Triple rapid detection method for NDV, AIV and IBV based on microfluidic chip and LAMP isothermal amplification technology

Abstract

The invention discloses an NDV (Newcastle Disease Virus), AIV (Avian Infectious Virus) and IBV (Infectious Bursal Virus) triple rapid detection method based on a micro-fluidic chip and an LAMP (Loop-Mediated Isothermal The method comprises the following steps: (1) preparing a working solution from LAMP primers designed for NDV, AIV and IBV according to a specific final concentration, embedding the working solution in a micro-fluidic chip reaction chamber, and drying and fixing the working solution; (2) mixing a nucleic acid sample to be detected with the reaction liquid, adding the mixture into a chip sample adding hole, sealing and removing bubbles; and (3) pressing the reaction liquid into each reaction chamber through centrifugation, carrying out LAMP amplification for 30 minutes at a constant temperature of 63.5 DEG C, and collecting fluorescence signals in real time during the amplification so as to realize rapid detection of the target virus. According to the present invention, the highly integrated design of the microfluidic chip and the efficient, rapid and constant temperature characteristics of the LAMP amplification technology are integrated so as to achieve the synchronous, rapid and sensitive detection of the three important poultry respiratory viruses;

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular biology, and particularly relates to a NDV, AIV and IBV triple rapid detection method based on a microfluidic chip and LAMP constant temperature amplification technology. BACKGROUND

[0002] With the continuous development of the poultry breeding industry, the prevention and control of poultry diseases is becoming increasingly important. Among them, Newcastle disease virus (NDV), avian influenza virus (AIV) and chicken infectious bronchitis virus (IBV) are the main pathogens that endanger the health of poultry, causing serious economic losses to the breeding industry. Therefore, rapid and accurate detection of these viruses is of great significance for effective prevention and control of disease transmission and protection of poultry health.

[0003] Currently, the commonly used detection methods mainly include polymerase chain reaction (PCR), fluorescent quantitative PCR and nucleic acid probe technology, etc. In China, PCR technology has been widely used in clinical detection, but it still has some obvious limitations. First, PCR detection relies on expensive equipment, limiting its application in small farms and grassroots detection institutions. Second, the PCR detection process is complex and time-consuming, usually taking more than 1 hour, which is difficult to meet the needs of on-site rapid diagnosis. Although fluorescent quantitative PCR has higher sensitivity and specificity, it requires higher technical requirements for operators, the addition process is complicated, and it is strongly dependent on equipment, which is not conducive to large-scale promotion.

[0004] In addition, in practical application, the existing detection methods also face many other problems. For example, during the sample addition process, aerosol pollution is easy to occur, leading to the occurrence of false positive results. At the same time, with the increasing number of mixed infection cases, traditional single pathogen detection methods have been difficult to meet the needs of simultaneous detection of multiple pathogens. Although conventional PCR and fluorescent quantitative PCR have high accuracy and sensitivity, in the application scene of multiple detection, repeated experiments, large amounts of reagents and occupation of more instrument resources are often required, which significantly increases the detection cost and work burden.

[0005] Therefore, it is urgent to develop a more efficient, convenient and accurate new detection technology to meet the actual needs of rapid diagnosis and on-site application of poultry diseases. SUMMARY

[0006] Therefore, the application provides a NDV, AIV and IBV triple rapid detection method based on a microfluidic chip and LAMP constant temperature amplification technology.

[0007] To achieve the above object, the application provides the following technical scheme.

[0008] The application provides a NDV, AIV and IBV triple rapid detection method based on a microfluidic chip and LAMP constant temperature amplification technology, characterized by comprising the following steps.

[0009] (1) Microfluidic chip manufacturing: LAMP specific primers designed for NDV, AIV and IBV are prepared into a storage solution with a concentration of 100 μmol / L, and the primer working solution is configured with a final concentration of F3 / B3: 0.2 μM, FIP / BIP: 1.6 μM, LF / LB: 0.8 μM; after the above-mentioned primer working solution is fully mixed (vortex oscillation for 10 s, instantaneous centrifugation for 5 s), it is embedded in each reaction chamber of the microfluidic chip, and after drying treatment, a fixed detection area is formed;

[0010] (2) Sample adding and sealing: after the LAMP reaction system containing the nucleic acid sample to be detected is fully mixed, it is added to the sample adding hole of the microfluidic chip in step (1), the sample adding hole and the air hole are sealed with a sealing film, and the sealing film is completely attached to the surface of the chip by using a scraping piece to remove air bubbles;

[0011] (3) Amplification and analysis: the microfluidic chip packaged in step (2) is placed in a matched detection instrument, low-speed centrifugation (1600 r / min, 10 s) and high-speed centrifugation (4600 r / min, 30 s) are sequentially performed, so that the reaction liquid is uniformly distributed to each reaction chamber; then LAMP constant temperature amplification reaction is carried out under constant temperature condition of 63.5℃, the reaction time is 30 min, and the fluorescence signal is collected in real time during the amplification process for result analysis.

[0012] Preferably, the LAMP primers include six primer sets designed for the respective target genes of NDV, AIV and IBV: F3, B3, FIP, BIP, LF and LB, wherein FIP and BIP are internal primers, F3 and B3 are external primers, and LF and LB are loop primers.

[0013] Preferably, the LAMP primers include six primer sets designed for the target gene of NDV: the nucleotide sequence of the F3 forward primer is shown in SEQ ID NO. 1, the nucleotide sequence of the B3 reverse primer is shown in SEQ ID NO. 2; the nucleotide sequence of the FIP internal forward primer is shown in SEQ ID NO. 3, the nucleotide sequence of the BIP internal reverse primer is shown in SEQ ID NO. 4; the nucleotide sequence of the LF loop forward primer is shown in SEQ ID NO. 5, and the nucleotide sequence of the LB loop reverse primer is shown in SEQ ID NO. 6.

[0014] Preferably, the LAMP primers include six primer sets designed for the target gene of AIV: the nucleotide sequence of the F3 forward primer is shown in SEQ ID NO. 7, the nucleotide sequence of the B3 reverse primer is shown in SEQ ID NO. 8; the nucleotide sequence of the FIP internal forward primer is shown in SEQ ID NO. 9, the nucleotide sequence of the BIP internal reverse primer is shown in SEQ ID NO. 10; the nucleotide sequence of the LF loop forward primer is shown in SEQ ID NO. 11, and the nucleotide sequence of the LB loop reverse primer is shown in SEQ ID NO. 12.

[0015] Preferably, the LAMP primers include six primer sets designed for the target gene of IBV: the nucleotide sequence of the F3 forward primer is shown in SEQ ID NO. 13, the nucleotide sequence of the B3 reverse primer is shown in SEQ ID NO. 14; the nucleotide sequence of the FIP internal forward primer is shown in SEQ ID NO. 15, the nucleotide sequence of the BIP internal reverse primer is shown in SEQ ID NO. 16; the nucleotide sequence of the LF loop forward primer is shown in SEQ ID NO. 17, and the nucleotide sequence of the LB loop reverse primer is shown in SEQ ID NO. 18.

[0016] Preferably, the microfluidic chip is provided with a plurality of mutually isolated reaction chambers, and each reaction chamber is pre-embedded with a specific primer combination to achieve simultaneous detection of NDV, AIV and IBV.

[0017] Preferably, the sealing film is a heat-sensitive pressure-sensitive film or an adhesive film, which has good sealing performance and heat conductivity, ensuring the airtightness and temperature uniformity during the reaction.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] Based on the microfluidic chip and the constant temperature amplification method, the present application independently develops a triple rapid detection technology for chicken respiratory diseases (NDV, AIV and IBV), which has the following beneficial effects compared with the existing detection technology:

[0020] ① Reduce the detection cost: Traditional PCR detection relies on expensive equipment, increasing the detection cost. The present application uses microfluidic chip technology, and its supporting detection instrument is more affordable than traditional PCR instrument, reducing the purchase cost of detection equipment, so that more farms and primary detection institutions can afford the detection fee, thereby improving the popularization rate of detection technology and better serving the prevention and control of poultry diseases.

[0021] ② Shorten the detection time: The existing detection method takes a long time, which cannot meet the demand of rapid detection. The LAMP detection technology based on the microfluidic chip can quickly complete the reaction under constant temperature conditions, and the detection time is shortened to 30 minutes, which saves about half the time compared with the traditional method. This has important significance for rapid diagnosis of poultry diseases and timely prevention and control measures, which can effectively reduce the risk of disease transmission.

[0022] ③ Realize multi-target joint detection: The current detection method usually needs to detect different pathogens respectively when facing mixed infection, which is complicated and easy to cause cross contamination. The present application realizes "single sample adding, threefold joint detection", that is, integrating NDV, AIV and IBV three groups of pre-embedded primers on one microfluidic chip, which can detect multiple pathogens in the same sample at the same time. In addition, the sample adding hole is sealed with sealing film during operation, which eliminates the risk of false positive caused by pollution, improves the accuracy and reliability of detection, meets the demand of clinical rapid detection, and is especially suitable for clinical detection of mixed infection of chicken viruses.

[0023] ④ Improve the on-site applicability: The traditional detection method has high requirements for laboratory environment and equipment, which is not convenient for rapid detection in on-site environment such as farms. The microfluidic chip detector of the present application is equipped with lithium battery, which is convenient to carry and use, without the need of complex laboratory conditions and a large number of equipment. The system is easy to operate, which greatly improves the on-site applicability of the detection technology, and can quickly provide detection results for farms to take corresponding prevention and control measures in time.

[0024] In summary, the present application integrates the highly integrated design of microfluidic chip and the efficient, rapid and constant temperature characteristics of LAMP amplification technology, realizes the synchronous, rapid and sensitive detection of three important poultry respiratory viruses, and has good application prospect and popularization value. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 NDV, AIV, IBV sensitivity amplification results in the embodiments of the present application (A is NDV, B is AIV, and C is IBV);

[0026] Figure 2 NDV, AIV, IBV repeatability amplification results in the embodiments of the present application (A is NDV, B is AIV, and C is IBV);

[0027] Figure 3 NDV, AIV, IBV cross-specificity amplification results in the embodiments of the present application (A is NDV, B is AIV, and C is IBV);

[0028] Figure 4 NDV, AIV, IBV specificity amplification results in the embodiments of the present application (A is NDV, B is AIV, and C is IBV). DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0030] The experimental methods used in the following embodiments are conventional methods unless otherwise specified.

[0031] The materials and reagents used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0032] The present application will be further described below with reference to the drawings and specific embodiments:

[0033] EMBODIMENT

[0034] The samples used in the test of the present embodiment include positive samples of Newcastle Disease Virus (NDV), Avian Influenza Virus (AIV) and Infectious Bronchitis Virus (IBV); plasmid standards containing target sequences of NDV, AIV and IBV are constructed respectively, and 10-fold gradient dilution is performed to 10 9 ~ 10 1 copies / μL; in addition, 120 clinical sample nucleic acids from multiple chicken farms are selected for actual application evaluation.

[0035] 1. Primer design:

[0036] In this embodiment, six LAMP primer sets were designed for each of the target genes of Newcastle disease virus (NDV), avian influenza virus (AIV) and chicken infectious bronchitis virus (IBV), including outer primers F3 and B3, inner primers FIP and BIP, loop primers LF and LB. The primer sequences and related parameters for each virus are shown in Table 1.

[0037] Table 1. NDV, AIV and IBV LAMP detection primer sequences and related parameters

[0038]

[0039] 2. In this embodiment, the specific steps of the NDV, AIV and IBV triple rapid detection method based on microfluidic chip and LAMP isothermal amplification technology are as follows:

[0040] (1) Fabrication of microfluidic chip: The LAMP specific primers designed for NDV, AIV and IBV were prepared into a storage solution with a concentration of 100 μmol / L, and the primer working solution was configured with a final concentration of F3 / B3: 0.2 μM, FIP / BIP: 1.6 μM, LF / LB: 0.8 μM. After the above primer working solution was thoroughly mixed (vortexed for 10 s and centrifuged for 5 s), it was embedded in each reaction chamber of the microfluidic chip, and after drying treatment, a fixed detection area was formed;

[0041] (2) Sample addition and sealing: After the LAMP reaction system containing the nucleic acid sample to be tested was thoroughly mixed, it was added to the sample addition hole of the microfluidic chip of step (1), and the sample addition hole and the air hole were sealed with a sealing film, and the air bubbles were removed using a scraping film to ensure that the sealing film was completely attached to the surface of the chip;

[0042] (3) Amplification and analysis: The microfluidic chip packaged in step (2) was placed in the matching detection instrument, and low-speed centrifugation program (1600 r / min, 10 s) and high-speed centrifugation program (4600 r / min, 30 s) were sequentially performed to evenly distribute the reaction solution to each reaction chamber. Then, LAMP isothermal amplification reaction was carried out under constant temperature conditions of 63.5℃ for 30 min, and the fluorescence signal was collected in real time during the amplification process for result analysis.

[0043] 3. Detection and analysis:

[0044] 3.1 To ensure the accuracy of the detection results, the following four types of controls were set in the test:

[0045] Negative control: sample without target nucleic acid, added to the chip sample addition hole alone;

[0046] Positive control: A standard sample containing the target nucleic acid, used as a positive reference;

[0047] Blank control: No primers were added to the reaction wells to eliminate background interference;

[0048] Internal control: Primers for amplifying the internal control gene are pre-embedded in the reaction wells, and an internal control plasmid is added to the reaction solution to monitor whether the entire amplification process proceeds normally.

[0049] 3.2 Result Judgment Criteria

[0050] (a) Quality control standard: If the internal control well and the positive control well show an S-shaped amplification curve when the Tt value is ≤30, and the blank control has no amplification signal, the test is considered valid; otherwise, it is considered invalid.

[0051] (b) Sample result determination: Positive: Tt value of detection well ≤ 30 and obvious S-shaped amplification curve appears; Negative: Tt value of detection well > 30, or no obvious amplification curve.

[0052] 3.3 Sensitivity Test

[0053] NDV, AIV, and IBV standard plasmids were serially diluted 10-fold (10... 6 ~10 1 The samples were analyzed using microfluidic chip LAMP assays (copies / μL), with a blank control group included to exclude the influence of non-specific amplification. Results showed that the limits of detection for NDV, AIV, and IBV all reached 10 copies / μL. 2 copies / μL, such as Figure 1 As shown, from Figure 1 As can be seen from (AC): in 10 2 At a concentration of copies / μL, all three viruses produced significant fluorescence signal growth, while below this concentration, they could not be effectively amplified, indicating that the method has high sensitivity and reliability.

[0054] 3.4 Repeatability Test

[0055] To evaluate the repeatability of the microfluidic chip LAMP method, 10 4 Eight replicate experiments were performed using NDV, AIV, and IBV standard plasmid DNA (copies / μL) as templates; the amplification results are as follows: Figure 2 As shown in Table 2, the amplification Tt values ​​(time thresholds) and repeatability test CV values ​​(coefficient of variation) for each concentration are presented.

[0056] Table 2. Repeatability test results (Tt values) for NDV, AIV, and IBV

[0057] Number of repetitions NDV AIV IBV 1 10.58 14.21 13.46 2 10.38 14.12 13.32 3 10.34 14.20 13.67 4 10.32 14.48 13.97 5 10.56 14.25 14.10 6 10.64 14.04 13.70 7 10.62 13.98 13.95 8 10.60 14.18 13.71 CV / % 1.27 1.06 1.92

[0058] As can be seen from Table 2, the CV values of the method are all less than 2%, specifically, the CV value of NDV is 1.27%, the CV value of AIV is 1.06%, and the CV value of IBV is 1.92%, which indicates that the microfluidic chip LAMP method established in the application has good repeatability and stability, and is suitable for high-precision virus detection.

[0059] Figure 2 Figure for the repeated amplification results of NDV (A), AIV (B) and IBV (C). In 8 repeated tests: ① The fluorescence intensity of NDV changes with time shows high consistency (A), and all curves reach the plateau at about the same time point, indicating that the amplification reaction has good repeatability; specifically, the Tt value of NDV ranges from 10.32 to 10.64 min, and the CV value is 1.27% (as shown in Table 2), which further confirms the stability of the method. ② The fluorescence amplification curve of AIV also shows high consistency (B), and the fluorescence signal growth trend in 8 repeated tests is similar, the Tt value ranges from 13.98 to 14.48 min, and the CV value is 1.06% (Table 2), indicating that the detection of AIV also has good repeatability and reliability. ③ The fluorescence amplification curve of IBV also shows good repeatability (C), and all curves reach the maximum fluorescence intensity at similar time points, the Tt value ranges from 13.46 to 14.10 min, and the CV value is 1.92% (Table 2), which further verifies the stability and consistency of the method in the detection of different viruses. Figure 2 Figure 2 Figure 2

[0060] 3.5 Cross-specificity test

[0061] In order to evaluate the cross-specificity of the microfluidic chip LAMP method, NDV, AIV and IBV standard plasmid DNAs with a concentration of 10 5 copies / μL were selected as templates, and the corresponding primers were used for amplification test. As shown in Figure 3 Figure 3 A, Figure 3 B and Figure 3 C are the curves of the fluorescence intensity of NDV, AIV and IBV at 10 5 copies / μL with time, specifically:

[0062] Figure 3 In A, when NDV primers are used for amplification, only the NDV plasmid produces significant fluorescence signal growth, while the fluorescence signals of AIV and IBV plasmids remain at the baseline level, indicating that the NDV primers have good specificity.

[0063] Figure 3 ​​​​In B, only AIV plasmid produced significant fluorescence signal increase when using AIV primers for amplification, while the fluorescence signals of NDV and IBV plasmids remained at baseline level, which proved that AIV primers also had good specificity.

[0064] Figure 3 In C, only IBV plasmid produced significant fluorescence signal increase when using IBV primers for amplification, while the fluorescence signals of NDV and AIV plasmids remained at baseline level, which further verified the specificity of IBV primers.

[0065] As can be seen, each primer can only amplify the corresponding plasmid, indicating that the method has good cross-specificity.

[0066] 3.6 Specificity test

[0067] In order to evaluate the specificity of the microfluidic chip LAMP method for NDV, AIV and IBV, the genomes of other common viruses such as chicken infectious laryngotracheitis, Marek's disease, infectious bursal disease, avian leukemia, and avian rotavirus disease were selected as templates, and the chip embedded with NDV, AIV, and IBV primers was used for amplification test to analyze its specificity. As shown in Figure 4 , Figure 4 A, Figure 4 B and Figure 4 C are the curves of fluorescence intensity of NDV, AIV and IBV at 105 copies / μL over time, specifically:

[0068] Figure 4 In A, only the NDV positive sample produced a significant "S" shape fluorescence signal increase (red curve) when using NDV primers for amplification, while the fluorescence signals of other virus samples remained at baseline level (blue and green curves), indicating that NDV primers have good specificity.

[0069] Figure 4 In B, only the AIV positive sample produced a significant "S" shape fluorescence signal increase (red curve) when using AIV primers for amplification, while the fluorescence signals of other virus samples remained at baseline level (blue and green curves), which proved that AIV primers also have good specificity.

[0070] Figure 4 In C, only the IBV positive sample produced a significant "S" shape fluorescence signal increase (red curve) when using IBV primers for amplification, while the fluorescence signals of other virus samples remained at baseline level (blue and green curves), which further verified the specificity of IBV primers.

[0071] It can be seen that only NDV, AIV and IBV three positive samples appeared obvious "S" shape amplification curve, while other sample detection results were negative, no cross reaction, indicating that the method has good specificity for the three viruses.

[0072] 3.7 Actual sample detection and coincidence rate analysis

[0073] In order to evaluate the performance of microfluidic chip LAMP method in actual sample detection, 120 actual sample nucleic acids were selected, which were subjected to microfluidic chip LAMP amplification and fluorescent quantitative PCR amplification respectively, and the results of the two methods were compared. And the detection limit of NDV, AIV and IBV three viruses was determined, as shown in Table 3.

[0074] Table 3 Comparison of the sensitivity of isothermal amplification of three avian viruses and fluorescent quantitative PCR

[0075]

[0076] As can be seen from Table 3, the detection limit of NDV, AIV and IBV using microfluidic chip LAMP method is 10 3 copies / μL, 10 4 copies / μL and 10 3 copies / μL, which is basically the same as the detection limit of fluorescent quantitative PCR method, indicating that the microfluidic chip LAMP method has the same performance as the fluorescent quantitative PCR method in terms of sensitivity.

[0077] The above discloses a kind of based on microfluidic chip and LAMP constant temperature amplification technology NDV, AIV and IBV three combined rapid detection method is introduced in detail. Specific examples are applied in this paper to describe the principle and implementation mode of the present application, the above example is only used to help understand the method of the present application and its core idea. It should be pointed out that for ordinary skilled person in the art, without departing from the principle of the present application, the present application can be improved and modified, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A method for rapid detection of NDV, AIV and IBV based on microfluidic chip and LAMP isothermal amplification technology, characterized in that, Comprising the following steps: (1) Fabrication of microfluidic chip: LAMP specific primers designed for NDV, AIV and IBV were prepared into storage solution with a concentration of 100 μmol / L, and the primer working solution was configured with a final concentration of F3 / B3: 0.2 μM, FIP / BIP: 1.6 μM, LF / LB: 0.8 μM; after the above-mentioned primer working solution was mixed thoroughly (vortex oscillation for 10 s, instantaneous centrifugation for 5 s), it was embedded in each reaction chamber of the microfluidic chip, and after drying treatment, a fixed detection area was formed; (2) Sample addition and sealing: after the LAMP reaction system containing the nucleic acid sample to be tested was mixed thoroughly, it was added to the sample addition hole of the microfluidic chip in step (1), the sample addition hole and the air hole were sealed with a sealing film, and the sealing film was completely attached to the surface of the chip by using a scraping film to remove air bubbles; (3) Amplification and analysis: the microfluidic chip packaged in step (2) was placed in a matched detection instrument, and low-speed centrifugation program (1600 r / min, 10 s) and high-speed centrifugation program (4600 r / min, 30 s) were sequentially performed to uniformly distribute the reaction solution to each reaction chamber; then LAMP constant temperature amplification reaction was carried out under constant temperature condition of 63.5℃, the reaction time was 30 min, and the fluorescence signal was collected in real time during the amplification process for result analysis. The LAMP primers include six primer groups designed for respective target genes of NDV, AIV and IBV: F3, B3, FIP, BIP, LF and LB, wherein FIP and BIP are internal primers, F3 and B3 are external primers, and LF and LB are loop primers. 2.The NDV, AIV and IBV triple rapid detection method based on the microfluidic chip and LAMP constant temperature amplification technology according to claim 1, characterized in that, The LAMP primers include six primer groups designed for NDV target genes: the nucleotide sequence of the F3 forward primer is shown as SEQ ID NO. 1, the nucleotide sequence of the B3 reverse primer is shown as SEQ ID NO. 2; the nucleotide sequence of the FIP internal forward primer is shown as SEQ ID NO. 3, the nucleotide sequence of the BIP internal reverse primer is shown as SEQ ID NO. 4; the nucleotide sequence of the LF loop forward primer is shown as SEQ ID NO. 5, and the nucleotide sequence of the LB loop reverse primer is shown as SEQ ID NO.

6. 3.The NDV, AIV and IBV triple rapid detection method based on the microfluidic chip and LAMP constant temperature amplification technology according to claim 2, characterized in that, The LAMP primers include six primer groups designed for AIV target genes: the nucleotide sequence of the F3 forward primer is shown as SEQ ID NO. 7, the nucleotide sequence of the B3 reverse primer is shown as SEQ ID NO. 8; the nucleotide sequence of the FIP internal forward primer is shown as SEQ ID NO. 9, the nucleotide sequence of the BIP internal reverse primer is shown as SEQ ID NO. 10; the nucleotide sequence of the LF loop forward primer is shown as SEQ ID NO. 11, and the nucleotide sequence of the LB loop reverse primer is shown as SEQ ID NO.

12.

4. The method according to claim 2, wherein the method is used for detecting NDV, AIV and IBV simultaneously. ​ 5. A rapid triple detection method for NDV, AIV, and IBV based on microfluidic chip and LAMP isothermal amplification technology according to claim 2, characterized in that, The LAMP primer comprises six primer groups designed for IBV target genes: the nucleotide sequence of the F3 forward primer is shown as SEQ ID NO. 13, the nucleotide sequence of the B3 reverse primer is shown as SEQ ID NO. 14; the nucleotide sequence of the FIP internal forward primer is shown as SEQ ID NO. 15, the nucleotide sequence of the BIP internal reverse primer is shown as SEQ ID NO. 16; the nucleotide sequence of the LF loop forward primer is shown as SEQ ID NO. 17, and the nucleotide sequence of the LB loop reverse primer is shown as SEQ ID NO.

18. 6.The NDV, AIV and IBV triple rapid detection method based on a microfluidic chip and LAMP constant temperature amplification technology according to claim 1, wherein, The microfluidic chip is provided with a plurality of mutually isolated reaction chambers, and each reaction chamber is pre-embedded with a specific primer combination, so as to simultaneously detect NDV, AIV and IBV.

7. The method according to claim 1, wherein the method is used for detecting NDV, AIV and IBV simultaneously. The sealing film is a heat-sensitive pressure-sensitive film or a viscous film, which has good sealing performance and heat conductivity, and ensures the airtightness and temperature uniformity during the reaction process.

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