Duck tembusu virus nucleic acid detection kit and detection method based on RT-LAMP and CRISPR / Cas12a technologies

Abstract

The invention discloses a duck tembusu virus nucleic acid detection kit and a detection method based on a CRISPR-Cas12a system combined with RT-LAMP mediation, an RT-LAMP primer group and crRNA of the detection kit have high sensitivity and high specificity, and a detection result can be rapidly and sensitively presented under a blue light gel cutting instrument. The positive coincidence rate and the negative coincidence rate of duck tembusu virus detection results of the detection method and RT-qPCR and RT-PCR methods are both 100%, the operation method is simple and convenient, expensive equipment is not needed, low-cost and on-site rapid detection can be achieved, the method can be widely applied to various breeding enterprises, the economic benefits of the breeding industry are improved, and convenience is provided for early diagnosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and specifically relates to a duck Tembusu virus nucleic acid fluorescence visualization detection kit and a detection method based on RT-LAMP-CRISPR / Cas12a technology. Background Art

[0002] Duck Tembusu virus (DTMUV) is a newly emerged mosquito-borne virus of the genus Flavivirus in the family Flaviviridae. The disease caused by it is clinically characterized by decreased egg production in laying ducks and damage to the nervous system of ducklings. In 1955, researchers first discovered Tembusu virus in Culex tritaeniorhynchus. It was not until 2000 that cases of encephalitis and growth retardation in chicks caused by Tembusu virus infection were found in Malaysia. In 2010, DTMUV first broke out in laying ducks in Shanghai and other places in my country, causing significant economic losses to the domestic duck farming industry. At present, the infection hosts of DTMUV have expanded to meat ducks, Muscovy ducks, geese, laying hens, pigeons, sparrows and other poultry; DTMUV has various transmission routes. The virus can be transmitted through direct contact or aerosols in poultry groups, and even vertical transmission, which seriously threatens the healthy development of the poultry industry.

[0003] Since the outbreak of duck Tembusu virus disease, my country has successively developed a variety of vaccines such as DTMUV attenuated vaccines and inactivated vaccines. The investment and use of commercial vaccines have gradually changed the epidemic intensity of the disease from outbreaks to sporadic outbreaks. In order to better prevent duck Tembusu virus disease, a variety of clinical diagnosis or laboratory detection methods for DTMUV have been established in China. Among them, the laboratory detection methods for DTMUV mainly include virus isolation and identification, serological detection and molecular biology techniques. Virus isolation and identification is the "gold standard" for detecting viral infectious diseases and a classic method for testing DTMUV. It can accurately obtain the main information of the virus, such as its genotype and whether it has mutated. However, due to factors such as low viral load, difficult separation, large workload, high experimental conditions, long detection time and low success rate, this method is not conducive to the rapid detection of duck Tembusu virus disease. There are relatively many serological detection methods, such as indirect enzyme-linked immunosorbent assay (ELISA), competitive ELISA, colloidal gold immunochromatography strip method, virus neutralization test and hemagglutination inhibition test, which are mainly based on detecting specific antigens and antibodies in the serum of suspected cases. Neutralization test and hemagglutination inhibition test are traditional methods commonly used in epidemiological surveys, but these two methods require the collection of serum from the acute phase or the recovery phase for testing, which is time-consuming and laborious, and cannot achieve the purpose of early rapid diagnosis. The ELISA method can detect the specific duck Tembusu virus E protein and NS1 protein, but this method is complicated to operate, time-consuming and has low sensitivity, so it cannot be used as the best rapid diagnosis method. The colloidal gold immunochromatography strip method detects duck Tembusu virus particles or serum antibodies through a color reaction, and its detection results are intuitive, but the markers are unstable and have low sensitivity. Molecular biological detection methods mainly use reverse transcription-polymerase chain reaction (RT-PCR) technology. RT-PCR is currently considered to be an effective early diagnosis method in etiological diagnosis methods. The reverse transcription real-time fluorescence PCR developed in recent years can achieve closed-tube detection by adding SYBR Green I fluorescent dye or a specific TaqMan fluorescent probe to the reaction system. It not only maintains the sensitivity and other characteristics of traditional PCR technology, but also avoids false positive contamination and shortens the detection time. However, the PCR method requires expensive instruments and strict laboratory conditions, which limits its application in field testing.

[0004] Rapid nucleic acid detection plays an important role in early diagnosis of diseases and application of biotechnology, and is widely used in the fields of public health, livestock and poultry farming, and import and export quarantine. With the vigorous development of science and technology, CRISPR nucleic acid detection technology has gradually replaced the original detection technology that requires nucleic acid amplification, and has the significant advantages of strong specificity, high sensitivity, time saving and cost saving. For example, the most commonly used PCR or RT-PCR, compared with CRISPR detection technology, have higher requirements for experimental equipment and experimental personnel, and it is difficult to achieve on-site detection in the early stage of disease in livestock breeding areas, that is, point-of-care testing (POCT). Summary of the invention

[0005] The object of the present invention is to provide a duck Tembusu virus nucleic acid fluorescence visualization detection kit and a detection method based on RT-LAMP-CRISPR / Cas12a technology, and develop a detection kit and a detection method that can detect duck Tembusu virus nucleic acid quickly, sensitively, and highly specifically under a blue light gel cutting instrument by combining RT-LAMP technology with CRISPR-Cas12a detection and a fluorescent reporter gene for fluorescence visualization detection.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme: A duck Tembusu virus nucleic acid detection kit based on RT-LAMP and CRISPR / Cas12a technology, the kit comprising an RT-LAMP amplification system and a CRISPR / Cas12a detection system for duck Tembusu virus nucleic acid detection; The RT-LAMP amplification system includes an RT-LAMP primer set for duck Tembusu virus; the nucleotide sequence of the primer set is shown in SEQ ID NO: 14 to SEQ ID NO: 19; The CRISPR / Cas12a detection system includes specific crRNA for duck Tembusu virus, Cas12 protein and single-stranded DNA reporter molecules; The nucleotide sequence of the crRNA specific for duck Tembusu virus nucleic acid is shown in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 13; The single-stranded DNA reporter molecule is ROX-GTATCCAGTGCG-BHQ2.

[0007] Furthermore, the nucleotide sequence of the crRNA is shown in SEQ ID NO:10.

[0008] The application of the above-mentioned kit in detecting duck Tembusu virus nucleic acid products.

[0009] A method for detecting duck Tembusu virus nucleic acid, comprising the following steps: (1) Preparing duck Tembusu virus nucleic acid template; (2) Using the nucleic acid extracted in step (1) as a template, introducing the RT-LAMP primer set to amplify in a constant temperature metal bath under the reaction conditions of 65°C for 40 min to obtain an amplification product; the amplification system is 25 μL, including 1 μL 8U Bst II DNA large fragment polymerase, 2.5 μL 10× isothermal amplification buffer, 6 mM MgSO4, 1.4 mM dNTP Mix, and supplemented to 25 μL with nuclease-free water; (3) The amplification system obtained in step (2) was subjected to Cas12a restriction enzyme reaction, and the reaction system was 1 μM crRNA, 2 μL LAMP amplification product, 0.5 μM Cas12a, 20 μM single-stranded DNA reporter molecule ROX-GTATCCAGTGCG-BHQ2, 2 μL 10× CutSmart buffer, and supplemented to 20 μL with nuclease-free water; (4) Move the reaction product to a blue light gel cutting instrument and observe the fluorescence to identify the test results. If the reaction product shows orange fluorescence, it is positive; if the reaction product does not show any fluorescence, it is negative.

[0010] The beneficial effects of the present invention are: 1. The present invention provides a duck Tembusu virus nucleic acid detection kit and detection method based on the CRISPR-Cas12a system combined with reverse transcription loop-mediated isothermal amplification (RT-LAMP). The RT-LAMP primer set and crRNA of the detection kit have high sensitivity and high specificity.

[0011] 2. The positive and negative coincidence rates of the detection results of duck Tembusu virus by the detection method of the present invention and the RT-qPCR and RT-PCR methods are both 100%. The operation method of the present invention is simple, does not require expensive equipment, can achieve low-cost, on-site rapid detection, and can be widely used in major breeding companies to improve the economic benefits of the breeding industry and provide convenience for early diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 A: TA clone sequence alignment diagram for preparing the plasmid template for detecting the target gene NS3 of duck Tembusu virus; Figure 1 B: Schematic diagram of the duck Tembusu virus structure, crRNA-1, crRNA-2, crRNA-3, crRNA-4 and crRNA-5 can target the amplified fragment of the NS3 gene.

[0013] Figure 2 : Flowchart of duck Tembusu virus detection.

[0014] Figure 3 : Screening for highly active crRNA targeting the NS3 gene of duck Tembusu virus, using a ssDNA-reporter with a modification type of ROX-GTATCCAGTGCG-BHQ2; dsDNA represents the amplified product of the NS3 gene, among which NS3-crRNA-1, NS3-crRNA-2, NS3-crRNA-3, NS3-crRNA-4, and NS3-crRNA-5 can target the amplified fragment of the NS3 gene; detection under blue light refers to pictures taken with a mobile phone in a blue light gel cutting instrument; detection under ultraviolet light refers to pictures taken with a digital camera and related software in a ultraviolet gel imaging system.

[0015] Figure 4 : Screening for highly conserved crRNA targeting the duck Tembusu virus NS3 gene, using Jalview software to align 109 duck Tembusu virus NS3 sequences selected from the NCBI database that were targeted by highly active NS3-crRNA-1, NS3-crRNA-2 and NS3-crRNA-5.

[0016] Figure 5 : A schematic diagram for evaluating the sensitivity and specificity of the combination of RT-LAMP and CRISPR-Cas12a in detecting the NS3 gene of duck Tembusu virus in the present invention, wherein: A is the gel electrophoresis detection of RNA product of NS3 gene amplified by RT-LAMP and cloned by TA, lane 1: 2000 DNAMaker; lane 2: 1.93×10 8 copies / μL, Lane 3: 1.93×10 4 copies / μL, Lane 4: 1.93×10 3 copies / μL, Lane 5: 1.93×10 2 copies / μL, Lane 6: 1.93×10 1 copies / μL, Lane 7: 1.93×10 0 copies / μL, Lane 8: 1.93×10 -1 copies / μL, lane 9: non-template control (NTC); B is the blue light visualization detection using CRISPR-Cas12a using the RT-LAMP amplification product in Figure A; C is the gel electrophoresis detection of RNA product of TA cloned NS3 gene amplified by RT-LAMP and synthetic plasmid of duck reovirus, lane 1: 2000 DNA Maker; lane 2: NS3 gene standard plasmid 1.93×10 8copies / μL (DTMUV); Lane 3: duck reovirus synthetic plasmid 2.63×10 11 copies / μL (MDRV); Lane 4: no template control (NTC); D is the naked eye fluorescence visualization detection using CRISPR-Cas12a using the RT-LAMP amplification product in Figure C.

[0017] Figure 6 : RT-LAMP combined with CRISPR-Cas12a detected duck Tembusu virus simulated clinical samples, where the simulated samples included 8 duck embryo allantoic fluid RNAs and 11 chicken embryo fibroblast cell line (DF1) RNAs.

[0018] Figure 7 :RT-qPCR detection of serially diluted NS3 gene TA clone plasmid standards and duck Tembusu virus simulated clinical samples.

[0019] Figure 8 : RT-PCR detection of duck Tembusu virus in simulated clinical samples.

[0020] Fig. 9 : Compare Figure 6 , Figure 7 and Figure 8 Venn diagram of the test results in . DETAILED DESCRIPTION

[0021] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific embodiments, but the present invention is not limited thereto. The test methods used in the following examples are conventional methods unless otherwise specified; all materials and reagents can be purchased from commercial sources unless otherwise specified.

[0022] Example 1 Preparation of plasmid template for duck Tembusu virus NS3 target gene The genome sequence of duck Tembusu virus (KX977555.1) was downloaded from the NCBI database. Primer pairs for amplifying partial NS3 gene fragments were designed using primer 5 software (Table 1). The partial NS3 gene fragment amplified by PCR was cloned into the pCE2 TA / Blunt-Zero vector vector. The steps for preparing a standard plasmid template for detecting duck Tembusu virus NS3 gene were as follows: (1) PCR amplification of NS3 gene fragment: Prepare 50 μL PCR reaction solution, including 25 μL 2× Taq Master Mix, 10 pmoL of upstream and downstream primers NS3-PCR-F and NS3-PCR-R, 1 μL (about 10 ng) of cDNA template with sequence number KX977555.1, and DEPC water to 50 μL. The PCR reaction program was set as follows: first 95℃ for 3 min, then 95℃ for 15 s, 60℃ for 15 s, 72℃ for 50 s, and so on for 30 cycles, and finally 72℃ for 5 min to obtain the NS3 gene amplification fragment.

[0023] (2) TA cloning of NS3 gene amplification product: Prepare 5 μL TA cloning reaction solution, including 1 μL 5×TA-Zero CloningMix (NoviZan Biotechnology Co., Ltd., C601-01), 1 μL NS3 gene amplification fragment, and 3 μL nuclease-free water. Incubate in a 25°C metal bath for 5 min, and clone the NS3 gene amplification fragment into the pCE2 TA / Blunt-Zero vector. After the reaction is completed, gently mix the above reaction solution with 50 μL of competent cells DH5α competent cells (Vazyme #C502), let stand on ice for 5 min, heat shock at 42°C for 45 s, cool on ice for 3 min, spread on LB solid plate, incubate in a 37°C constant temperature incubator overnight, and identify positive clones the next day. Figure 1 As shown in A, the positive clone was correctly identified. The positive clone strain was added to a centrifuge tube containing 50 mL LB liquid culture medium and shaken at 200 r / min in a 37°C constant temperature incubator overnight. The next day, the turbid bacterial solution was subjected to plasmid extraction using an endotoxin-free plasmid extraction kit (Omega Bio-Tek, D6950-01). The extracted plasmid was the duck Tembusu virus NS3 gene standard plasmid.

[0024] Table 1. PCR primers and primer pairs for crRNA in vitro transcription template amplification Example 2 Screening for highly active and highly conserved crRNA targeting duck Tembusu virus NS3 gene For the PCR amplification region in Example 1, 5 crRNAs were designed using CRISPR-offinder software (https: / / sourceforge.net / projects / crispr-offinder-v1-2 / ), PAM was TTTV, and the crRNA empty vector was used as a template to design primer pairs for in vitro transcription (Table 1). The steps are as follows: (1) In vitro transcription of crRNA: T7-crRNA-F (SEQ ID NO: 3) and different crRNA-R primers (SEQ ID NO: 4~8) were combined to directly amplify the template for in vitro transcription of crRNA by PCR technology. The experimental reaction system was 50 μL, including 2× Taq Master Mix 25 μL, 20 pmoL of upstream and downstream primers, and DEPC water was added to 50 μL. The reaction conditions were: first 95℃ for 3min, then 95℃ for 15s, 60℃ for 15s, and 72℃ for 10s for 30 cycles, and finally 72℃ for 5min. The obtained PCR product was run on 3% agarose gel, and the target band was recovered using a DNA recovery kit (NoviZan Biotechnology Co., Ltd., DC301-01). Using the recovered DNA as a template, according to HiScribe TM Quick T7 High Yield RNA Synthesis Kit (New England Biolabs, E2050S) was used to synthesize crRNAs (Table 2). The reaction conditions were: 37°C for about 6 h. The transcribed crRNAs were purified by the phenol-chloroform method, and after the concentration was determined, they were aliquoted and frozen at -80°C for long-term storage.

[0025] Table 2 crRNA sequences (2) Cas12a enzyme cleavage reaction: In a 20 μL reaction system, the purified crRNA in step (2) (final concentration 0.5 μM), Cas12a (final concentration 0.25 μM), the product recovered from the 3 μL PCR amplification gel in step (1), the single-stranded DNA reporter molecule ssDNA-reporter (ROX-GTATCCAGTGCG-BHQ2) (20 μM), and 2 μL of 10×CutSmart buffer were added to a centrifuge tube with DEPC water to 20 μL. The negative control was set to not add the purified crRNA in step (1). The positive control was set to add the purified crRNA in step (1). The reaction was carried out at 37°C for 15 min, and then the reaction was terminated at 98°C for 2 min to obtain a reaction solution; wherein the sequence of ssDNA-reporter is shown in SEQ ID NO: 24.

[0026] (3) Result detection and judgment: directly place the centrifuge tube containing the reaction solution in a blue light gel cutting instrument or UV gel imaging instrument to detect the change in fluorescence intensity. Figure 3As shown, using ssDNA-reporter (ROX-GTATCCAGTGCG-BHQ2), the signal was detected by fluorescence visualization, indicating that it can be used for CRISPR-Cas12a nucleic acid detection experiments. Comparing the five crRNAs targeting the NS3 gene of duck Tembusu virus, when the PCR amplification product of the NS3 gene was used as the template of the target gene, the signals of NS3-crRNA-1, NS3-crRNA-2 and NS3-crRNA-5 were the strongest.

[0027] (4) The NS3 gene sequence of duck Tembusu virus and 109 viral genome sequences containing the fragment were downloaded from the NCBI database, and the sequence regions of highly active NS3-crRNA-1, NS3-crRNA-2 and NS3-crRNA-5 were compared using Jalview software. The results are as follows: Figure 4 As shown, in the region including the PAM sequence, NS3-crRNA-2 is the most conserved, that is, NS3-crRNA-2 is a highly active and highly conserved crRNA targeting the NS3 gene of duck Tembusu virus.

[0028] Example 3 Sensitivity and specificity of RT-LAMP combined with CRISPR-Cas12a for detecting duck Tembusu virus nucleic acid The sensitivity of the RT-LAMP-CRISPR / Cas12a blue light visualization method for detecting duck Tembusu virus nucleic acid was evaluated. The plasmid template of the NS3 target gene of duck Tembusu virus was gradient diluted to prepare a gradient template. After the gradient template was amplified by RT-LAMP, CRISPR / Cas12a blue light visualization detection was performed to determine the detection limit of RT-LAMP and CRISPR / Cas12a technology for blue light visualization detection of duck Tembusu virus nucleic acid. A plasmid containing a partial fragment of the duck reovirus σC gene was synthesized in the whole genome, and the synthetic plasmid and the plasmid of the duck Tembusu virus NS3 gene were co-detected by the RT-LAMP-CRISPR / Cas12a method to determine the specificity of the method. The experimental process is as follows: Table 3. RT-LAMP amplification primer pairs (1) RT-LAMP amplification sensitivity detection: The NS3 gene standard plasmid of duck Tembusu virus obtained in step (2) of Example 1 was diluted to 1.93×10 8 copies / ml, 1.93×10 4 copies / ml, 1.93×10 3 copies / ml, 1.93×10 2 copies / ml, 1.93×10 1 copies / ml, 1.93×100 copies / ml, 1.93×10 -1 The DNA plasmid of NS3 gene with different dilution multiples was used as template, and NS3-1-F / NS3-1-R, NS3-2-F / NS3-2-R, NS3-3-F / NS3-3-R were used as primers for amplification (Table 3); the amplification system was 25 μL, including 8U Bst II DNA large fragment polymerase 1 μL, 10× isothermal amplification buffer 2.5 μL, MgSO4 6mM, dNTP Mix 1.4mM each, primer Mix 2.5 μL (10×primer: NS3-1-F / NS3-1-R 0.5 μL, NS3-2-F / NS3-2-R 4 μL, NS3-3-F / NS3-3-R 2 μL), template 1 μL, and nuclease-free water was added to 25 μL. The reaction conditions were: 65℃, 40 min. After the reaction was completed, 5 μL of the amplified product was taken and identified by electrophoresis on a 2% agarose gel.

[0029] (2) Evaluation of the specificity of RT-LAMP and CRISPR-Cas12a combined to detect the NS3 gene of duck Tembusu virus: The partial sequence of the σC gene of duck reovirus (KX831117.1) was downloaded from the NCBI database, and the pUC57 plasmid containing the partial sequence of the σC gene of duck reovirus was sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for whole genome synthesis. Wuhan Tianyi Huayu Gene Technology Co., Ltd. used EcoRV rapid endonuclease to clone the sequence into the 3'EcoRV site of the pUC57 vector, and finally obtained the duck reovirus standard plasmid. Using the 1.93×10 8 NS3 gene standard plasmid, duck reovirus σC gene standard plasmid and nuclease-free water were used as templates. NS3-1-F / NS3-1-R, NS3-2-F / NS3-2-R, NS3-3-F / NS3-3-R were used as primers for amplification (Table 3); the amplification system was 25 μL, including 8U Bst II DNA large fragment polymerase 1 μL, 10× isothermal amplification buffer 2.5 μL, MgSO4 6mM, dNTP Mix 1.4mM each, primer Mix 2.5 μL (10×primer: NS3-1-F / NS3-1-R 0.5 μL, NS3-2-F / NS3-2-R 4 μL, NS3-3-F / NS3-3-R 2 μL), template 1 μL, and supplemented to 25 μL with nuclease-free water. The reaction conditions were: 65℃ 40min. After the reaction was completed, 5 μL of the amplified product was taken and identified by electrophoresis on a 2% agarose gel.

[0030] (3) Preparation of crRNA by in vitro transcription: T7-crRNA-F and NS3-crRNA-2-R are used as primer sets, wherein T7-crRNA-F contains the T7 sequence and the scaffold sequence required for Cas12a, and NS3-crRNA-2-R includes the crRNA-2 spacer sequence and the 15bp superimposed complementary scaffold sequence, so there is no need to add a template, and the in vitro transcribed template can be amplified directly by PCR. The reaction system of the specific steps is 50 μL, including 25 μL of 2×Taq Master Mix, 20 pmoL of upstream and downstream primers, and DEPC water is supplemented to 50 μL. The reaction procedure is: first 95°C for 3 min, then 95°C for 15 s, 60°C for 15 s, and 72°C for 10 s for 30 cycles, and finally 72°C for 5 min. The PCR product was electrophoresed on a 2% agarose gel, and the target band was recovered using a DNA recovery kit (Novizan Biotechnology Co., Ltd., DC301-01). Use HiScribe TM QuickT7 High Yield RNA Synthesis Kit (NEB) was used to synthesize NS3-crRNA-2 at 37°C for about 6 hours. The transcribed NS3-crRNA-2 was purified by the phenol-chloroform method. First, the DNA in the in vitro transcription product needed to be removed. 2 μL of DNase I (Thermo Fisher Scientific (China) Co., Ltd., EN0521) and 2 μL of 10×Reaction Buffer Thermo Fisher Scientific (China) Co., Ltd., EN0521) were added to 20 μL of in vitro transcription product. After gently flicking to mix, the mixture was placed in a 37°C metal bath for incubation for 15 minutes. The reaction solution was transferred to a new enzyme-free 1.5 mL centrifuge tube, and 160 μL of DEPC water and 20 μL of sodium acetate (Sinopharm Chemical Reagent Co., Ltd., Catalog No. 6131-90-4) solution (3 M) and 200 μL of 1:1 phenol chloroform isoamyl alcohol (Sinopharm Chemical Reagent Co., Ltd., catalog number 58-90-2), shake up and down to mix, centrifuge at 12000r / min for 5 min at 4°C, and take the supernatant; add 2 times the volume of pre-cooled anhydrous ethanol (64-17-5, Sinopharm Chemical Reagent Co., Ltd.) to the supernatant, incubate in a -20°C refrigerator for 1h, then centrifuge at 12000r / min for 5min at 4°C, and discard the supernatant; add 500 μL of pre-cooled 75% ethanol to the RNA precipitate, centrifuge at 12000r / min for 5min at 4°C, and discard the supernatant; air-dry the RNA precipitate under the clean bench for 5min, add 50 μL of DEPC water to the center of the column to dissolve the RNA, and after measuring the concentration, aliquot and freeze at -80°C for long-term storage.

[0031] (4) Cas12a enzyme digestion reaction: In a 20 μL reaction system, add the RT-LAMP amplification product (2 μL) in step (1), NS3-crRNA-2 (1 μM) purified in step (2), Cas12a (0.5 μM), 20 μM ssDNA-reporter (ROX-GTATCCAGTGCG-BHQ2), and 10×CutSmart buffer (2 μL). Make up to 20 μL with nuclease-free water. React at 37°C for 15 min, and then terminate the reaction at 98°C for 2 min.

[0032] (5) Result detection and judgment: The reaction solution is directly placed in a blue light gel cutting instrument or UV gel imaging system for detection, and the changes in the strength of the blue light visualization detection signal after amplification using different copy templates are compared; this is used to determine the sensitivity of nucleic acid detection.

[0033] (6) The results are as follows Figure 5 As shown in the figure, the RT-LAMP amplification product was detected by gel electrophoresis. It can be seen that the lowest sensitivity of RT-LAMP amplification gel electrophoresis for detecting duck Tembusu virus NS3 gene corresponds to the NS3 gene DNA template dilution concentration of 1.93×10 1 Copies / μl ( Figure 5 A); RT-LAMP amplification of NS3 gene gradient DNA template, RT-LAMP product was digested with Cas12a, and then detected by blue light gel cutting instrument or UV gel imaging system, and the minimum detection limit was 1.93×10 1 copies / μl, that is, the detection sensitivity reached 1.93×10 1 Copies / μl ( Figure 5 B). Figure 5 In C, the RT-LAMP or LAMP amplification products were detected by gel electrophoresis. It can be seen that only the duck Tembusu virus as a template group (DTMUY) showed obvious ladder-like bands, while the duck reovirus as a template group (MDRV) only had regular bands of plasmid templates, which was related to the large amount of plasmid loading. The no-template control group (NTC) did not have any bands. This shows that the detection technology based on CRISPR-Cas12a for detecting duck Tembusu virus nucleic acid is highly specific.

[0034] Example 4 Preparation and collection of samples of duck embryos and DF1 passaged cells infected with duck Tembusu virus Prepare and collect samples of duck embryos and DF1 passage cells for evaluating the accuracy of LAMP-CRISPR / Cas12a in detecting duck DTMUV nucleic acid, prepare duck embryo eggs that meet the conditions for virus injection, including no maternal antibodies, and can survive to 13 days of age (the eggs around this age have the most allantoic fluid, which is convenient for collection), etc. After the virus dilutions are infected with duck embryos and DF1 passage cells, observe for 2-3 days, and collect the total RNA of duck embryo allantoic fluid and cell culture when the samples show death or cytopathic effects. The experimental process is as follows: (1) Preparation and collection of samples of duck embryos infected with duck Tembusu virus: 8-day-old Cherry Valley duck eggs purchased from Taisi Poultry Business Department in Nanle County were placed in a constant temperature incubator at 37.8°C for 2 days. The 10-day-old eggs were candled in a dark room, and the survival of the duck embryo was determined based on the presence or absence of fetal movement and clear blood vessels. The blood vessels and duck embryo positions of the surviving eggs were marked, and the injection points were marked to avoid large blood vessels. A total of 8 10-day-old eggs that met the injection conditions were randomly selected and injected with 200 μL of duck Tembusu dilution as the infection group. The duck Tembusu dilution was made from duck Tembusu virus stock solution (donated by Fujian Academy of Agricultural Sciences, stock solution EID 50 1×10 -4.5 ) was diluted 3 times with PBS; EID 50 Determination: First, the virus suspension to be determined is diluted in equal proportions from high concentration to low concentration. Subsequently, each dilution of the virus suspension is inoculated into highly susceptible SPF (specific pathogen-free) duck embryos, with each duck embryo inoculated with 200 μL of virus suspension. After inoculation, the duck embryos are placed in an incubator with constant temperature and humidity for incubation, and the growth and pathological conditions of the duck embryos are regularly observed and recorded. Based on the observed data, the Reed-Muench method is used to calculate the EID 50 , that is, find the virus dilution multiple that infects 50% of duck embryos. The other three eggs were injected with 200 μL of PBS as the control group. Take iodine tincture and 75% alcohol to disinfect the injection area, use pointed tweezers to pierce a small hole at the injection point of the egg, use a disposable small syringe to draw 200 μL of the above-mentioned duck Tembusu dilution or PBS to pierce the small hole about 0.6 cm, and inject gently. Finally, use a transparent fixed dressing to seal the small hole and mark it. Continue to incubate and observe at 37.8℃ for 2-3 days. Among them, the duck embryos that died within 1 day were non-specific deaths. After the duck embryos in the infected group died (fetal movement disappeared and blood vessels were inconvenient to distinguish), all duck embryos were placed in a 4℃ refrigerator for 6h to coagulate the blood and kill the duck embryos in the control group. The duck embryo allantoic fluid was collected and frozen at -80℃ for long-term storage. The naming of duck embryo samples is shown in Table 4.

[0035] (2) Preparation and collection of DF1 passage cell samples: DF1 passage cells with a cell density of 30% were plated into 11 wells of two 6-well plates (9 wells for infection group and 2 wells for control group) and 2 mL of 10% serum concentration 1640 culture medium (5 mL of fetal bovine serum + 500 μL of double antibody (penicillin-streptomycin solution) + 44.5 mL of 1640 culture medium) was added to each well. The cells were cultured in a 39°C constant temperature biochemical incubator with 5% CO2. When the cell density grew to about 90%, the infection group was inoculated with 0.1 TCID 50 Dilution of duck Tembusu virus (TCID 50 For 10 -3.8 / 0.1mL), where TCID 50 Assay: First, DF-1 cells were seeded into 96-well cell culture plates, and 100 μL of 10% serum-concentrated 1640 medium was added to each well. The cells were incubated in a 39°C incubator containing 5% CO2 until the cell density reached about 80%. Then, 1640 medium was used in 1.5 mL EP tubes to -1 Up to 10 -10 The thoroughly mixed virus solution to be tested was gradiently diluted within the dilution range, ensuring that the pipette tip was changed each time the liquid was pipetted to ensure the accuracy of the dilution. Then the waste liquid in the 96-well plate was discarded, the cells were washed with PBS, and 100 μL of virus dilution solution was added to each well. Each dilution was repeated for 8 wells, and the 11th and 12th columns were added with an equal amount of 1640 culture medium as the control group. The culture plate was gently shaken during the 2-hour period of virus infection of the cells, and then the cells were washed twice with PBS, and 2% serum concentration of 1640 culture medium (1 mL fetal bovine serum + 500 μL double antibody + 48.5 mL 1640 culture medium) was added for continued culture. The number of wells with cytopathic effect (CPE) was observed and recorded daily until the cells in the lowest dilution well stopped being diseased and the cells in the control group began to age. Finally, the TCID of the virus to be tested was calculated according to the Reed-Muench method. 50 Value. The control group was inoculated with the same volume of PBS. After incubation at 37°C for 2 hours, the cells were replaced with 1640 cell culture medium with a 2% serum concentration (1 mL fetal bovine serum + 500 μL double antibody + 48.5 mL 1640 culture medium) for continuous culture. The cell cultures of the infection group were collected at 6 hpi, 12 hpi, 24 hpi, 36 hpi, 48 hpi, 60 hpi, and 72 hpi, respectively. A total of 3 wells were set up in the 48 hpi infection group, and the cell cultures of the control group were collected 48 hours later. The collected cell cultures were aliquoted and frozen at -80°C for long-term storage. The cell sample names are shown in Table 4.

[0036] Table 4 Preparation and collection of samples of duck embryos and DF1 passaged cells infected with duck Tembusu virus (3) Extraction of total RNA and reverse transcription of each sample: The total RNA of each sample was extracted using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 (Bao-Ri Yi Biotechnology (Beijing) Co., Ltd., Cat. No. 9766) (Table 4). The reaction time was 20 min. Since carrier RNA was added during the extraction process, the RNA concentration was not determined by electrophoresis or absorbance. The RNA of each sample was frozen at -80°C. The reaction system for reverse transcription was 20 μL, including 2× NovoScript ® Plus 1st Strand cDNA Synthesis SuperMix 10μL, RNA template (total RNA extracted from each sample) 2 μL, gDNA Purge 1μL and RNase Free Water 7 μL. The reaction procedure is 50℃ 15min, 85℃ 5s. After the reaction, the cDNA concentration of each sample was measured and temporarily frozen at -20℃.

[0037] Example 5 Evaluation of the accuracy of RT-LAMP and CRISPR / Cas12a technology in detecting duck Tembusu virus nucleic acid Whether the technology for detecting duck Tembusu virus nucleic acid based on RT-LAMP and CRISPR-Cas12a technology is accurate needs to be compared with the conventional technology for detecting duck Tembusu virus nucleic acid at this stage. The cDNA of each sample preserved in Example 4 was detected by RT-LAMP-CRISPR / Cas12a, RT-qPCR and RT-PCR, respectively, and the detection results of the three methods were summarized and compared. The experimental process is as follows: Table 5 RT-qPCR and RT-PCR primer pairs (1) Preparation of crRNA by in vitro transcription: T7-crRNA-F and NS3-crRNA-2-R (Table 1) are used as primer sets, where T7-crRNA-F contains the T7 sequence and the scaffold sequence required for Cas12a, and NS3-crRNA-2-R includes the crRNA-2 spacer sequence and a 15bp overlapping complementary scaffold sequence, so there is no need to add a template, and the in vitro transcribed template can be amplified directly by PCR. The reaction system of the specific steps is 50 μL, including 2× Taq Master Mix 25μL, 20pmoL of upstream and downstream primers, and DEPC water is supplemented to 50 μL. The reaction procedure is: first 95℃ 3min, then 95℃ 15s, 60℃ 15s, 72℃ 10s for 30 cycles, and finally 72℃ extension for 5min. The PCR product was electrophoresed on a 3% agarose gel, and the target band was recovered using the Novezan gel recovery kit. HiScribe TM NS3-crRNA-2 was synthesized using Quick T7 High Yield RNASynthesis Kit (NEB) at 37°C for about 6 hours. The transcribed NS3-crRNA-2 was purified using the phenol-chloroform method, and after the concentration was determined, it was aliquoted and frozen at -80°C for long-term storage.

[0038] (2) RT-LAMP-CRISPR / Cas12a detection of duck Tembusu virus nucleic acid: After fully oscillating and mixing the cDNA of each sample in Example 4, 2 μL of each sample was taken as a template, and LAMP isothermal amplification was performed with the primer pairs in Table 3. The specific amplification system was 25 μL of the amplification system, including 8U Bst II DNA large fragment polymerase 1 μL, 10× isothermal amplification buffer 2.5 μL, MgSO4 6mM, dNTP Mix 1.4mM each, primer Mix 2.5 μL (10×primer: NS3-1-F / NS3-1-R 0.5 μL, NS3-2-F / NS3-2-R 4 μL, NS3-3-F / NS3-3-R 2 μL), and each sample cDNA template 2 μL (about 10 ng), supplemented to 25 μL with nuclease-free water. The reaction conditions were: 65°C, 40 min. After the reaction, take 2 μL of LAMP amplification product for CRISPR-Cas12a digestion reaction: add NS3-crRNA-2 (1 μM) purified in step (1), LAMP product (2 μL), Cas12a (0.5 μM), 20 μM ssDNA-reporter (ROX-GTATCCAGTGCG-BHQ2), 10×CutSmart buffer (2 μL) to the 20 μL reaction system, and add nuclease-free water to 20 μL. React at 37 °C for 15 min, then terminate the reaction at 98 °C for 2 min.

[0039] The PCR tube containing the above CRISPR-Cas12a enzyme digestion reaction termination solution was directly placed in a blue light gel cutting instrument or UV gel imaging system to observe the changes in the intensity of the fluorescence visualization detection signal to evaluate the feasibility of the established RT-LAMP and CRISPR / Cas12a technology two-step fluorescence visualization detection method for duck Tembusu virus. Figure 6 As shown, among the duck embryo and DF1 passage cell samples, except for DF1 passage cell sample 1 (cell sample 1), the other results were consistent with the expected results of whether or not the virus was infected (i.e., the sample group infected with duck Tembusu virus showed fluorescence under LAMP-CRISPR-Cas12a detection), while DF1 passage cell sample 1 (cell sample 1) was a cell culture collected 6 hours after virus infection. At this time, duck Tembusu virus may not have begun to replicate in large quantities in the cells, so duck Tembusu virus nucleic acid could not be detected.

[0040] (3) RT-qPCR detection of duck Tembusu virus nucleic acid: The plasmid of duck Tembusu virus NS3 target gene was diluted in enzyme-free water to obtain a final concentration of 1.88×10 -1copies / ml, 1.88×10 0 copies / ml, 1.88×10 2 copies / ml, 1.88×10 4 copies / ml, 1.88×10 6 copies / ml, 1.88×10 8 copies / mL and 1.88×10 10 7 standard plasmids with 19 copies / mL. qPCR was performed using 7 standard plasmids and 19 sample cDNAs as templates. The reaction system was 20 μL, including 2×NovoStart ® SYBR qPCR SuperMix Plus 10μL, upstream and downstream primers NS3-RTqPCR-F and NS3-RTqPCR-F (Table 5) 0.5μM each, template 2μL, RNase Free Water 6μL. Reaction procedure: The first step is 95℃1min, the second step is 95℃20s, 60℃20s and 72℃30s for 45 cycles. Result determination: Calculate the mean CT value of each group, draw a predicted standard curve based on the mean CT value of the standard plasmid, substitute the CT value of each sample into the standard curve, and calculate the viral nucleic acid copy concentration of each sample. By default, CT≥35 indicates viral nucleic acid negative, and CT<35 indicates viral nucleic acid positive. The results are as follows Figure 7 As shown, the standard curve is y=-1.408ln(x)+40.902, the number of positive samples is 13, and the number of negative samples is 6, and the test result is 100% consistent with the LAMP-CRISPR-Cas12a test result. Therefore, the method for detecting duck Tembusu virus nucleic acid by LAMP-CRISPR-Cas12a involved in the present invention is comparable to conventional RT-qPCR.

[0041] (4) RT-PCR detection of duck Tembusu virus nucleic acid: After thoroughly shaking and mixing the cDNA of each sample, take 2 μL of each as a template, and use NS5-RTPCR-F and NS5-RTPCR-R (Table 5) as primers for PCR amplification reaction. Prepare a 50 μL reaction system, including 25 μL of 2×Taq Master Mix, 10 pmoL each of NS5-RTPCR-F and NS5-RTPCR-R (Table 5), 2 μL of sample cDNA template (about 10 ng), and DEPC water to make up to 50 μL. The PCR reaction program is set as follows: first 95°C for 3 minutes, then 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 50 seconds for 35 cycles, and finally 72°C for extension for 5 minutes. After the reaction is completed, take 5 μL of the amplified product and perform electrophoresis on a 2% agarose gel for identification. The results are as follows. Figure 8As shown, the number of positive samples was 13, while the number of negative samples was 6, and the test results were 100% consistent with the RT-LAMP-CRISPR / Cas12a test results.

[0042] (5) Summarize and compare the test results of the three methods: Fig. 9 As shown, the consistency of the detection results of the three detection methods was 100%, and the test results were basically consistent with the expected results, which shows that the primer set of RT-LAMP-CRISPR / Cas12a for detecting duck Tembusu virus nucleic acid is accurate.

[0043] In summary, the present invention provides a duck Tembusu virus nucleic acid detection kit and detection method based on RT-LAMP and CRISPR / Cas12a technology. During the implementation of the method, it first takes about 20 minutes to extract the duck Tembusu virus RNA that may exist in each sample and reverse transcribe it into cDNA, and then use the primer set to perform CRISPR fluorescence visualization detection on each cDNA in a constant temperature metal bath. The constant temperature metal bath can be replaced with a constant temperature metal bath with a temperature range of 25-100°C, so that the duck Tembusu virus nucleic acid can be quickly detected on site within 100 minutes.

[0044] The above-mentioned embodiments merely express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the present invention.

Claims

1. A duck Tembusu virus nucleic acid detection kit based on RT-LAMP and CRISPR / Cas12a technology, characterized in that: The kit includes an RT-LAMP amplification system and a CRISPR / Cas12a detection system for duck Tembusu virus nucleic acid detection; The RT-LAMP amplification system includes an RT-LAMP primer set for duck Tembusu virus; the nucleotide sequence of the primer set is shown in SEQ ID NO: 14 to SEQ ID NO: 19; The CRISPR / Cas12a detection system includes specific crRNA for duck Tembusu virus, Cas12 protein and single-stranded DNA reporter molecules; The nucleotide sequence of the crRNA specific for duck Tembusu virus nucleic acid is shown in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 13; The single-stranded DNA reporter molecule is ROX-GTATCCAGTGCG-BHQ2.

2. The duck Tembusu virus nucleic acid detection kit according to claim 1, characterized in that: The nucleotide sequence of the crRNA is shown in SEQ ID NO:

10.

3. Use of the kit as claimed in claim 1 in detecting duck Tembusu virus nucleic acid products.

4. A method for detecting duck Tembusu virus nucleic acid using the kit according to claim 1, characterized in that: The following steps are involved: (1) Preparing duck Tembusu virus nucleic acid template; (2) Using the nucleic acid extracted in step (1) as a template, introducing the RT-LAMP primer set to amplify in a constant temperature metal bath under the reaction conditions of 65°C for 40 min to obtain an amplification product; the amplification system is 25 μL, including 1 μL 8U Bst II DNA large fragment polymerase, 2.5 μL 10× isothermal amplification buffer, 6 mM MgSO4, 1.4 mM dNTP Mix, and supplemented to 25 μL with nuclease-free water; (3) The amplification system obtained in step (2) was subjected to Cas12a restriction enzyme reaction. The reaction system was 1 μM crRNA, 2 μL LAMP amplification product, 0.5 μM Cas12a, 20 μM single-stranded DNA reporter molecule ROX-GTATCCAGTGCG-BHQ2, 2 μL 10×CutSmart buffer, and supplemented to 20 μL with nuclease-free water. The reaction was carried out at 37°C for 15 minutes, and then at 98°C for 2 minutes; (4) Move the reaction product to a blue light gel cutting instrument and observe the fluorescence to identify the test results. If the reaction product shows orange fluorescence, it is positive; if the reaction product does not show any fluorescence, it is negative.

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