Method for detecting chicken infectious bursal disease virus based on RAA-CRISPR / cas13a-LFD

Abstract

The invention relates to the technical field of virus detection, and particularly discloses a method for detecting a chicken infectious bursal disease virus based on RAA-CRISPR / Cas13a-LFD. The sequence of the upstream primer for detecting the infectious bursal disease virus is as shown in SEQ ID NO: 1, the sequence of the downstream primer is as shown in SEQ ID NO: 2, and the sequence of the specific crRNA is as shown in SEQ ID NO: 3. According to the method provided by the invention, the target gene can be effectively amplified by reacting for 40 minutes under the condition of 37 DEG C, and a test result can be judged through LFD naked eye observation; the specificity is good, and the lowest detection limit can reach 100 copies / mu L; good repeatability and stability are realized; the coincidence rate of clinical detection reaches 98.33%, and the kit can be used for clinical rapid diagnosis and prevention and control of the infectious bursal disease (IBD).

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to a method for detecting infectious bursal disease virus of chickens based on RAA-CRISPR / Cas13a-LFD. Background Art

[0002] Infectious bursal disease (IBD) is an acute, highly contagious and immunosuppressive infectious disease caused by the infectious bursal disease virus (IBDV) that mainly infects chicks. The disease mainly infects chickens within 3-6 weeks of age, mainly invading the central immune organ of chicks - the bursa of Fabricius, thereby causing immunosuppression, and then causing vaccine immunization failure or secondary other viral and bacterial diseases. Severe cases can lead to mass deaths of chickens, causing huge economic losses to the poultry industry. There are two serotypes of IBDV. Serotype 1 includes all virus strains, and only serotype I is pathogenic to chickens. Serotype 2 is neither pathogenic nor protective against serotype 1. The virus is mainly excreted with the feces of sick chickens, and then contaminates feed, water and the surrounding environment, thereby infecting chickens in the same chicken house. The infection transmission rate is extremely high and can spread rapidly between sick chickens, latently infected chickens, and susceptible chickens. Therefore, early prevention is crucial to reducing the risk of disease transmission and controlling the epidemic in a timely manner, and rapid detection of IBDV plays a key role in promoting early prevention and control.

[0003] At present, there are many methods for detecting IBDV, such as virus isolation and identification, polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP) and enzyme-linked immunosorbent assay (ELISA). Although the commonly used virus isolation and identification methods are relatively accurate, they are cumbersome to operate and have high requirements for culture conditions. The ELISA method has good specificity, but low sensitivity and complex operation. LAMP can react at a constant temperature of 60-65℃, has strong specificity and high amplification efficiency, but the primer design is relatively complex. Although PCR and qPCR methods have high sensitivity, they often rely on expensive equipment and professionals, have long reaction times, cumbersome operation steps, and are often prone to non-specific reactions. They are not suitable for on-site rapid diagnosis of IBDV. Therefore, the development of a rapid, easy-to-operate, highly specific and highly sensitive rapid detection method for infectious bursal disease virus in chickens is of great significance for the early prevention and treatment of infectious bursal disease in chickens. Summary of the invention

[0004] In view of the problems of existing methods for detecting infectious bursal disease virus of chickens, such as long time consumption, poor specificity, low sensitivity and unsuitability for grassroots and on-site detection, the present invention provides a method for detecting infectious bursal disease virus of chickens based on RAA-CRISPR / Cas13a-LFD. The primers provided by the present invention can realize visual detection of infectious bursal disease virus of chickens, and the detection has high accuracy, short time consumption, good specificity and high sensitivity, which provides a basis for rapid, simple and accurate diagnosis of IBDV and meets the needs of grassroots and on-site detection.

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

[0006] In the first aspect, the present invention provides a nucleic acid molecule composition for detecting infectious bursal disease virus of chickens based on RAA-CRISPR / Cas13a-LFD, comprising a primer pair and a specific crRNA, wherein the sequence of the primer pair is as follows:

[0007] Upstream primer IBDV-RAA-F: 5′-TCTGCAACAGCCAACATCAACGACAAAATT-3′ (SEQ ID NO: 1),

[0008] Downstream primer IBDV-RAA-R: 5′-TGCTGTCACATGTGGCTACCATTTTTGGGT C-3′ (SEQ ID NO: 2);

[0009] The sequence of the specific crRNA is:

[0010] IBDV-crRNA: 5′-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCAUAUGAUGUGGGUAAGCUGAGGACGGU-3′ (SEQ ID NO: 3).

[0011] Compared with the prior art, the nucleic acid molecule composition for detecting infectious bursal disease virus provided by the present invention combines RAA, CRISPR / Cas13a and lateral flow test strip (LFD), designs RAA primers and specific crRNA according to the conservative sequence of VP2 gene of IBDV, and can accurately detect infectious bursal disease virus of chickens by using the primers and specific crRNA, and has no cross-amplification reaction with Marek's virus (MDV), avian leukosis virus (ALV), Newcastle disease virus (NDV), chicken anemia virus (CAV), infectious bronchitis virus (IBV) and the like, and has strong specificity; the minimum detection limit for IBDV is 10 0 copies / μL(10 -2fg / μL), which can realize the early and rapid diagnosis of IBDV. At the same time, the detection accuracy is high, the clinical detection compliance rate reaches 98.33%, the detection time is significantly shortened, and no special instruments and professional operators are required. It realizes the purpose of rapid visual detection of IBDV under non-laboratory conditions. It is suitable for health institutions or epidemic sites with relatively simple instrument conditions, can better meet clinical detection needs, and is of great significance to the prevention and control of IBDV epidemics.

[0012] Furthermore, a T7 promoter is provided at the 5′ end of the upstream primer IBDV-RAA-F.

[0013] The sequence of the T7 promoter is: GAAATTAATACGACTCACTATAGGG.

[0014] In a second aspect, the present invention further provides use of any of the above-mentioned nucleic acid molecule compositions in non-diagnostic detection of infectious bursal disease virus.

[0015] In a third aspect, the present invention further provides a kit for detecting infectious bursal disease virus of chickens, comprising any of the nucleic acid molecule compositions described above.

[0016] Preferably, the kit also includes Cas13a protein and a reagent or kit for RAA amplification.

[0017] In a fourth aspect, the present invention also provides the use of the above-mentioned kit in non-diagnostic detection of infectious bursal disease virus.

[0018] In a fifth aspect, the present invention also provides a method for detecting infectious bursal disease virus of chickens using the above kit, the specific operation being:

[0019] S1, extracting the genome of infectious bursal disease virus of chicken as a template, and performing RAA amplification using a primer pair consisting of an upstream primer IBDV-RAA-F and a downstream primer IBDV-RAA-R of the kit;

[0020] S2, adding the RAA amplification product to the CRISPR-Cas13a system for incubation; the CRISPR-Cas13a system includes the specific crRNA in the kit;

[0021] S3, detection of CRISPR-Cas13a incubation products using LFD.

[0022] The above method for detecting infectious bursal disease virus in chickens is time-saving, simple to operate, and has intuitive reaction results. It can be used for the rapid clinical diagnosis and prevention and control of infectious bursal disease virus in chickens, and can greatly reduce the occurrence of misdiagnosis and omission of IBDV infection, which is of great significance for purifying IBDV.

[0023] The detection principle of the RAA-CRISPR / Cas13a-LFD method provided by the present invention is as follows Figure 1 As shown. Nucleic acid is extracted from the sample, and a DNA molecule with a T7 promoter is obtained after rapid amplification through the RAA reaction. A large amount of single-stranded RNA (ssRNA) containing the target sequence is obtained using the T7 in vitro transcription system. When the target sequence is present, the target RNA specifically binds to the crRNA-Cas13a protein complex, activating the "incidental cleavage" activity of the Cas13a protein, cutting the 6-FAM-Biotin reporter molecule, and the complex of the FAM end of the reporter molecule and the colloidal gold particles labeled with the FAM antibody is bound by the secondary antibody, and the T line appears, which is positive; when the target sequence is not present, Cas13a will not be activated, and the cleavage function will not be performed. The streptavidin at the C line captures the Biotin end of the complex of the reporter molecule and the colloidal gold particles labeled with the FAM antibody, and only the C line appears, which is negative.

[0024] Preferably, in S1, the RAA amplification system includes the following reagents and amounts: 32.9 μL of basic buffer A, 2.0 μL of upstream primer and downstream primer respectively, 2 μL of cDNA template, 2.5 μL of basic buffer B, and purified water supplemented to 50 μL; wherein the concentrations of the upstream primer and downstream primer are both 10 μmol / L, and the concentration of the DNA template is 115.4 μg / mL.

[0025] Furthermore, the basic buffer A is A buffer for RAA amplification, the main component of which is polyethylene glycol; the basic buffer B is B buffer for RAA amplification, the main component of which is Mg 2+ .

[0026] Preferably, in S2, the CRISPR-Cas13a system includes the following reagents and dosages: 25mmol / L NTPbuffer mix 4μL, 40U / μL RNase inhibitor 2μL, 50nmol / L Cas13a 4μL, 25ng / μL crRNA 2μL, RNA dual-labeled probe 2μL, 5000U / mL T7 RNA polymerase mix 1μL, 1mol / L magnesium chloride solution 0.5μL, 1mol / L HEPES buffer 1μL, enzyme-free sterile water 28.5μL and RAA amplification product 5μL.

[0027] The above-mentioned preferred reaction conditions can further improve the detection efficiency of infectious bursal disease virus.

[0028] Preferably, in S1, the reaction temperature of the RAA amplification is 37° C., and the reaction time is 20 min.

[0029] Preferably, in S2, the incubation temperature is 37° C. and the incubation time is 20 min.

[0030] Furthermore, when the CRISPR-Cas13a incubation product was detected using LFD, two red bands appeared on LFD for positive results, one in the quality control area and one in the detection area. The positive result indicated that the amplified product contained the nucleic acid fragment to be detected; a red band appeared in the quality control area of ​​LFD and there was no red band in the detection area, indicating a negative result. The negative result indicated that the amplified product did not contain the detection fragment.

[0031] The present invention designs specific crRNA and RAA primers according to the relatively conservative sequence of infectious bursal disease virus of chickens. When crRNA recognizes the target sequence, the Cas13a protein is activated to cut the reporter probe. With the use of lateral flow test strips (LFD) to read the test results, the purpose of simple, efficient and visual detection of IBDV is achieved. The RAA-CRISPR / Cas13a-LFD method provided by the present invention can achieve effective amplification of the target gene under 37°C conditions and react for 40 minutes. The test results can be determined by naked eye observation of LFD; the specificity is good, and there is no cross reaction with Marek's disease virus (MDV), avian leukosis virus (ALV), Newcastle disease virus (NDV), chicken anemia virus (CAV), and infectious bronchitis virus (IBV); the minimum detection limit can reach 10 0 copies / μL; has good repeatability and stability; clinical detection compliance rate reaches 98.33%. In summary, the present invention establishes a simple, efficient, accurate, and visually detectable IBDV detection method, which can be used for clinical rapid diagnosis and prevention of infectious bursal disease (IBD) in chickens. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the detection principle of the RAA-CRISPR / Cas13a-LFD method for detecting infectious bursal disease virus of chickens provided by the present invention;

[0033] Figure 2 is a primer screening result diagram in Example 1 of the present invention;

[0034] Figure 3 1 is a graph showing the results of the optimization of the RAA-CRISPR / Cas13a-LFD method for detecting infectious bursal disease virus in chickens in Example 1 of the present invention; wherein A is the screening of RAA amplification time; B is the screening of the optimal Cas13a concentration; C is the screening of the optimal crRNA concentration; and D is the screening of the optimal CRISPR reaction time;

[0035] Figure 4: is a specificity test result diagram of the RAA-CRISPR / Cas13a-LFD method in Example 2 of the present invention; NC is a negative control;

[0036] Figure 5 : This is a sensitivity test result diagram of the RAA-CRISPR / Cas13a-LFD method in Example 3 of the present invention; wherein A, B, and C are the results of RAA-CRISPR / Cas13a-LFD, PCR method, and qPCR method for detecting IBDV, respectively;

[0037] Figure 6 This is a diagram of the repeatability test results of the RAA-CRISPR / Cas13a-LFD method in Example 4 of the present invention; NC is a negative control. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0039] In order to better illustrate the embodiments of the present invention, further examples are given below.

[0040] Example 1

[0041] 1. Materials and Methods

[0042] 1.1 Reagents and instruments

[0043] Virus genome DNA / RNA extraction kit and plasmid extraction kit were purchased from Tiangen Biochemical Technology Co., Ltd. DH5α competent cells were purchased from Beijing Bomade Gene Technology Co., Ltd. Column RNA rapid concentration and purification kit and SanPrep column DNA gel recovery kit were purchased from Shanghai Shenggong Biotechnology Co., Ltd. RNA isothermal amplification reaction kit was purchased from Suzhou Jinnuo Biomedical Technology Co., Ltd. 2×Universal Blue SYBR Green qPCR Master Mix was purchased from Wuhan Saiweier Biotechnology Co., Ltd., T-Vector pMDTM 20 vector and RNaseInhibitor were purchased from TaKaRa. NTP buffer mix, T7 RNA polymerase mix, and Standard Taqbuffer (10×) were purchased from New England Biolabs. HEPES was purchased from Solarbio. MgCl2 was purchased from Coolaber. RNA FAM and Biotin dual-labeled probes and Cas12 / Cas13a test strips were purchased from Guangzhou Bolais Technology Biological Co., Ltd. Cas13a protein was purchased from Meige Biotechnology.

[0044] The thermostatic amplification instrument was purchased from Jiangsu Qitian Biotechnology Co., Ltd.

[0045] 1.2. Nucleic acid extraction

[0046] IBDV, ALV, MDV, NDV, and CAV are all strains preserved in our laboratory, and IBV (AV1511) was purchased from the China Veterinary Drug Administration. DNA of MDV and CAV and RNA of IBDV, ALV, NDV, and IBV were extracted using a viral genomic DNA / RNA extraction kit, and the RNA of IBDV, ALV, NDV, and IBV was reverse transcribed into cDNA and stored at -20°C for future use.

[0047] 1.3. Primer and crRNA design and primer screening

[0048] According to the conserved region in the VP2 gene sequence of IBDV logged in GenBank (accession number: DQ202329.1), three pairs of specific RAA primers were designed, IBDV-RAA-F1 (SEQ ID NO: 4), IBDV-RAA-F2 (SEQ ID NO: 5), IBDV-RAA-F3 (SEQ ID NO: 1), IBDV-RAA-R1 (SEQ ID NO: 2), IBDV-RAA-R2 (SEQ ID NO: 6), IBDV-RAA-R3 (SEQ ID NO: 7), and the T7 RNA polymerase initiation sequence (GAAATTAATACGACT CACTATAGGG, SEQ ID NO: 8) was added to the 5' end of the upstream primer. In addition, crRNA was designed in the conserved sequence of the VP2 gene, and crRNA consisted of a LwaCas13a fixed stem-loop structure and a target sequence of 28 nt in length, which was included in the primer amplification region. Primers, crRNA in vitro transcription template (crRNA-IVT, SEQ ID NO: 9) and T7-3G oligonucleotide were synthesized by Shanghai Shenggong Biotechnology Co., Ltd. All nucleic acid sequences used in the experiment are shown in Table 1. IBDV-PCR-F (SEQ ID NO: 10) and IBDV-PCR-R (SEQ ID NO: 11) in the table were used in the PCR-agarose electrophoresis sensitivity verification of this experiment.

[0049] Table 1 Primers and crRNA

[0050]

[0051]

[0052] Using IBDV cDNA as template, RAA amplification was performed. The reaction system was 50 μL, including 32.9 μL Abuffer, 8.6 μL purified water, 2.0 μL upstream primer (10 μM), 2.0 μL downstream primer (10 μM), 2 μL cDNA template (115.4 μg / mL), and 2.5 μL Bbuffer. Amplification was performed at 37°C for 30 min. The amplified product was purified and the results were observed by 1.5% agarose electrophoresis to screen the best primer combination. The results are shown in Figure 2 shown.

[0053] The results showed that dimers appeared in the amplification of IBDV-RAA-F1 / R1, F1 / R2, and F1 / R3, and the amplification efficiency of IBDV-RAA-F3 / R1, F3 / R2, and F3 / R3 primer combinations was higher than that of other primer combinations. Among them, the F3 / R1 primer combination did not show nonspecific amplification. Therefore, the IBDV-RAA-F3 / R1 combination was selected as the best primer.

[0054] 1.4 In vitro transcription and purification of crRNA

[0055] Mix 10 μL of reaction system: crRNA-IVT 1 μL (100 μM), T7-3G oligonucleotide 1 μL (100 μM), Standard Taq buffer (10×) 1 μL, and ultrapure water 7 μL, denature at 95 °C for 5 min in a PCR instrument, and then slowly cool to 4 °C at 0.1 °C / s. After the reaction, mix 10 μL of reaction product with 10 μL of NTP buffer mixture, 2 μL of T7 RNA polymerase mixture, and 17 μL of ultrapure water, transcribe overnight at 37 °C, and slowly cool to 4 °C at 0.1 °C / s. After the in vitro transcription is completed, purify the crRNA using a column-type RNA rapid concentration and purification kit, measure the concentration, and store at -80 °C for use.

[0056] 1.5 Kit for detecting infectious bursal disease virus in chickens Establishment of a method for detecting infectious bursal disease virus in chickens

[0057] The above-mentioned kit includes a RAA reaction system and a CRISPR / Cas13a-LFD reaction system.

[0058] Extract the genomic DNA of the virus to be tested as a template and prepare a 50μL RAA reaction system: A buffer 32.9μL, purified water 8.6μL, upstream primer 2.0μL (10μM), downstream primer 2.0μL (10μM), cDNA template 2μL (115.4μg / mL), Bbuffer 2.5μL. First, add the above reaction reagents to the reaction tube unit containing 3.45mg RAA dry powder in sequence, cover the tube, mix thoroughly by turning upside down 8-10 times, centrifuge at low speed for 10s, place in a constant temperature amplification instrument, and amplify at 37°C for 30min.

[0059] Prepare 50μL CRISPR-Cas13a-LFD reaction system: 25mmol / L NTP buffer mix 4μL, 40U / μLRNase inhibitor 2μL, 80nmol / L Cas13a 4μL, 80ng / μL crRNA 2μL, 10μmol / L RNA dual-labeled probe 2μL, 5000U / mL T7 RNA polymerase mix 1μL, 1mol / L magnesium chloride solution 0.5μL, 1mol / L HEPES buffer 1μL, enzyme-free sterile water 28.5μL and RAA amplification product 5μL. Mix the above reactants thoroughly and incubate at 37℃ for 25min. Then, add 50μL of the reaction product to LFD and observe the results within 3-5min.

[0060] When two strips appear on the test strip, one in the quality control area and the other in the test area, the result is recorded as positive. When there is only one red strip in the quality control area, the result is recorded as negative.

[0061] Table 2 Primers and specific crRNA

[0062]

[0063] 1.5 Optimization of RAA-CRISPR / Cas13a-LFD method for detecting infectious bursal disease virus in chickens

[0064] The above reaction system was used to optimize the factors affecting the amplification efficiency, including RAA amplification time, Cas13a concentration, crRNA concentration and CRISPR reaction time.

[0065] The RAA amplification time was set to start from 10 min and increase in a gradient of every 5 min. When a clear detection line appeared, this time was selected as the optimal RAA amplification time.

[0066] The Cas13a concentration was set to 40 nmol / L and increased in a gradient of 10 nmol / L. The other factors remained unchanged. When a clear detection line appeared, this concentration was selected as the optimal Cas13a concentration.

[0067] At the optimal Cas13a concentration, the crRNA concentration was set to 10 ng / μL and increased in a gradient of 5 ng / μL. The other factors remained unchanged. When a clear line appeared, this concentration was selected as the optimal crRNA concentration.

[0068] At the optimal Cas13a concentration and the optimal crRNA concentration, the CRISPR reaction time was set to 10 minutes, and the gradient was increased every 5 minutes until a clear line appeared. This time was selected as the optimal CRISPR reaction time. Figure 3 shown.

[0069] The test results show that the optimal reaction conditions for RAA-CRISPR / Cas13a-LFD are RAA amplification time of 20 minutes, crRNA concentration of 25ng / μL, and CRISPR reaction time of 20 minutes. When the Cas13a concentration is 50nmol / L and 60nmol / L, clear detection lines appear, and the optimal Cas13a concentration is 50nmol / L.

[0070] Example 2

[0071] Specificity test

[0072] Primers IBDV-RAA-F, IBDV-RAA-R and specific crRNA were used, DNA of MDV and CAV and cDNA of IBDV, ALV, NDV and IBV were used as templates, ddH2O was set as a negative control, and RAA-CRISPR / Cas13a-LFD detection was performed under the above optimized conditions to observe whether red bands appeared in the detection area to verify the specificity of the reaction.

[0073] Test results such as Figure 4 As shown in the figure, it can be seen that only the test result of IBDV is positive, and no detection line appears for other viruses and negative controls. It proves that the RAA-CRISPR / Cas13a-LFD detection method has good specificity for detecting ALV.

[0074] Example 3

[0075] Sensitivity test

[0076] 3.1 Construction of standard plasmid

[0077] PCR amplification was performed using IBDV cDNA as a template, with upstream primer ALV-PCR-F (5'-ACTGACAGATGTTAGCTACA-3', SEQ ID NO: 10) and downstream primer ALV-PCR-R (5'-GCAGTTATGGTGTAGACTCT-3', SEQ ID NO: 11).

[0078] The PCR reaction system was 25 μL, including 12.5 μL of 2×Taq Mix, 0.5 μL of upstream primer (10 μM), 0.5 μL of downstream primer (10 μM), 1 μL of DNA template (115.4 μg / mL), and 10.5 μL of ddH2O. The reaction program was: 94°C for 5 min, 94°C for 30 s, 47°C for 30 s, 72°C for 30 s, 35 cycles, and 72°C for 5 min extension.

[0079] The PCR product was verified by 2% agarose gel electrophoresis, and the DNA was recovered by using the SanPrep column DNA gel recovery kit. The recovered DNA was connected to the T-Vector PMD 20 vector and introduced into DH5α competent cells. After overnight culture, blue-white screening was performed, and colonies were selected and plasmids were extracted using a plasmid mini kit. Standard plasmids were sequenced by Shanghai Sangon Biotechnology Co., Ltd.

[0080] According to Moore's law, the number of DNA copies per unit volume of plasmid is calculated as follows:

[0081] Plasmid copy number (copies / μL) = [plasmid concentration (g / μL) × 10 -9 ×6.02×1023 ] / {[vector length (bp) + fragment length (bp)] × 660 g / mol}

[0082] The constructed standard plasmid was diluted to 10 9 -10 0 copies / μL, stored at -20℃ for future use.

[0083] 3.2 Sensitivity testing

[0084] The IBDV standard plasmid was diluted to different concentrations (10 7 -10 0 copies / μL) as a template and ddH2O as a negative control, and PCR-agarose gel electrophoresis, qPCR and RAA-CRISPR / Cas13a-LFD methods were used to determine the minimum detection limit.

[0085] The PCR reaction system was 25 μL, including 2× Taq Mix 12.5 μL, PCR upstream primer (10 μM) 0.5 μL, PCR downstream primer (10 μM) 0.5 μL, template (10 7 -10 0 The reaction procedure was as follows: 94°C for 5 min, 94°C for 30 s, 47°C for 30 s, 72°C for 30 s, 35 cycles, and 72°C for 5 min extension. The PCR product was subjected to agarose gel electrophoresis to determine the minimum detection limit.

[0086] The qPCR reaction system was 20 μL, including 2×Universal Blue SYBR Green qPCR Master Mix 10 μL, PCR upstream primer (10 μM) 0.4 μL, PCR downstream primer (10 μM) 0.4 μL, template (10 6 -10 0 The reaction procedure was as follows: 95°C for 30 s, 95°C for 15 s, 60°C for 30 s, and the results were observed after 40 cycles.

[0087] RAA-CRISPR / Cas13a-LFD detection method 5 -10 0 The optimized reaction system was used as a template to determine the minimum detection limit of the method.

[0088] The sensitivity evaluation results showed that the minimum detection limit of the PCR method was 10 3The minimum detection limit of the qPCR method is 10 1 copies / μL, the minimum detection limit of RAA-CRISPR / Cas13a-LFD is 10 0 copies / μL, which is 10 times more sensitive than the qPCR method and 1000 times more sensitive than the PCR method. Figure 5 shown.

[0089] Example 4

[0090] Repeatability test

[0091] The optimized RAA-CRISPR / Cas13a-LFD method was used to dilute the IBDV standard plasmid at three different concentrations (10 5 copies / μL, 10 3 copies / μL, 10 0 The repeatability of the method was tested by using 1000 copies / μL) as the template and repeating the experiment three times for each concentration, and setting ddH2O as the negative control. Figure 6 shown.

[0092] The results of the repeatability test showed that standard plasmids of different concentrations were all positive, and the control groups were all negative, indicating that the detection method has good repeatability and stability.

[0093] Example 5

[0094] Clinical sample testing

[0095] The 120 samples of bursa and spleen of sick chickens initially diagnosed with IBD based on clinical symptoms were tested by PCR-agarose electrophoresis and RAA-CRISPR / Cas13a-LFD respectively. The coincidence rate of the two methods was calculated based on the PCR method. The results are shown in Table 3.

[0096] Coincidence rate (%) = (number of all positive samples in the two methods compared + number of all negative samples) / total number of samples × 100%

[0097] Table 3 Clinical sample test results

[0098]

[0099] The results of IBDV clinical sample testing showed that RAA-CRISPR / Cas13a-LFD detected 28 positive samples, with a consistency rate of 98.33% with the PCR-agarose electrophoresis method. After sequencing the target fragments of the 28 positive samples detected by RAA-CRISPR / Cas13a-LFD, it was determined that they were all IBDV positive, indicating that the RAA-CRISPR / Cas13a-LFD method has more accurate detection capabilities and can be used for clinical testing.

[0100] In summary, the present invention has established a RAA-CRISPR / Cas13a-LFD method for rapid detection of infectious bursal disease virus in chickens, and the results are determined by using lateral flow test strips. The RAA-CRISPR / Cas13a-LFD method can complete the detection of the target gene in only 40 minutes. The targeted cutting ability of Cas13a combined with crRNA makes this method highly specific. The combination of RAA technology and CRISPR / Cas system improves the sensitivity of detection, and the minimum detection limit can reach 10 0 copies / μL, which is 10 times more sensitive than the qPCR method and 1000 times more sensitive than the PCR method. It realizes the rapid visual detection of infectious bursal disease virus in non-laboratory conditions. It has the advantages of being fast, sensitive, convenient and highly accurate. It provides a reliable technology for the diagnosis and prevention of infectious bursal disease in chickens and has broad application prospects.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A nucleic acid molecule composition for detecting infectious bursal disease virus of chickens based on RAA-CRISPR / Cas13a-LFD, characterized in that: It includes a primer pair and a specific crRNA, and the sequence of the primer pair is as follows: Upstream primer IBDV-RAA-F: 5′-TCTGCAACAGCCAACATCAACGACAAAATT-3′; Downstream primer IBDV-RAA-R: 5′-TGCTGTCACATGTGGCTACCATTTTTGGGT C-3′; The sequence of the specific crRNA is: IBDV-crRNA: 5′-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCAUAUGAUGUGGGUAAGCUGAGGACGGU-3′.

2. Use of the nucleic acid molecule composition according to claim 1 in non-diagnostic detection of infectious bursal disease virus.

3. A kit for detecting infectious bursal disease virus of chickens, characterized in that: The invention comprises the nucleic acid molecule composition according to claim 1.

4. The kit according to claim 3, characterized in that The kit also includes a Cas13a protein and a reagent or kit for RAA amplification.

5. Use of the kit according to claim 3 or 4 in non-diagnostic detection of infectious bursal disease virus.

6. A method for detecting infectious bursal disease virus of chickens using the kit according to claim 3 or 4, characterized in that: The specific operations are: S1, extracting the genome of infectious bursal disease virus of chicken as a template, and performing RAA amplification using a primer pair consisting of an upstream primer IBDV-RAA-F and a downstream primer IBDV-RAA-R of the kit; S2, adding the RAA amplification product to the CRISPR-Cas13a system for incubation; the CRISPR-Cas13a system includes the specific crRNA in the kit; S3, detection of CRISPR-Cas13a incubation products using LFD.

7. The method for detecting infectious bursal disease virus of chickens according to claim 6, characterized in that: In S1, the RAA amplification system includes the following reagents and dosages: 32.9 μL of basic buffer A, 2.0 μL of upstream primer and downstream primer respectively, 2 μL of cDNA template, 2.5 μL of basic buffer B, and purified water supplemented to 50 μL; wherein, the concentrations of the upstream primer and downstream primer are both 10 μmol / L, and the concentration of the DNA template is 115.4 μg / mL.

8. The method for detecting infectious bursal disease virus of chickens according to claim 6, characterized in that: In S2, the CRISPR-Cas13a system includes the following reagents and dosages: 25mmol / L NTP buffer mix 4μL, 40U / μL RNase inhibitor 2μL, 50nmol / L Cas13a 4μL, 25ng / μL crRNA 2μL, RNA dual-labeled probe 2μL, 5000U / mL T7RNA polymerase mix 1μL, 1mol / L magnesium chloride solution 0.5μL, 1mol / L HEPES buffer 1μL, enzyme-free sterile water 28.5μL and RAA amplification product 5μL.

9. The method for detecting infectious bursal disease virus of chickens according to claim 6, characterized in that: In S1, the reaction temperature of the RAA amplification is 37° C., and the reaction time is 20 min.

10. The method for detecting infectious bursal disease virus of chickens according to claim 6, characterized in that: In S2, the incubation temperature is 37° C. and the incubation time is 20 min.

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