the establishment of a temporary virus detection laboratory.

[0028] Furthermore, in step S2, the LAMP amplification working solution is prepared by vortexing and mixing 112 μL RNase Free ddH2O, 20 μL buffer, 28 μL 6 mM dNTP solution, 8 μL 8U / μL Bst DNA polymerase, 8 μL 10×SYBR GreenⅠDNADye fluorescent dye and an additional 8 μL 10 mM MgSO4 solution at 4°C, and can be stored for a long time at 4°C in the dark; when using the LAMP amplification working solution for the reaction, take 3 μL of the nucleic acid sample in step S1 and add it to 30 μL LAMP amplification working solution and vortex mix.

[0029] Preferably, when processing RNA samples, the samples after the reverse transcription step can be directly added to the LAMP amplification working solution.

[0030] Furthermore, in step S3, the primer pre-embedding process of the disposable centrifugal microfluidic chip is as follows: (1) preparing a primer pre-embedding combination liquid: mixing four primers of each virus at a volume ratio of FIP / BIP:TE Buffer:F3 / B3=8:4:1 to obtain a 1× primer combination liquid; taking 3.3 μL of the 1× primer combination liquid, adding 32.2 μL of TE buffer, and further adding 2 μL of 10×SYBR GreenⅠDNADye fluorescent dye, vortexing and defoaming to obtain a primer pre-embedding combination liquid; (2) primer pre-embedding of the reaction pool: injecting 2.5 μL of the primer pre-embedding combination liquid of each virus into the reaction pool, drying in a constant temperature vacuum drying oven at 37°C for 10 min, and then taking out and sticking a bottom film to seal.

[0031] Further, in step S4, the reaction conditions of the loop-mediated isothermal amplification reaction are 62° C. to 70° C. for 30 to 40 min. More preferably, the reaction temperature is set to 65° C. for 40 min.

[0032] Preferably, all reagents and chips included in the present invention should be stored at 4°C in a dark environment. Before the reaction, the operating temperature should be avoided to exceed 35°C, and during the reaction, the reaction temperature should be avoided to exceed 70°C. Experimental operations that do not meet the above temperature factors will directly affect the operation of the LAMP amplification working solution and produce erroneous interpretation of the reaction results.

[0033] It is understandable that, under the conditions of the above loop-mediated isothermal amplification reaction, the amount of each component in the LAMP amplification working solution can be appropriately adjusted, but cannot be defaulted. The above LAMP amplification working solution components have been screened, and the amplification effect is the best and the result interpretation is accurate.

[0034] Compared with the prior art, the above technical solution has the following beneficial effects:

[0035] Loop-mediated isothermal amplification (LAMP) is an isothermal amplification method. With the help of the strand displacement activity of Bst DNA polymerase, high-sensitivity and rapid amplification can be performed under simple experimental conditions. The reaction amplifies the target through four or six primers to produce products containing multiple stem-loop structures and DNA double strands with repeated folded cauliflower structures. The raw materials of the reaction are simple and the cost is much lower than PCR. The reaction is carried out under isothermal conditions and the detection time is less than 1 hour. The applicant screened and mixed the raw material ratio of the LAMP reaction to obtain a working solution that can be stably stored at 4°C. And through sequence alignment, the LAMP amplification primer sequences of VSV, NiV, EEEV and WEEV were designed, and at the same time, with the centrifugal microfluidic chip, the four viral nucleic acids can be detected simultaneously with one sample addition. There is no non-specific amplification in the detection process, and there is no mutual interference between the signals of different channels, which solves the problem of multiple detection of equine viruses. The detection method of the present invention has strong specificity, short reaction time, safe and simple operation, high sensitivity and low cost, and is suitable for establishing temporary virus detection laboratories and on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The results are shown for single-plex detection of four viruses using LAMP amplification working solution;

[0037] Figure 2 The results of sensitivity testing of four viruses using LAMP amplification working solution;

[0038] Figure 3 The figure is a test result diagram of independent repeatability verification of the same concentration of VSV, NiV, EEEV and WEEV plasmids using the LAMP microfluidic chip and LAMP amplification working solution in the content of the invention;

[0039] Figure 4 A schematic diagram of the principle of using the disposable centrifugal microfluidic chip of the present invention to perform multiple detection of four equine viruses, namely, VSV, NiV, EEEV and WEEV;

[0040] Figure 5 FIG. 1 is a diagram showing the detection results of multiple samples extracted from cell culture fluid in one embodiment of the present invention. DETAILED DESCRIPTION

[0041] The advantages of the present invention are further described below in conjunction with the accompanying drawings and specific embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0042] Embodiment 1

[0043] The primer pre-embedding process of the disposable centrifugal microfluidic chip is as follows:

[0044] 1. Preparation of primer pre-embedded combination solution

[0045] The combination solution includes primer combination solution, RNase Free ddH2O, and 10×SYBR GreenⅠDNA Dye fluorescent dye. The specific preparation process is as follows.

[0046] (1) The four primers for each virus were mixed at a volume ratio of FIP / BIP:TE Buffer:F3 / B3=8:4:1 to obtain a 1× primer combination solution.

[0047] (2) Take 3.3 μL of 1× primer combination solution and add 32.2 μL of TE buffer. Add 2 μL of 10×SYBR Green I DNA Dye fluorescent dye, vortex and mix thoroughly to remove bubbles to obtain the primer pre-embedded combination solution.

[0048] The combined solution can be stored at 4°C in the dark for a short time and at -20°C in the dark for a long time. Repeated freezing and thawing should be avoided during storage.

[0049] 2. Primer pre-embedding in the reaction pool

[0050] 2.5 μL of the primer pre-embedded combination solution of each virus was injected into the reaction pool, dried in a constant temperature vacuum drying oven at 37°C for 10 minutes, and then taken out and pasted with a base film for sealing.

[0051] Embodiment 2

[0052] In this example, the following LAMP amplification working solution was used to perform single-plex detection of four viruses:

[0053] The LAMP amplification working solution is prepared by vortexing and mixing 112 μL RNase Free ddH2O, 20 μL buffer, 28 μL 6 mM dNTP solution, 8 μL 8 U / μL Bst DNA polymerase, 8 μL 10×SYBR GreenⅠDNA Dye fluorescent dye and an additional 8 μL 10 mM MgSO4 solution at 4°C and can be stored for a long time at 4°C in the dark. When using the LAMP amplification working solution for reaction, take 3 μL of nucleic acid sample and add it to 30 μL LAMP amplification working solution and vortex and mix.

[0054] The test results are as follows Figure 1 shown.

[0055] By testing the plasmid models of four different equine viruses, VSV, NiV, EEEV, and WEEV, it was found that the corresponding reaction pools had fluorescent signals and could produce positive reports, while other reaction wells had no amplification signals.

[0056] Embodiment 3

[0057] This example uses the LAMP amplification working solution in the content description to perform sensitivity detection on four viruses:

[0058] The LAMP amplification working solution is prepared by vortexing and mixing 112 μL RNase Free ddH2O, 20 μL buffer, 28 μL 6 mM dNTP solution, 8 μL 8 U / μL Bst DNA polymerase, 8 μL 10×SYBR GreenⅠDNA Dye fluorescent dye and an additional 8 μL 10 mM MgSO4 solution at 4°C and can be stored for a long time at 4°C in the dark. When using the LAMP amplification working solution for reaction, take 3 μL of nucleic acid sample and add it to 30 μL LAMP amplification working solution and vortex and mix.

[0059] The test results are as follows Figure 2 shown.

[0060] By 10 4 copies / μL, 10 3 copies / μL, 10 2 The minimum detection limit of VSV for the detection of four equine viruses can reach 10 copies / μL and 10 copies / μL, and the remaining minimum detection limits can reach 100 copies / μL.

[0061] Embodiment 4

[0062] This example uses the LAMP microfluidic chip and LAMP amplification working solution in the invention content to independently test VSV, NiV, EEEV and WEEV plasmids of the same concentration five times, and records the Tt value of the reaction to calculate the coefficient of variation. Figure 3 As shown, the coefficient of variation of the Tt values ​​of each plasmid was less than 10%, and the repeatability was good.

[0063] Embodiment 5

[0064] This embodiment uses the LAMP microfluidic chip kit in the invention content to detect multiple viral nucleic acid samples extracted from cell culture fluid, such as Figure 4 As shown, it includes steps S1 to S5:

[0065] Step S1: Use a fully automatic nucleic acid extractor to extract and purify nucleic acid samples, or use other nucleic acid samples extracted or reverse transcribed. The nucleic acid samples include any one or more of four different equine viruses: VSV, NiV, EEEV, and WEEV.

[0066] Step S2: Take the nucleic acid in step S1 and add it to the LAMP amplification working solution, vortex mix it, and use the loop-mediated isothermal amplification primer combination with nucleotide sequences as shown in SEQ ID: 1 to SEQ ID: 16 to perform a loop-mediated isothermal amplification reaction.

[0067] The LAMP amplification working solution is prepared by vortexing and mixing at 4°C with 112μL RNase Free ddH2O, 20μL buffer, 28μL 6mM dNTP solution, 8μL 8U / μL Bst DNA polymerase, 8μL 10×SYBR GreenⅠDNA Dye fluorescent dye, and an additional 8μL 10mM MgSO4 solution. It can be stored at 4°C in the dark for a long time. When using the LAMP amplification working solution for reaction, take 3μL of the nucleic acid sample in step S1 and add it to 30μL LAMP amplification working solution and vortex and mix. When processing RNA samples, the samples that have undergone the reverse transcription step can be directly added to the LAMP amplification working solution.

[0068] Step S3: injecting the mixed solution in step S2 into the loading well of the disposable microfluidic chip, in which the LAMP primer composition (the primer composition comprises 16 inner and outer primers with nucleotide sequences as shown in SEQ ID: 1 to SEQ ID: 16) and SYBR Green I DNA Dye nucleic acid dye mixture are pre-buried.

[0069] The primer pre-embedding process of the disposable centrifugal microfluidic chip is as follows: (1) preparing the primer pre-embedding combination liquid: mixing the four primers of each virus at a volume ratio of FIP / BIP:TE Buffer:F3 / B3=8:4:1 to obtain a 1× primer combination liquid; taking 3.3 μL of the 1× primer combination liquid, adding 32.2 μL of TE buffer, and further adding 2 μL of 10×SYBR GreenⅠDNADye fluorescent dye, vortexing and defoaming to obtain the primer pre-embedding combination liquid; (2) primer pre-embedding of the reaction pool: injecting 2.5 μL of the primer pre-embedding combination liquid of each virus into the reaction pool, drying in a constant temperature vacuum drying oven at 37°C for 10 minutes, and then taking out and sticking the bottom film to seal.

[0070] Step S4: Use aluminum foil to seal the sample well and place it in a centrifugal microfluidic nucleic acid amplifier for loop-mediated isothermal amplification reaction; the reaction conditions of the loop-mediated isothermal amplification reaction are 65° C. and 40 min. The results are displayed in the form of an amplification curve drawn with real-time fluorescence data.

[0071] Step S5: Set the threshold and output the result report.

[0072] The results are as follows Figure 5As shown in the figure, only the reaction pool corresponding to the virus-positive sample has a fluorescent signal and can generate a positive report. The other reaction wells have no amplification signal.

[0073] It should be noted that the embodiments of the present invention have better practicability and do not impose any form of limitation on the present invention. Any technician familiar with the field may use the technical content disclosed above to change or modify it into an equivalent effective embodiment. However, any modification or equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A loop-mediated isothermal amplification primer composition for joint detection of multiple equine viruses, characterized in that: The multiple equine viruses include vesicular stomatitis virus, eastern equine encephalomyelitis virus, western equine encephalomyelitis virus and Nipah virus. The primer composition comprises 16 inner and outer primers with nucleotide sequences as shown in SEQ ID: 1 to SEQ ID:

16.

2. A disposable centrifugal microfluidic chip for joint detection of multiple equine viruses, characterized in that: The multiple equine viruses include vesicular stomatitis virus, eastern equine encephalomyelitis virus, western equine encephalomyelitis virus and Nipah virus. The disposable centrifugal microfluidic chip is pre-embedded with a fluorescent dye and the loop-mediated isothermal amplification primer composition according to claim 1.

3. The disposable centrifugal microfluidic chip for joint detection of multiple equine viruses as claimed in claim 2, characterized in that: The microfluidic chip comprises 8 loading holes, each loading hole corresponds to 4 channels, the end of the channel is a reaction pool, the microfluidic chip is sealed by a bottom membrane to form a chamber, the loading holes are sealed by an aluminum foil membrane, and the fluorescent dye and the loop-mediated isothermal amplification primer composition are pre-buried in the reaction pool by a vacuum drying method.

4. A LAMP microfluidic chip kit for joint detection of multiple equine viruses, characterized in that: The multiple equine viruses include vesicular stomatitis virus, Eastern equine encephalomyelitis virus, Western equine encephalomyelitis virus and Nipah virus; the LAMP microfluidic chip kit includes: a disposable centrifugal microfluidic chip as described in claim 2 or 3 and a LAMP amplification working solution; the LAMP amplification working solution includes a buffer system, a fluorescent dye and / or a color developer, Bst DNA polymerase and dNTP, and the buffer system includes Tris-HCl, (NH4)2SO4, KCl, MgSO4, and Triton X-100.

5. The LAMP microfluidic chip kit according to claim 4, characterized in that: The LAMP amplification working solution includes: 20mM Tris-HCl, 10mM (NH4)2SO4, 10mM KCl, 2mM MgSO4, 0.1% Triton X-100; 8U / μL BstDNA polymerase; 6mM dNTP; SYBR Green Ⅰ DNA Dye.

6. The LAMP microfluidic chip kit according to claim 5, characterized in that: The fluorescent dye pre-embedded in the disposable centrifugal microfluidic chip is the same as the fluorescent dye in the LAMP amplification working solution, which is SYBR Green Ⅰ DNA Dye.

7. A LAMP microfluidic chip kit nucleic acid detection method for joint detection of multiple equine viruses, characterized in that: The plurality of equine viruses include vesicular stomatitis virus, eastern equine encephalitis virus, western equine encephalitis virus and Nipah virus; the detection method comprises the following steps: Step S1: extracting a purified nucleic acid sample using a fully automatic nucleic acid extractor, or using a nucleic acid sample obtained by other extraction or reverse transcription; Step S2: taking the nucleic acid in step S1 and adding it to the LAMP amplification working solution, vortexing and mixing, and performing a loop-mediated isothermal amplification reaction using the loop-mediated isothermal amplification primer composition as claimed in claim 1; Step S3: injecting the mixed solution in step S2 into the loading well of the disposable microfluidic chip pre-buried with the LAMP primer composition and SYBR Green I DNA Dye nucleic acid dye mixture as claimed in claim 2 or 3; Step S4: Use an aluminum foil membrane to seal the sample loading hole and place it in a centrifugal microfluidic nucleic acid amplifier to perform a loop-mediated isothermal amplification reaction; Step S5: Set the threshold and output the result report.

8. The LAMP microfluidic chip kit nucleic acid detection method for joint detection of multiple equine viruses as claimed in claim 7, characterized in that: In step S2, the LAMP amplification working solution is prepared by vortexing and mixing 112 μL RNase Free ddH2O, 20 μL buffer, 28 μL 6 mM dNTP solution, 8 μL 8 U / μL Bst DNA polymerase, 8 μL 10×SYBR Green Ⅰ DNA Dye fluorescent dye and an additional 8 μL 10 mM MgSO4 solution at 4°C; when using the LAMP amplification working solution for reaction, take 3 μL of the nucleic acid sample in step S1 and add it to 30 μL LAMP amplification working solution and vortex and mix.

9. The LAMP microfluidic chip kit nucleic acid detection method for joint detection of multiple equine viruses as claimed in claim 7, characterized in that: In step S3, the primer pre-embedding process of the disposable centrifugal microfluidic chip is as follows: (1) preparing a primer pre-embedding combination liquid: mixing four primers of each virus at a volume ratio of FIP / BIP:TE Buffer:F3 / B3=8:4:1 to obtain a 1× primer combination liquid; taking 3.3 μL of the 1× primer combination liquid, adding 32.2 μL of TE buffer, and further adding 2 μL of 10×SYBR Green Ⅰ DNA Dye fluorescent dye, vortexing and defoaming to obtain a primer pre-embedding combination liquid; (2) primer pre-embedding of the reaction pool: injecting 2.5 μL of the primer pre-embedding combination liquid of each virus into the reaction pool, drying in a constant temperature vacuum drying oven at 37°C for 10 minutes, and then taking out and sticking a bottom film to seal.

10. The LAMP microfluidic chip kit nucleic acid detection method for simultaneously detecting vesicular stomatitis virus, eastern equine encephalomyelitis virus, western equine encephalomyelitis virus and Nipah virus as claimed in claim 7, characterized in that: In step S4, the reaction conditions of the loop-mediated isothermal amplification reaction are 62° C. to 70° C. for 30 to 40 min.

Images