A system and kit for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a, and its application

Abstract

The present application belongs to the biological field and discloses a system for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a. The system includes a first RPA primer pair and a second RPA primer pair designed for the Fiber2 gene and Hexon gene of avian adenovirus FAdV-4, respectively, Cas12a protein, crRNA-Fiber2, crRNA-Hexon, and ssDNA-reporter; in addition, the present application also discloses a kit for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a, which includes the above system as well as buffer, RNase-free H2O, RNase inhibitor, and DNA plasmid.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a system, kit, and application for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a. Background Art

[0002] Avian adenovirus disease is a common, highly pathogenic, and contagious disease in poultry. Avian adenovirus (FAdV) is a pathogenic organism comprising three major groups: serogroups I, II, and III. These serogroups can cause hydropericardial effusion syndrome (HPS) in chickens, inclusion body hepatitis in chickens, hemorrhagic enteritis in turkeys, splenomegaly in chickens, and egg production decline syndrome in chickens, respectively. FAd V-I is divided into five species and 12 serotypes, including A (serotype 1), B (serotype 5), C (serotypes 4 and 10), D (serotypes 2, 3, 9, and 11), and E (serotypes 6, 7, 8a, and 8b). Over the years, clinical cases of poultry infection with group I adenovirus have increased dramatically. Among these, HPS (HPS), caused by serotype 4 (FAdV-4), is characterized by rapid onset, rapid spread, and high mortality, resulting in significant economic losses for the domestic poultry industry.

[0003] The disease is most common in broiler and growing chickens between 20 and 60 days of age, and is also common in laying hens up to 300 days of age. FAdV-4 is a virus with a distinct age-specific pathogenicity. Its pathogenicity is highest in young chickens, with a mortality rate as high as 100%, but this rate gradually decreases with age. The virus can be transmitted not only vertically but also horizontally. The main characteristic pathological changes of this disease are the accumulation of clear, translucent, or pale yellow, watery or jelly-like fluid in the pericardium of deceased chickens, and myocardial weakness.

[0004] Chinese patent application 202210842143.1 discloses a CRISPR-Cas13 system, a kit, and a method for detecting FAdV-4, wherein the CRISPR-Cas13 system includes a Cas13a nuclease, a guide RNA probe, and an FB-RNA reporter molecule; the guide RNA probe targets the Hexon gene of avian adenovirus serotype 4, and the Cas13a nuclease activates the enzyme activity after recognizing the target gene with the guide RNA probe, and cuts the FB-RNA reporter molecule to release a detection signal.

[0005] This detection system uses a two-step method for detection, and the entire process takes about 70 minutes. The primer and probe combination it provides can detect a minimum plasmid copy number of 10 copies / μL.

[0006] The problem that this solution needs to solve is: how to make a rapid, sensitive, simple and specific FAdV-4 test kit that can be used on site. Summary of the Invention

[0007] The purpose of this application is to provide a system and kit for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a, which binds to crRNA to guide the Cas12a protein to perform positive cleavage of viral nucleic acid, while also having the ability to non-specifically cleave ssDNA.

[0008] To achieve the above objectives, this paper provides a system for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a, characterized in that the system for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a includes a first RPA primer pair, a second RPA primer pair, Cas12a protein, crRNA-Fiber2, crRNA-Hexon, ssDNA-reporter, and DNA plasmid;

[0009] The first RPA primer pair includes an upstream primer RPA-FI-F and a downstream primer RPA-FI-R, and the first RPA primer pair is designed for the Fiber2 gene of fowl adenovirus FAdV-4;

[0010] The second RPA primer pair includes an upstream primer RPA-HE-F and a downstream primer RPA-HE-R, and the second RPA primer pair is designed for the Hexon gene of fowl adenovirus FAdV-4;

[0011] The crRNA-Fiber2 is designed for the Fiber2 gene of fowl adenovirus FAdV-4;

[0012] The crRNA-Hexon is designed for the Hexon gene of fowl adenovirus FAdV-4;

[0013] In the working process of the CRISPR-Cas12a-based system for detecting avian adenovirus FAdV-4, when crRNA-Fiber2 or crRNA-Hexon forms a ternary complex with DNA plasmid and Cas12a protein, the Cas12a protein is guided to forward-cleave the amplification products of the first RPA primer pair and the second RPA primer pair. At the same time, the Cas12a protein spontaneously reverse-cuts the ssDNA-reporter and releases the fluorescent group.

[0014] Preferably, the sequence of the upstream primer RPA-FI-F in the first RPA primer pair is shown as SEQ ID NO.1;

[0015] The sequence of the downstream primer RPA-FI-R of the first RPA primer pair is shown in SEQ ID NO.2;

[0016] The sequence of the upstream primer RPA-HE-F of the second RPA primer pair is shown in SEQ ID NO.3;

[0017] The sequence of the downstream primer RPA-HE-R of the second RPA primer pair is shown in SEQ ID NO.4.

[0018] Preferably, the sequence of the crRNA-Fiber2 upstream primer crRNA-Fiber2-F is as shown in SEQ ID NO.5;

[0019] The sequence of the crRNA-Fiber2 downstream primer crRNA-Fiber2-R is shown in SEQ ID NO.6;

[0020] The sequence of the crRNA-Hexon upstream primer crRNA-Hexon-F is shown in SEQ ID NO.7;

[0021] The sequence of the crRNA-Hexon downstream primer crRNA-Hexon-R is shown in SEQ ID NO.8.

[0022] Preferably, the ssDNA-reporter is a single-stranded nucleotide sequence labeled with FAM at the 5' end and BHQ1 at the 3' end, or a single-stranded nucleotide sequence labeled with FAM at the 5' end and Biotin at the 3' end.

[0023] Preferably, the ssDNA-reporter is ssDNA-reporter-CLA or ssDNA-reporter-LFA, and the nucleotide sequence of the ssDNA-reporter-CLA is 5'-FAM-TTATT-BHQ1-3';

[0024] The nucleotide sequence of the ssDNA-reporter-LFA is 5'-FAM-TTTTTTTTTTTTTTT-Biotin-3'.

[0025] In addition, a kit for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a is also disclosed, which comprises any of the above-mentioned systems for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a.

[0026] Preferably, the kit further comprises Buffer, RNase-free H2O, and RNase Inhibitor.

[0027] The beneficial effects of this application are:

[0028] The present application provides a system and kit for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a. The kit can be used for on-site detection and is rapid, sensitive, simple, and can specifically target the FAdV-4 virus. The Cas12a protein in the system combines with crRNA and DNA plasmid to form an RNP complex, and the crRNA guides the complex to perform forward cleavage of the FAdV-4 viral nucleic acid. At the same time, it also reversely cuts the ssDNA-reporter labeled with a fluorescent group to achieve detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Screening fluorescence results for 8 pairs of crRNA;

[0030] Figure 2 This is the electrophoresis result of screening 7 pairs of RPA primers in one tube method;

[0031] Figure 3 Electrophoresis results of three pairs of RPA primers screened for the two-step method;

[0032] Figure 4 The fluorescence results of ssDNA at different concentrations;

[0033] Figure 5 is the fluorescence value of ssDNA at different concentrations;

[0034] Figure 6 is the fluorescence value of different ratios of Lbcas12a and crRNA;

[0035] Figure 7 To specifically detect fluorescence results;

[0036] Figure 8 To specifically detect the fluorescence value;

[0037] Figure 9 This is the sensitivity test result of the one-step method. Three replicates were made for each dilution factor. From tube 1 to tube 11, the results are: 7.17*10 9 copy / ul, 7.17*10 8 copy / ul, 7.17*10 7 copy / ul, 7.17*10 6 copy / ul, 7.17*10 5 copy / ul, 7.17*10 4 copy / ul, 7.17*10 3 copy / ul, 7.17*10 2 copy / ul, 7.17*10 1 copy / ul, 7.17*100 copy / ul, 7.17*10 -1 copy / ul, negative control;

[0038] Figure 10 To detect the baseline fluorescence results in one step, three replicate wells were made for each dilution factor, which were 7.84*10 2 *1 / 2copy / ul, 196copy / ul, 98copy / ul, 49copy / ul, negative control;

[0039] Figure 11 The fluorescence detection results of the two-step method are 1.40*10 from left to right. 10 copy / ul, 1.40*10 9 copy / ul, 1.40*10 8 copy / ul, 1.40*10 7 copy / ul, 1.40*10 6 copy / ul, 1.40*10 5 copy / ul, 1.40*10 4 copy / ul, 1.40*10 3 copy / ul, 1.40*10 2 copy / ul, 1.40*10 1 copy / ul, 7copy / ul, 3.5copy / ul, 1.75copy / ul, 0.875copy / ul;

[0040] Figure 12 This is the test result of the two-step sensitivity test strip;

[0041] Figure 13 This is the test result of the two-step sensitivity test strip;

[0042] Figure 14 The fluorescence results of 19 clinically positive samples were detected by one tube method;

[0043] Figure 15 The fluorescence results of 19 clinically positive samples were detected using a two-step method;

[0044] Figure 16 The fluorescence results of 50 random samples were detected by one tube method;

[0045] Figure 17 Results of 50 random samples tested by qPCR. DETAILED DESCRIPTION

[0046] In the description of this application, it should be noted that if specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0047] Example 1

[0048] Design and screening of RPA primer sets

[0049] Targeting the FAdV4 fiber2 gene (GenBank accession no. MW509553.1), we used snapgene for sequence alignment and analysis of conserved regions to design seven pairs of RPA primers, which were synthesized by Sangon Biotechnology (PAGE purified). After testing and screening, the following set was finally selected:

[0050] RPA-FI-F: 5'-AAGTCCCAGCCTCAACACCTACAATGCCACGA-3';

[0051] RPA-FI-R: 5'-CCATTTGGCATTGGCGGAGTTGAGGTCCCCAG-3';

[0052] The amplified fragment size was 152 bp;

[0053] Three pairs of RPA primers were designed for the FAdV4 Hexon gene (GenBank accession no. MN102413.1) and synthesized by Qingke Biotechnology (PAGE purification). After testing and screening, the final set was selected as follows:

[0054] RPA-HE-F: 5'-ATTAAACTCTACACCAACAACACCGCCGCGAAC-3';

[0055] RPA-HE-R: 5'-CGTCGAAGCCGGAGATGCTAAACTTGTAACGGTC-3';

[0056] The amplified fragment size was 162 bp;

[0057] Example 2

[0058] Design and screening of crRNA

[0059] The PAM sequence recognized by Cas12a is TTTN. In the two-step method, a TTTN-type PAM sequence is selected. In the one-tube method, a suboptimal PAM sequence is selected instead of a TTTN-type PAM sequence. Eight pairs of crRNAs were designed and synthesized and purified by GeneWeichi. The crRNA sequences are shown in Table 1:

[0060] Table 1

[0061]

[0062]

[0063] according to Figure 1 As shown in the fluorescence results, the crRNA-Fiber2 primer set was designed based on the crRNA1 primer set, which includes the following primers:

[0064] crRNA-Fiber2-F: 5'-GAAATTAATACGACTCACTATAGGGtaatttctactaagtgtagatCTTGAAATTGGACAGCGCCACCA-3';

[0065] crRNA-Fiber2-R: 5'-TGGTGGCGCTGTCCAATTTCAAGatctacacttagtagaaattaCCCTATAGTGAGTCGTATTAATTTC-3';

[0066] The crRNA-Hexon primer set was designed using software and contains the following primers:

[0067] crRNA-Hexon-F: 5'-GAAATTAATACGACTCACTATAGGGTAATTTCTACTAAGTGTAGATGTAGGAGGGTACCGTGCCGAAGT-3';

[0068] crRNA-Hexon-R: 5'-ACTTCGGCACGGTACCCTCCTACATCTACACTTAGTAGAAATTACCCTATAGTGAGTCGTATTAATTTC-3';

[0069] Annealing system: upstream primer 10ul, downstream primer 10ul, buffer 10ul, ddH2O 20ul; PCR program settings: 95℃ 2min, 95℃ options increment -0.1℃ 8s, 700X, storage at 4℃.

[0070] The DNA double strand was transcribed into RNA using the T7 in vitro transcription kit (HiScribe T7 Quick High Yield RNA Synthesis Kit), purified using the NucleoSpin RNA Clean-up kit (Macherey-Nagel), and the concentration was measured on a NanoDrop. The purified crRNA was frozen and stored at -80°C. The T7 transcription process was as follows:

[0071] The annealed crRNA1-5 was transcribed in vitro using Novoprotein's T7 high yield transcript kit according to the instructions. The DNA template concentration was 1 μg / μl and the transcription was carried out at 37°C for 16 h. The components and contents of the transcription kit are shown in Table 2:

[0072] Table 2

[0073] Element Volume (20 μl) Reaction Buffer 2 μl ATP / GTP / CTP / UTP Solution 1.5 μl each DNA TemplateX 0.5~1.0μg T7 RNA Polymerase Mix 2 μl <![CDATA[RNase free H2O]]> Up to 20μl

[0074] Example 3

[0075] Establishment of the RPA-CRISPR-Cas12a method

[0076] 1. The content of each component detected by the RPA-CRISPR-Cas12a one-tube method is:

[0077] RPA component 18ul (including 10uM RPA-FI-F 2ul, 10uM RPA-FI-R 2ul, ABuffer 29.4ul, RNase-free H2O 14.1ul), Lbcas12a 100nM, crRNA 75nM, ssDNA-reporter-CLA 400nM, RNase Inhibitor 0.5ul, B Buffer 2ul, plasmid DNA 2ul, and RNase-free H2O were added to 28ul, mixed, and reacted at 37°C in the dark for 40min.

[0078] 2. Detection by the RPA-CRISPR-Cas12a two-step method:

[0079] RPA amplification reaction system: 10uM RPA-HE-F 2ul, 10uM RPA-HE-R 2ul, A buffer 29.4ul, RNase-free H2O 11.1ul, B buffer 2.5ul, plasmid DNA 1.5ul, reaction at 37℃ for 25min.

[0080] The CRISPR-Cas12a cleavage reaction system is as follows: RNase inhibitor 0.5ul, ssDNA-reporter-CLA400nM / ssDNA-reporter-LFA 400nM, crRNA 100nM, Lbcas12a 100nM, NE Buffer 2.1 2ul, RPA amplification product 3ul, RNase-free H2O added to 20ul, and reacted at 37°C in the dark for 25min.

[0081] Example 4

[0082] Screening the crRNA with the highest efficiency: According to the method of Example 3, 8 pairs of crRNA were screened and the crRNA with better results was selected. The fluorescence results were as follows Figure 1 As shown, crRNA1 showed better results and higher sensitivity among all crRNAs.

[0083] Example 5

[0084] Screening RPA primer sets: According to the RPA amplification method in Example 3, 10 pairs of RPA primers were screened, and the RPA amplification products were subjected to 1.5% agarose gel electrophoresis to observe the position and brightness of the bands. The results are as follows: Figure 2-3 As shown, RPA primer 3 and RPA primer 1 were selected.

[0085] Example 6

[0086] Exploring ssDNA concentration: According to the method in Example 3, four groups of ssDNA-reporter concentrations were set, namely 100nM for group D, 200nM for group C, 300nM for group B, and 400nM for group A. The optimal concentration was screened. The results are as follows: Figure 4-5 As shown, the optimal concentration for group A was 400 nM.

[0087] Example 7

[0088] Exploration of the ratio of Lbcas12a to crRNA: According to the method of Example 3, four groups of Lbcas12a and crRNA concentration ratios were set, namely:

[0089] Experimental group 1: Lbcas12a: crRNA = 100 nm: 100 nm;

[0090] Experimental group 2: Lbcas12a: crRNA = 100 nm: 200 nm;

[0091] Experimental group 3: Lbcas12a: crRNA = 50 nm: 100 nm;

[0092] Experimental group 4: Lbcas12a: crRNA = 50 nm: 50 nm;

[0093] The results are as follows Figure 6 As shown, the ratio shown in experimental group 1 is the best.

[0094] Example 8

[0095] RPA-CRISPR-Cas12a detects FAdV-4 specificity analysis: According to the method of Example 3, the detection includes chicken infectious bronchitis virus (IBV), chicken infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), chicken Marek's virus (MDV), avian adenovirus serotype 8b and avian adenovirus serotype 11. The results are as follows Figure 7-8 As shown, no fluorescence was detected, indicating specificity.

[0096] Example 9

[0097] RPA-CRISPR-Cas12a sensitivity detection:

[0098] 1. One-tube method

[0099] Dilute the plasmid standard: dilute the plasmid standard containing the FAdV-4 fiber2 gene fragment with RNase-free H2O from 7.84*10 10 copy / ul is diluted tenfold to 7.84*10 2 copy / ul, and then dilute half to 49copy / ul;

[0100] The diluted plasmid was tested according to the one-tube method of Example 3. The results are as follows: Figure 9-10 As shown, the minimum detection limit is 98 copy / ul.

[0101] 2. Two-step method

[0102] Dilute the plasmid standard: dilute the plasmid standard containing the FAdV-4 hexon gene fragment with RNase-free H2O to 1.40*10 11 copy / ul ten-fold dilution to 1.40*10 1 copy / ul, and then dilute half to 0.875copy / ul;

[0103] The diluted plasmid was detected by the two-step method of Example 3, and the results were checked by fluorescence and lateral flow chromatography (colloidal gold method). Figure 11-13 As shown, the minimum detection limit of lateral flow assay (colloidal gold method) is 14 copy / ul, and the minimum detection limit of fluorescence assay is 7 copy / ul.

[0104] Example 10

[0105] RPA-CRISPR-Cas12a detection of clinical samples: According to the method of Example 3, 19 positive samples infected with FAdV-4 were tested using the one-tube method and the two-step method, and a negative control was set for each group. The results are as follows Figure 14-15 As shown, all were positive.

[0106] Example 11

[0107] RPA-CRISPR-Cas12a repeatability test: According to the method of Example 3, 50 samples to be tested were tested using the one-tube method and fluorescent quantitative PCR, including 38 positive samples (positive tissue lesions and allantoic fluid) and 12 negative samples. The results are as follows: Figure 16 、 17 As shown in Table 3, the results of the one-tube method test were 12 negative and 38 positive; the results of the qPCR test were 12 negative and 38 positive, and the consistency rate of the two was 100%. The test results are shown in Table 3:

[0108] Table 3

[0109]

[0110] The above embodiments are preferred implementation modes of the present application, but the implementation modes of the present application are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present application shall be considered as equivalent replacement methods and shall be included in the scope of protection of the present application.

Claims

1. A reagent for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a, characterized in that The CRISPR-Cas12a-based system for detecting avian adenovirus FAdV-4 includes a first RPA primer pair, a second RPA primer pair, Cas12a protein, crRNA-Fiber2, crRNA-Hexon, ssDNA-reporter, and a DNA plasmid; The first RPA primer pair includes an upstream primer RPA-FI-F and a downstream primer RPA-FI-R, and the first RPA primer pair is designed for the Fiber2 gene of fowl adenovirus FAdV-4; The second RPA primer pair includes an upstream primer RPA-HE-F and a downstream primer RPA-HE-R, and the second RPA primer pair is designed for the Hexon gene of fowl adenovirus FAdV-4; The crRNA-Fiber2 is designed for the Fiber2 gene of fowl adenovirus FAdV-4; The crRNA-Hexon is designed for the Hexon gene of fowl adenovirus FAdV-4; In the working process of the CRISPR-Cas12a-based system for detecting avian adenovirus FAdV-4, when crRNA-Fiber2 or crRNA-Hexon forms a ternary complex with the DNA plasmid and Cas12a protein, the Cas12a protein is guided to forward-cleave the amplified products of the first RPA primer pair and the second RPA primer pair, while the Cas12a protein spontaneously reverse-cleaves the ssDNA-reporter and releases the fluorescent group; The sequence of the upstream primer RPA-FI-F in the first RPA primer pair is shown in SEQ ID NO. 1; The sequence of the downstream primer RPA-FI-R of the first RPA primer pair is shown in SEQ ID NO. 2; The sequence of the upstream primer RPA-HE-F of the second RPA primer pair is shown in SEQ ID NO. 3; The sequence of the downstream primer RPA-HE-R of the second RPA primer pair is shown in SEQ ID NO. 4; The sequence of the crRNA-Fiber2 upstream primer crRNA-Fiber2-F is shown in SEQ ID NO. 5; The sequence of the crRNA-Fiber2 downstream primer crRNA-Fiber2-R is shown in SEQ ID NO. 6; The sequence of the crRNA-Hexon upstream primer crRNA-Hexon-F is shown in SEQ ID NO. 7; The sequence of the crRNA-Hexon downstream primer crRNA-Hexon-R is shown in SEQ ID NO.

8.

2. The reagent for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a according to claim 1, characterized in that The ssDNA-reporter is a single-stranded nucleotide sequence labeled with FAM at the 5' end and BHQ1 at the 3' end, or a single-stranded nucleotide sequence labeled with FAM at the 5' end and Biotin at the 3' end.

3. The reagent for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a according to claim 2, characterized in that The ssDNA-reporter is ssDNA-reporter-CLA or ssDNA-reporter-LFA, and the nucleotide sequence of the ssDNA-reporter-CLA is 5'-FAM-TTATT-BHQ1-3'; The nucleotide sequence of the ssDNA-reporter-LFA is 5'-FAM-TTTTTTTTTTTTTTT-Biotin-3'.

4. A kit for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a, characterized in that: The invention comprises a reagent for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a according to any one of claims 1 to 3.

5. The kit for detecting avian adenovirus FAdV-4 based on CRISPR-Cas12a according to claim 4, characterized in that Also includes Buffer, RNase-free H2O, and RNase Inhibitor.

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