LAMP-CRISPR / Cas12a-based kit for visually detecting duck circovirus and application of LAMP-CRISPR / Cas12a-based kit

Abstract

The invention relates to a reagent combination and a kit for visually detecting duck circovirus based on LAMP-CRISPR / Cas12a and application of the reagent combination and the kit, the reagent combination comprises LAMP amplification primers and crRNA, the LAMP amplification primers comprise two outer primers F3-1 and B3-1 and two inner primers FIP-1 and BIP-1, the crRNA is any one of crRNA-1, crRNA-2 and crRNA-3, and the sequences of the LAMP amplification primers and the crRNA are shown as SEQ ID NO.1-4 and SEQ ID NO.13-15. The invention further discloses a kit for visually detecting duck circovirus based on LAMP-CRISPR / Cas12a. According to the method, the LAMP-CRISPR / Cas12a detection system is established by using the reagent combination, so that the rapid detection of the duck circovirus is realized. The detection method established by the invention does not need an expensive thermal cycle instrument, is simple to operate, and can achieve the purpose of rapid visual detection.

Description

Technical Field

[0001] The invention belongs to the technical field of livestock and poultry disease diagnosis, and relates to the detection of duck circovirus, and specifically to a kit and a detection method for realizing rapid visual detection of duck circovirus by combining a LAMP amplification method and a CRPSPR / Cas12a detection method. Background Art

[0002] Duck circovirus (DuCV) belongs to the Circoviridae family and the Circovirus genus. The virus has no envelope and is icosahedral in structure. Its genome is a single-stranded circular DNA with a length of 1988nt-1996nt. Clinically, ducks infected with circovirus are mainly manifested by growth retardation, emaciation, and feather disorder. The weight of infected ducks is more than 30% lighter than that of uninfected ducks in the same batch. In addition, the infected ducks gradually become dead ducks or defective ducks. They are immunosuppressive diseases with damage to immune organs and severe reduction of lymphocytes. The decline in immunity increases the susceptibility to other diseases and is prone to secondary infection and multiple infection. In recent years, the infection rate of duck circovirus in duck flocks has been on the rise, posing a serious threat to the healthy development of large-scale farms. With the genetic evolution of the virus, there are reports of cross-species infection of geese, which is one of the important viruses that endanger duck farms.

[0003] Loop-mediated isothermal amplification (LAMP) is a nucleic acid isothermal amplification technology developed by Notomi et al. in 2000. It can amplify nucleic acids with high specificity, sensitivity and high rate under isothermal conditions without the need for precision instruments. Its principle is to use four primers in combination with DNA polymerase with strong chain displacement activity to produce multi-ring amplification products. By adding two circular primers, amplification can be further accelerated, providing high-efficiency amplification in a short time. This technology is widely used in rapid on-site testing (Point of Care Testing, POCT) in the fields of environmental monitoring, food safety testing, early disease detection and diagnosis.

[0004] Clustered regularly interspaced shortpalindromic repeats (CRISPR) have been widely used as molecular diagnostic tools in recent years to detect DNA and RNA with high sensitivity and specificity. The key role of the CRISPR / Cas system in infectious disease detection is to identify pathogens through Cas proteins and perform nucleic acid-based diagnosis. The Cas9 system only has target cleavage activity, while the Cas12, 13, and 14 systems also have non-target cleavage activity after identifying the target. Through this activity, non-target single-stranded DNA (ssDNA) can be cleaved. The fluorescent labeled probes in the system can be cleaved to obtain fluorescence or flow readings when detecting nucleic acids. A variety of CRISPR / Cas diagnostic tools have been made. This study combines LAMP technology with the CRISPR / Cas12a system to establish a high-specificity, high-sensitivity, and high-rate duck circovirus nucleic acid instant detection technology. Summary of the invention

[0005] The object of the present invention is to provide a reagent combination (including a LAMP amplification primer set, a crRNA combination) and a kit for visually detecting duck circovirus based on the LAMP-CRISPR / Cas12a system, so as to realize on-site rapid detection of duck circovirus.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A reagent combination for visual detection of duck circovirus based on LAMP-CRISPR / Cas12a, comprising a LAMP amplification primer and crRNA;

[0008] The LAMP amplification primers include two outer primers F3-1 and B3-1, and two inner primers FIP-1 and BIP-1; the nucleotide sequences of the outer primers F3-1 and B3-1 are respectively:

[0009] F3-1:5'-ATCGTCGAGACGCAACGT-3',

[0010] B3-1: 5'-ATCATACGGCAACCAACCAT-3';

[0011] The nucleotide sequences of the inner primers FIP-1 and BIP-1 are respectively:

[0012] FIP-1: 5'-TGCATAACGGCTCTTTCCGGTTTGGAAGACCGAAGTCATCG-3',

[0013] BIP-1: 5'-TTACAAACCACGCGGGAAGTGGAGTCGTCCATGACGACTACG-3';

[0014] The crRNA is any one of the three oligonucleotides targeting the duck circovirus genome (i.e., any one of crRNA-1, crRNA-2, and crRNA-3), and the nucleotide sequences of crRNA-1, crRNA-2, and crRNA-3 are respectively:

[0015] crRNA-1: 5'-UAAUUUCUACUAAGUGUAGAUCCGCCGAAAACAAGUAUUACAAA-3',

[0016] crRNA-2: 5'-UAAUUUCUACUAAGUGUAGAUGGCGGGAAAUUCAAAUGCAUAAC-3',

[0017] crRNA-3: 5'-UAAUUUCUACUAAGUGUAGUAAUUUCCCGCCGAAAACAAGUAU-3'.

[0018] Preferably, the crRNA is crRNA-1.

[0019] The reagent combination further comprises a ssDNA probe, the nucleotide sequence of the ssDNA probe is 5'-TTATT-3', the 5' end of the ssDNA probe is labeled with FAM and the 3' end is labeled with BHQ, or the 5' end of the ssDNA probe is labeled with FAM and the 3' end is labeled with Biotin.

[0020] The present invention also provides a kit for visually detecting duck circovirus nucleic acid based on LAMP-CRISPR / Cas12a, wherein the kit comprises a LAMP amplification system and a CRISPR / Cas12a detection system;

[0021] The LAMP amplification system includes an outer primer pair F3-1 and B3-1, and an inner primer pair FIP-1 and BIP-1; the nucleotide sequences of the outer primer pair F3-1 and B3-1 are respectively:

[0022] F3-1:5'-ATCGTCGAGACGCAACGT-3',

[0023] B3-1: 5'-ATCATACGGCAACCAACCAT-3';

[0024] The nucleotide sequences of the inner primers FIP-1 and BIP-1 are:

[0025] FIP-1: 5'-TGCATAACGGCTCTTTCCGGTTTGGAAGACCGAAGTCATCG-3',

[0026] BIP-1: 5'-TTACAAACCACGCGGGAAGTGGAGTCGTCCATGACGACTACG-3';

[0027] The CRISPR / Cas12a detection system includes crRNA, which is any one of three oligonucleotides targeting the duck circovirus genome (i.e., any one of crRNA-1, crRNA-2, and crRNA-3), and the nucleotide sequences of crRNA-1, crRNA-2, and crRNA-3 are respectively:

[0028] crRNA-1: 5'-UAAUUUCUACUAAGUGUAGAUCCGCCGAAAACAAGUAUUACAAA-3',

[0029] crRNA-2: 5'-UAAUUUCUACUAAGUGUAGAUGGCGGGAAAUUCAAAUGCAUAAC-3',

[0030] crRNA-3: 5'-UAAUUUCUACUAAGUGUAGUAAUUUCCCGCCGAAAACAAGUAU-3'.

[0031] Preferably, the crRNA is crRNA-1

[0032] The LAMP amplification system also includes 2×LAMP Master Mix, DNA Polymerase, ddH 2 O and test samples, negative and positive control samples.

[0033] The CRISPR / Cas12a detection system also includes Cas12a Reaction buffer (10×), LbaCas12a, LAMP amplification product, ssDNA probe and ddH 2 O.

[0034] The nucleotide sequence of the ssDNA probe is 5'-TTATT-3'. The 5' end of the ssDNA probe is labeled with FAM and the 3' end is labeled with BHQ, or the 5' end of the ssDNA probe is labeled with FAM and the 3' end is labeled with Biotin.

[0035] The kit is used in the non-diagnostic detection of duck circovirus. The nucleic acid of the sample to be tested is amplified using the aforementioned LAMP amplification system, and the amplified product is then added to the CRISPR / Cas12a detection system to detect the nucleic acid. The kit can be used to visually detect duck circovirus based on LAMP-CRISPR / Cas12a.

[0036] A method for visually detecting duck circovirus based on LAMP-CRISPR / Cas12a, specifically comprising the following steps:

[0037] (1) Extract DNA from the sample to be tested;

[0038] (2) using the DNA extracted in step (1) as a template, amplifying in a LAMP amplification system;

[0039] (3) performing enzyme digestion and fluorescence detection on the LAMP amplification product obtained in step (2) in a CRISPR / Cas12a detection system;

[0040] The total volume of the LAMP amplification system was 25 μL, including 12.5 μL 2×LAMP Master Mix, 0.5 μL DNA Polymerase, 0.5 μL each of the outer primers F3-1 and B3-1 to a final concentration of 0.2 μM, 2 μL each of the inner primers FIP-1 and BIP-1 to a final concentration of 0.8 μM, 1 μL of the sample DNA to be tested, and ddH 2 O was added to 25 μL;

[0041] The total volume of the CRISPR / Cas12a detection system is 20 μL, including 2 μL of Cas12a Reaction buffer (10×), 1 μL of Lba Cas12a to a final concentration of 150 nM, 1 μL of crRNA-1 (1 μM) to a final concentration of 50 nM, 1.2 μL of ssDNA (4 μM) to a final concentration of 240 nM, and 1 μL of LAMP amplification product, supplemented with ddH 2 0 to 20 μL.

[0042] In the CRISPR / Cas12a detection system, the concentration ratio of LbaCas12a protein to crRNA is 1:0.5-4, preferably, the concentration of LbaCas12a is 150 nM, the concentration of crRNA is 50 nM, and the concentration of ssDNA probe is 240 nM.

[0043] Compared with the prior art, the advantages of the present invention are: the present invention uses the reagent combination (including LAMP amplification primers and crRNA) to establish a LAMP-CRISPR / Cas12a detection system, thereby realizing rapid detection of duck circovirus.

[0044] The method for visual detection of duck circovirus based on LAMP-CRISPR / Cas12a established in the present invention has the characteristics of high sensitivity and strong specificity. According to the results of the sensitivity test, the detection limit of the detection method is 2.88×10 0 Copies / μL. According to the specificity test results, only duck circovirus was positive, and the other viruses and negative controls were not detected, indicating that this method has high specificity.

[0045] The LAMP-CRISPR / Cas12a detection method established in the present invention was used to detect 32 duck tissue samples suspected of being infected with duck circovirus. At the same time, the TaqMan qPCR (invention patent number ZL202010748507.0) established in this laboratory was used for comparison. The results showed that the positive detection rate of the LAMP-CRISPR / Cas12a detection method established in the present invention was the same as that of TaqMan qPCR, which was 42.75% (14 / 32), indicating that the consistency rate of the LAMP-CRISPR / Cas12a method and the TaqManqPCR detection results was 100%.

[0046] The detection method established by the present invention does not require expensive thermal cyclers and is simple to operate, and can achieve the purpose of rapid visual detection. Compared with the TaqMan qPCR detection method, the detection method established by the present invention is more suitable for use at the grassroots level. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the principle of LAMP-CRISPR / Cas12a detection of duck circovirus.

[0048] Figure 2LAMP primer screening and amplification condition optimization results; wherein, A: LAMP amplification results of primers shown in sequence SEQ ID NO.1-12, wherein LAMP-1 is the amplification result of the amplification system established by primers F3-1, B3-1, FIP-1, and BIP-1, and NTC-1 is the corresponding negative control; LAMP-2 is the amplification result of the amplification system established by primers F3-2, B3-2, FIP-2, and BIP-2, and NTC-2 is the corresponding negative control; LAMP-3 is the amplification result of the amplification system established by primers F3-3, B3-3, FIP-3, and BIP-3, and NTC-3 is the corresponding negative control; | B: LAMP incubation temperature optimization; C: LAMP incubation time optimization.

[0049] Figure 3 crRNA screening results; A: Blue light and UV gel imaging system captures CRISPR / Cas12a reaction images under different crRNAs; B: Real-time fluorescence quantitative PCR instrument collects fluorescence intensity in real time under different crRNAs.

[0050] Figure 4 Optimization results of CRISPR / Cas12a-mediated cleavage experiment; A: CRISPR / Cas12a reaction images at different crRNA concentrations in blue light and UV gel imaging system; B: Real-time fluorescence quantitative PCR instrument observing fluorescence intensity and reaction rate at different crRNA concentrations; C: CRISPR / Cas12a reaction images at different Lba Cas12a concentrations in blue light and UV gel imaging system; D: Real-time fluorescence quantitative PCR instrument observing fluorescence intensity and reaction rate at different Lba Cas12a concentrations; E: CRISPR / Cas12a reaction images at different temperatures in blue light and UV gel imaging system; F: Real-time fluorescence quantitative PCR instrument observing fluorescence intensity and reaction rate at different temperatures.

[0051] Figure 5 Specificity and sensitivity of CRISPR / Cas12a in detecting duck circovirus; A: CRISPR / Cas12a specific detection in blue light and UV gel imaging system; B: fluorescence intensity of CRISPR / Cas12a specific detection collected in real time by real-time fluorescence quantitative PCR instrument; C: CRISPR / Cas12a sensitivity detection image in blue light and UV gel imaging system; D: fluorescence intensity of sensitivity detection collected in real time by real-time fluorescence quantitative PCR instrument. DETAILED DESCRIPTION

[0052] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0053] Main experimental materials: 2×LAMP Master Mix; DNA / RNA extraction kit; Lba Cas12a protein.

[0054] Main instruments: FQD-96A fluorescence quantitative PCR instrument; H203-100C dry constant temperature metal bath; nucleic acid electrophoresis instrument; gel imaging system.

[0055] Example 1 Design and screening of LAMP primers and optimization of conditions

[0056] 1.1 Design of LAMP primers

[0057] The rep sequences of duck circovirus were downloaded from the GenBank database (accession numbers: OK094642.1, OR842553.1, ON756217.1, PP860902.1, OQ657183.1, OR842552.1, OR842551.1, Q657184.1, OR134505.1, OR134472.1). Sequence analysis and alignment were performed using MegAlign software (LaserGene 7.0 software, DNAStar). LAMP primers were designed based on the highly conserved regions of the rep gene. The primer sequences are shown in Table 1. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0058] Table 1 LAMP amplification primers

[0059]

[0060] 1.2 Establishment of LAMP amplification system

[0061] The duck circovirus positive sample nucleic acid stored in our laboratory was used as a template, and ddH 2 O is a negative control, and LAMP amplification was performed using the LAMP primers in Table 1 above.

[0062] The LAMP amplification system is shown in Table 2. After the reaction system is prepared, vortex and shake to mix, then centrifuge instantly, react in a metal bath at 65 °C for 1 h, and inactivate at 80 °C for 10 min. After the reaction is completed, take 9 μL of sample and add 1 μL 10×LoadingBuffer, and electrophoresed by 2% agarose gel. Then, refer to the LAMP amplification system described in Table 2 and replace the primers F3-1, B3-1, FIP-1, and BIP-1 with F3-2, B3-2, FIP-2, BIP-2 or F3-3, B3-3, FIP-3, and BIP-3, and react according to the above method. The results are as follows: Figure 2A is shown in the electrophoresis diagram of LAMP-1, LAMP-2, and LAMP-3. According to the electrophoresis results, the primers designed by the present invention can successfully amplify clear waterfall-like bands, but the LAMP primer group composed of primers F3-1, B3-1, FIP-1, and BIP-1 has the best effect. Therefore, primers F3-1, B3-1, FIP-1, and BIP-1 are selected as LAMP amplification primers, and the LAMP amplification conditions are optimized in the subsequent process.

[0063] Table 2 LAMP amplification system

[0064]

[0065] 1.3 Optimization of LAMP amplification system

[0066] The temperature and amplification time in the above LAMP amplification system were optimized. The optimal LAMP amplification temperature was found to be 68°C ( Figure 2 B), time is 40 minutes ( Figure 2 C).

[0067] Example 2 Establishment of LAMP-CRISPR / Cas12a detection system

[0068] 2.1 crRNA design

[0069] For the sequence of the product amplified by the LAMP primer set, find the detection site 23 nt after PAM (TTTV, V is G, A or C).

[0070] AGAU) plus the target sequence, i.e., 23 nt after the PAM sequence, and three crRNAs were designed as shown in Table 3.

[0071] Table 3 crRNA sequences

[0072]

[0073] The above LAMP amplification positive product was used as a template and ddH 2O is a negative control, and the crRNA in Table 3 above is used to perform CRISPR / Cas12a detection. The CRISPR / Cas12a detection system is shown in Table 4, where the ssDNA probe sequence is 5'-FAM-TTATT-BHQ-3', and the 5' end of the ssDNA probe is labeled with 6-FAM and the 3' end is labeled with BHQ-1. After the reaction system is prepared, vortex and shake to mix, then centrifuge instantly, place in a fluorescent quantitative PCR instrument at 37°C, react for 10 min, read the fluorescence value data, compare the fluorescence intensity and reaction rate, and use a gel imaging system to observe the fluorescence using blue light to screen out the optimal crRNA. The results are shown in Figure 3 As shown in A, all three crRNAs can activate ssDNA cleavage in the complex within the system and release fluorescent signals, thus realizing rapid visual detection of duck circovirus on site. Figure 3 As shown in B, the fluorescence signal value measured by the detection reaction solution of the crRNA-1 group is higher than that of the crRNA-2 and 3 groups, indicating that the fluorescence signal of the crRNA-1 group is the strongest.

[0074] Table 4 Components of the test system

[0075]

[0076] Example 3 Optimization of LAMP-CRISPR / Cas12a detection system

[0077] In order to achieve rapid detection of the LAMP-CRISPR / Cas12a detection system, the concentrations of Cas12a and crRNA were optimized based on the above crRNA screening. The above LAMP amplification product was used as a template and sterilized ddH 2 O was used as a negative control for CRISPR / Cas12a detection experiments. The reaction temperature was fixed at 37 °C, and four final concentrations of crRNA and Lba Cas12a were set at 25, 50, 100, 150, and 200 nM. The reaction was performed using a real-time fluorescence quantitative PCR instrument, and the fluorescence signal was collected every 30 s for 30 cycles, and then observed with blue light or ultraviolet light. By comparing the fluorescence intensity and reaction rate through the amplification curve, it was determined that the optimal final reaction concentrations of crRNA and Lba Cas12a in the detection system were 50 nM ( Figure 4 A and 4B) and 150 nM ( Figure 4 C and 4D), the optimal LAMP-CRISPR / Cas12a detection system is shown in the following table; the components of the fixed detection system remain unchanged, and the detection is carried out at 35℃ to 39℃, and the optimal reaction temperature is determined to be 37℃ ( Figure 4 E and 4F).

[0078]

[0079] Example 4 Specificity and sensitivity test of LAMP-CRISPR / Cas12a detection method

[0080] In order to verify the specificity of the CRISPR / Cas12a-mediated cleavage assay, the method established in this study was used to simultaneously detect duck circovirus (DuCV), duck plague virus (DPV), avian adenovirus type 4 (FAdV-4), duck hepatitis A virus type 3 (DHAV-3), and duck Tembusu virus (DTMUV). 2 O served as a negative control.

[0081] The specific method of the specific experiment is:

[0082] (1) Extracting DNA from samples to be tested: extracting DNA from duck circovirus (DuCV), duck plague virus (DPV), avian adenovirus type 4 (FAdV-4), duck hepatitis A virus type 3 (DHAV-3), and duck Tembusu virus (DTMUV) to obtain DNA from samples to be tested;

[0083] (2) Using the DNA extracted in step (1) as a template, amplification was performed in the optimized LAMP amplification system; wherein the total volume of the LAMP amplification system was 25 μL, 12.5 μL 2×LAMP Master Mix, 0.5 μL DNA Polymerase, 0.5 μL each of the outer primers F3-1 and B3-1, to a final concentration of 0.2 μM, 2 μL each of the inner primers FIP-1 and BIP-1, to a final concentration of 0.8 μM, 1 μL of the sample DNA to be tested, and ddH 2 O was added to 25 μL; the optimal LAMP amplification temperature was 68°C and the time was 40 minutes;

[0084] (3) The LAMP amplification product obtained in step (2) was subjected to enzyme digestion and fluorescence detection in the optimized CRISPR / Cas12a detection system; wherein the total volume of the CRISPR / Cas12a detection system is 20 μL, including 2 μL of Cas12a reaction buffer (10×), 1 μL of Lba Cas12a to a final concentration of 150 nM, 1 μL of crRNA-1 (1 μM) to a final concentration of 50 nM, 1.2 μL of ssDNA (4 μM) to a final concentration of 240 nM, and 1 μL of LAMP amplification product, supplemented with ddH 2 O to 20 μL. The optimal reaction temperature is 37°C

[0085] The specific test results showed that only duck circovirus was positive, while the other viruses and negative controls were not detected ( Figure 5 A, 5B), indicating that this method has high specificity.

[0086] To determine the sensitivity of this method, a pair of primers for amplifying the rep gene fragment (upstream: 5'-TGG AAC GCC TCC GTC ACC TGA T-3', downstream: 5'-GTC GGT AAT TCT CAG CAA ATCAT-3') were designed based on the LAMP amplification fragment. The amplified fragment was 236 bp in length and cloned into the pUC57 vector as a standard plasmid according to the conventional method and named pUC57-rep. The copy number of the recombinant plasmid pUC57-rep extracted from the positive clone was 2.88×10 9 Copies / μL. pUC57-rep was diluted 10-fold, from 2.88×10 9 Copies / μL diluted to 2.88×10 -1 Copies / μL. 10-fold serial dilution of plasmid (2.88×10 9 ~2.88×10 -1 Copies / μL) as a template with sterilized ddH 2 O was used as a negative control and tested under optimized conditions to verify the sensitivity of the detection method. The results of the sensitivity test showed that the detection limit of the detection method was 2.88×10 0 Copies / μL( Figure 5 C, 5D).

[0087] Example 5 Clinical sample testing

[0088] In order to further evaluate the consistency of the LAMP-CRISPR / Cas12a detection method with the existing detection methods, the LAMP-CRISPR / Cas12a method established in this study (refer to the operation method of the specific experiment) was used to detect 32 duck tissue samples suspected of being infected with duck circovirus, and the TaqMan qPCR (invention patent number ZL202010748507.0) established in this laboratory was used for comparison. The results are shown in Table 5. The positive detection rate of the LAMP-CRISPR / Cas12a detection method established in the present invention was the same as that of TaqMan qPCR, which was 42.75% (14 / 32), indicating that the consistency rate of the LAMP-CRISPR / Cas12a method with the TaqMan qPCR detection results was 100%.

[0089] Table 5 Clinical sample test results

[0090]

[0091] From the above results, it can be seen that the LAMP-CRISPR / Cas12a detection system established using the LAMP primer set and crRNA of the present invention can achieve high specificity, sensitivity, visualization and rapid on-site detection of duck circovirus-positive samples, which is of great value for the detection and prevention and control of duck circovirus.

[0092] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A reagent combination for visual detection of duck circovirus based on LAMP-CRISPR / Cas12a, characterized in that: It includes LAMP amplification primers and crRNA; The LAMP amplification primers include an outer primer pair F3-1 and B3-1, and an inner primer pair FIP-1 and BIP-1; the nucleotide sequences of the outer primer pair F3-1 and B3-1 are respectively: F3-1:5'-ATCGTCGAGACGCAACGT-3', B3-1: 5'-ATCATACGGCAACCAACCAT-3'; The nucleotide sequences of the inner primers FIP-1 and BIP-1 are: FIP-1: 5'-TGCATAACGGCTCTTTCCGGTTTGGAAGACCGAAGTCATCG-3', BIP-1: 5'-TTACAAACCACGCGGGAAGTGGAGTCGTCCATGACGACTACG-3'; The crRNA is any one of crRNA-1, crRNA-2 and crRNA-3, and the nucleotide sequences of crRNA-1, crRNA-2 and crRNA-3 are respectively: crRNA-1: 5'-UAAUUUCUACUAAGUGUAGAUCCGCCGAAAACAAGUAUUACAAA-3', crRNA-2: 5'-UAAUUUCUACUAAGUGUAGAUGGCGGGAAAUUCAAAUGCAUAAC-3', crRNA-3: 5'-UAAUUUCUACUAAGUGUAGUAAUUUCCCGCCGAAAACAAGUAU-3'.

2. The reagent combination according to claim 1, characterized in that: The crRNA is crRNA-1.

3. The reagent combination according to claim 1, characterized in that: It also includes a ssDNA probe, the nucleotide sequence of which is 5'-TTATT-3'.

4. A kit for visual detection of duck circovirus based on LAMP-CRISPR / Cas12a, characterized in that: The kit includes a LAMP amplification system and a CRISPR / Cas12a detection system; The LAMP amplification system includes an outer primer pair F3-1 and B3-1, and an inner primer pair FIP-1 and BIP-1; the nucleotide sequences of the outer primer pair F3-1 and B3-1 are respectively: F3-1:5'-ATCGTCGAGACGCAACGT-3', B3-1: 5'-ATCATACGGCAACCAACCAT-3'; The nucleotide sequences of the inner primers FIP-1 and BIP-1 are: FIP-1: 5'-TGCATAACGGCTCTTTCCGGTTTGGAAGACCGAAGTCATCG-3', BIP-1: 5'-TTACAAACCACGCGGGAAGTGGAGTCGTCCATGACGACTACG-3'; The CRISPR / Cas12a detection system includes crRNA, wherein the crRNA is any one of crRNA-1, crRNA-2 and crRNA-3, and the nucleotide sequences of crRNA-1, crRNA-2 and crRNA-3 are respectively: crRNA-1: 5'-UAAUUUCUACUAAGUGUAGAUCCGCCGAAAACAAGUAUUACAAA-3', crRNA-2: 5'-UAAUUUCUACUAAGUGUAGAUGGCGGGAAAUUCAAAUGCAUAAC-3', crRNA-3: 5'-UAAUUUCUACUAAGUGUAGUAAUUUCCCGCCGAAAACAAGUAU-3'.

5. The kit according to claim 4, characterized in that: The crRNA is crRNA-1.

6. The kit according to claim 4, characterized in that: The LAMP amplification system also includes 2×LAMP Master Mix, DNA Polymerase, ddH2O, samples to be tested, and negative and positive control samples.

7. The kit according to claim 4, characterized in that The CRISPR / Cas12a detection system also includes Cas12a Reaction buffer (10×), Lba Cas12a, LAMP amplification product, ssDNA probe and ddH2O.

8. The kit according to claim 7, characterized in that: The nucleotide sequence of the ssDNA probe is 5'-TTATT-3'.

9. Use of the kit as claimed in claim 8 in the detection of duck circovirus for non-diagnostic purposes.

10. The use according to claim 9, characterized in that: The method for visually detecting duck circovirus using the kit comprises the following steps: (1) Extract DNA from the sample to be tested; (2) using the DNA extracted in step (1) as a template, amplifying in a LAMP amplification system; (3) performing enzyme digestion and fluorescence detection on the LAMP amplification product obtained in step (2) in a CRISPR / Cas12a detection system; The total volume of the LAMP amplification system is 25 μL, 12.5 μL 2×LAMP Master Mix, 0.5 μL DNA Polymerase, 0.5 μL each of the outer primers F3-1 and B3-1, to a final concentration of 0.2 μM, 2 μL each of the inner primers FIP-1 and BIP-1, to a final concentration of 0.8 μM, 1 μL of the sample DNA to be tested, and ddH2O supplemented to 25 μL; The total volume of the CRISPR / Cas12a detection system is 20 μL, including 2 μL of Cas12a Reaction buffer (10×), 1 μL of Lba Cas12a to a final concentration of 150 nM, 1 μL of crRNA-1 (1 μM) to a final concentration of 50 nM, 1.2 μL of ssDNA (4 μM) to a final concentration of 240 nM, 1 μL of LAMP amplification product, and ddH2O supplemented to 20 μL.

Images