Rapid detection method for avian metapneumovirus based on CRISPR-Cas13a system

Abstract

The present invention discloses a rapid detection method for avian metapneumovirus based on the CRISPR-Cas13a system, which relates to the technical field of avian disease detection. More specifically, it discloses a primer set for detecting avian metapneumovirus based on the CRISPR-Cas13a system, and the primer set includes an isothermal amplification primer pair and a crRNA sequence. The detection method of the present invention can specifically detect avian metapneumovirus, with a sensitivity of 2 copies, and has the advantages of good specificity, high sensitivity, good repeatability, simple and rapid operation, and does not rely on professional technicians and specific instrument equipment, etc. It is suitable for grass-roots and on-site applications, can rapidly detect avian metapneumovirus nucleic acid molecules, and provides an efficient and accurate detection method for determining early infection of avian metapneumovirus in production.

Description

Technical Field

[0001] The present invention relates to the technical field of avian disease detection, and more specifically, to a rapid detection method for avian metapneumovirus based on the CRISPR-Cas13a system. Background Art

[0002] Avian metapneumovirus (aMPV) belongs to the Paramyxoviridae family, Pneumovirinae subfamily, and Metapneumovirus genus. Turkeys, chickens, and ducks of different ages can be infected with aMPV. After poultry are infected with aMPV, secondary infections often occur, such as Escherichia coli infection, which can cause swollen head syndrome and a series of neurological symptoms, such as torticollis and opisthotonos. In addition, aMPV can proliferate in reproductive and urinary tract tissues, leading to the degeneration of the oviduct and ovary of breeding chickens, resulting in a decrease in egg production and the production of deformed eggs, causing great harm to the poultry industry.

[0003] Currently, the main molecular biology detection methods for aMPV are RT-PCR and real-time fluorescence quantitative PCR. However, the above methods have the disadvantages of long detection time, cumbersome steps, high cost, dependence on professional technicians and specific instrument equipment, and high requirements for the detection environment, and are not suitable for clinical on-site detection.

[0004] Therefore, developing a method that does not rely on expensive detection instruments and can achieve efficient, rapid, and on-site detection of avian metapneumovirus is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a rapid detection method for avian metapneumovirus based on the CRISPR-Cas13a system. This method has the advantages of good specificity, high sensitivity, good repeatability, simple and rapid operation, and does not rely on professional technicians and specific instrument equipment. It is suitable for grass-roots and on-site applications, can rapidly detect avian metapneumovirus nucleic acid molecules, and provides an efficient and accurate detection method for determining early avian metapneumovirus infection in production.

[0006] To achieve the above object, the present invention adopts the following technical scheme:

[0007] A primer group for detecting avian metapneumovirus based on the CRISPR-Cas13a system, the primer group includes an isothermal amplification primer pair and a crRNA sequence;

[0008] Moreover, the nucleotide sequences of the isothermal amplification primer pair are as shown in SEQ ID NO.3 and SEQ ID NO.5, and the nucleotide sequence of the crRNA sequence is as shown in SEQ ID NO.11.

[0009] Another object of the present invention is to provide a kit for detecting avian metapneumovirus based on the CRISPR-Cas13a system, and the kit includes the above-mentioned primer set.

[0010] Preferably, the kit further includes LwaCas13a protein, fluorescent RNA reporter molecule, T7 RNA polymerase, NTPmix, RNase inhibitor, buffer.

[0011] More preferably, the sequence of the fluorescent RNA reporter molecule is 5'-FAM-UUUUUU-BHQ1-3', and the final concentration of the buffer is 30.6 mmol / L HEPES + 15.3 mmol / L MgCl2 aqueous solution.

[0012] Another object of the present invention is to provide the application of the above-mentioned primer set or the above-mentioned kit in the preparation of avian metapneumovirus detection products.

[0013] Another object of the present invention is to provide a method for detecting avian metapneumovirus based on the CRISPR-Cas13a system, including the following steps:

[0014] S1: Extract the RNA of the sample to be tested and reverse transcribe it into cDNA;

[0015] S2: Perform isothermal amplification on the cDNA to be tested, and the primers are the sequences shown in SEQ ID NO.3 and SEQ ID NO.5;

[0016] S3: Detect the isothermal amplification product obtained in S2 by using the CRISPR-Cas13a reaction system, and the crRNA in the CRISPR / Cas13a system is the sequence shown in SEQ ID NO.11;

[0017] S4: Result determination, perform biostatistical analysis on the difference between the fluorescence value at the reaction end point and the starting fluorescence value of the experimental group and the difference between the fluorescence value at the reaction end point and the starting fluorescence value of the negative control group. If there is a difference, the test result is positive; if there is no difference, the test result is negative.

[0018] Preferably, the isothermal amplification system in S2 is as shown in Table 2 of the specification, and the condition of the isothermal amplification is 39°C for 20 min.

[0019] Preferably, the CRISPR-Cas13a reaction system in S3 is as shown in Table 3 of the specification, and the condition of the CRISPR-Cas13a reaction is 37°C for 40 min.

[0020] Beneficial effects: The detection method of the present invention can specifically detect avian metapneumovirus, with a sensitivity of 2 copies. It has the advantages of good specificity, high sensitivity, good repeatability, simple and rapid operation, and does not rely on professional technicians and specific instrument equipment. It is suitable for grass-roots and on-site applications, can quickly detect the nucleic acid molecules of avian metapneumovirus, and provides an efficient and accurate detection method for determining early infection of avian metapneumovirus in production. Brief description of the drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0022] Figure 1 It is a schematic diagram for screening isothermal amplification primers. From top to bottom, the 3 rows are the upstream primers T71F, T72F, and T73F in sequence. From left to right, the 7 columns are the downstream primers 1R, 2R, 3R, 4R, 5R, 6R, and 7R in sequence.

[0023] Figure 2 It is a schematic diagram of the cross-reaction between isothermal amplification primers and crRNA primers. From top to bottom, 21 rows represent 21 combinations of isothermal amplification primers. From left to right, 4 columns represent 4 kinds of crRNA. The darker the color of the color block, the stronger the fluorescence signal generated after the reaction of the isothermal amplification primer pair and the crRNA combination.

[0024] Figure 3 It is the influence of different concentrations of LwaCas13a protein on the CRISPR-Cas13a detection reaction. The curves in the figure represent the reaction fluorescence curves when the final concentration of LwaCas13 protein is 10 nmol, 20 nmol, 40 nmol, 80 nmol, and 160 nmol.

[0025] Figure 4 It is the influence of different concentrations of crRNA on the CRISPR-Cas13a detection reaction. The curves in the figure represent the reaction fluorescence curves when the final concentration of crRNA is 10 nmol, 20 nmol, 40 nmol, 80 nmol, and 160 nmol.

[0026] Figure 5 It is the influence of different concentrations of NTP mix on the CRISPR-Cas13a detection reaction. The curves in the figure represent the reaction fluorescence curves when the final concentration of NTP mix is 0.5 mmol, 1 mmol, 2 mmol, 4 mmol, and 8 mmol.

[0027] Figure 6The effects of adding different amounts of T7 RNA polymerase on the CRISPR-Cas13a detection reaction are shown in the figure. The curves in the figure represent the fluorescence curves of the reaction when the addition amounts of T7 RNA polymerase are 0.2 μL, 0.4 μL, 0.6 μL, 0.8 μL, and 1.0 μL.

[0028] Figure 7 The effects of different concentrations of fluorescent RNA reporter molecules on the CRISPR-Cas13a detection reaction are shown in the figure. The curves in the figure represent the fluorescence curves of the reaction when the final concentrations of the fluorescent RNA reporter molecules are 10 nmol, 20 nmol, 40 nmol, 80 nmol, and 160 nmol.

[0029] Figure 8 The effects of different temperatures on the CRISPR-Cas13a detection reaction are shown in the figure. The curves in the figure represent the fluorescence curves of the reaction when the temperatures are 33 °C, 35 °C, 37 °C, 39 °C, and 41 °C.

[0030] Figure 9 The specific identification results of the detection system of the present invention.

[0031] Figure 10 The sensitivity detection results of the detection system of the present invention. Detailed implementation manners

[0032] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0033] A rapid detection method for avian metapneumovirus based on the CRISPR-Cas13a system includes the following steps:

[0034] S1: Extract the RNA of the sample to be detected and reverse transcribe it into cDNA;

[0035] S2: Perform isothermal amplification on the cDNA to be detected, and the primers are the sequences shown in SEQ ID NO.3 and SEQ ID NO.5;

[0036] S3: Use the CRISPR-Cas13a reaction system to detect the isothermal amplification product obtained in S2. In the CRISPR / Cas13a system, the crRNA is the sequence shown in SEQ ID NO.11;

[0037] S4: Result determination. Perform biostatistical analysis on the difference between the fluorescence value at the reaction end point and the starting fluorescence value of the experimental group and the difference between the fluorescence value at the reaction end point and the starting fluorescence value of the negative control group (using water as the template). If there is a difference, the test result is positive; if there is no difference, the test result is negative.

[0038] Example 1

[0039] 1. By comparing the avian metapneumovirus genome sequences published on NCBI using bioinformatics software, 3 primers with T7 promoters were designed upstream of the conserved sequences obtained by comparison, named T71F, T72F, and T73F respectively. 7 pairs of primers were designed downstream of the sequence, named 1R, 2R, 3R, 4R, 5R, 6R, and 7R respectively. The information of the upstream and downstream primers is shown in Table 1.

[0040] Table 1

[0041]

[0042]

[0043] 2. Using the clinical positive sample as the template for the isothermal amplification reaction, extract the RNA in the sample and reverse transcribe it into cDNA. React with a total of 21 primer combinations by cross-combining 3 upstream primers and 7 downstream primers. The reaction system is shown in Table 2 (using the AmpFuture kit), and the reaction conditions are 39°C for 20 min. The results are as shown in the appendix Figure 1 As shown, all 21 pairs of primer combinations can amplify the corresponding bands, so all of them are used for cross-reaction with the crRNA of the CRISPR-Cas13a system.

[0044] Table 2

[0045] Component Volume (μL) ABuffer 29.4 Forward Primer (10 μmol) 2 Reverse Primer (10 μmol) 2 <![CDATA[ddH2O]]> 12.1 DNA Template 2 BBuffer 2.5 Total Volume 50

[0046] 3. Configure the CRISPR-Cas13a reaction system according to Table 3. After mixing the components, place them in the ROCHE LightCycler 480 real-time fluorescence quantitative PCR instrument. Set the reaction program to 37°C for 40 min, detect the fluorescence signal every 10 s, and the detection channel is FAM.

[0047] Table 3

[0048]

[0049]

[0050] The crRNA sequences in Table 3 are shown in Table 1 (the underlined regions are the repeated sequences in crRNA). The fluorescent RNA reporter molecule sequence is 5'-FAM-UUUUUU-BHQ1-3'. The isothermal amplification product is obtained in Step 2. The final concentration of the buffer is 30.6 mmol / L HEPES + 15.3 mmol / L MgCl2 aqueous solution.

[0051] The results are shown in the appendix Figure 2 As shown, positive results were produced only in the reactions involving crRNA1 and crRNA2, while all the reactions involving crRNA3 and crRNA4 were negative. Moreover, there were significant differences in the detection effects among the positive combinations. The combination of T73F + 2R + crRNA1 produced the strongest fluorescent signal. Therefore, this combination is used in the kit of the present invention.

[0052] 4. The kit of the present invention includes: LwaCas13a protein (1 μmol / L), fluorescent RNA reporter molecule (10 μmol / L), crRNA (1 μmol / L), isothermal amplification upstream and downstream primers (10 μmol / L), buffer, T7 RNA polymerase (Sangon Biotech (Shanghai) Co., Ltd.), NTP mix (25 mmol / L), and RNA enzyme inhibitor (40 U / μL).

[0053] Among them, the fluorescent RNA reporter molecule sequence is 5'-FAM-UUUUUU-BHQ1-3'; the crRNA is crRNA1 (SEQ ID NO.11), the isothermal amplification upstream primer is T73F (SEQ ID NO.3), and the isothermal amplification downstream primer is 2R (SEQ ID NO.5); the final concentration of the buffer is 30.6 mmol / L HEPES + 15.3 mmol / L MgCl2 aqueous solution.

[0054] Kit optimization: The final concentrations of LwaCas13a protein, crRNA, NTP mix, T7 RNA polymerase, and fluorescent RNA reporter molecule in the reaction system were adjusted respectively to obtain the optimal reaction components, and the reaction temperature was optimized. The optimization results are shown in the appendix Figure 3 and the appendix Figure 4 and the appendix Figure 5 and the appendix Figure 6 and the appendix Figure 7 and the appendix Figure 8As shown in the figure, the best reaction components obtained by synthesis are: LwaCas13a protein (1 μmol / L): 2 μL, crRNA (1 μmol / L): 1 μL, fluorescent RNA reporter molecule (10 μmol / L): 1 μL, T7 RNA polymerase: 1 μL, NTPmix (25 mmol / L): 1 μL, RNase inhibitor (40 U / μL): 0.5 μL, isothermal amplification product: 2 μL, buffer: 16.5 μL (total 25 μL).

[0055] Example 2

[0056] 1. Specific detection

[0057] Positive samples of avian metapneumovirus, Newcastle disease virus, avian infectious bronchitis virus, infectious laryngotracheitis virus, infectious bursal disease virus, Marek's disease virus, avian leukosis virus, avian reovirus, Mycoplasma gallisepticum, Mycoplasma synoviae, Escherichia coli, Salmonella, and Avibacterium paragallinarum were used for detection to investigate the specificity of the detection method established in Example 1. The results are as shown in the appendix Figure 9 As shown in the figure, only the positive samples of avian metapneumovirus produced significantly different fluorescence patterns, while the fluorescence signals of other samples had no obvious difference from the negative control.

[0058] 2. Sensitivity detection

[0059] The gene sequence where the isothermal amplification primer is located was synthesized and ligated to the pUC-19T plasmid to prepare a positive standard quality plasmid. 2 μL of positive standard quality plasmids with concentrations of 1×10 5 -1×10 0 copies / μL were taken as samples respectively, and the method established in Example 1 was used for detection, with the empty pUC-19T plasmid as the negative control. The results are as shown in the appendix Figure 10 As shown in the figure, the lowest detectable sample of this method is 2 copies.

[0060] 3. Repeatability test

[0061] Five positive standard quality plasmids and empty plasmids with different concentrations were selected from the above steps for detection, and each sample was detected in 3 replicates to evaluate the within-batch repeatability of the detection system of the present invention. The above 6 samples were detected at three time points with an interval of 14 days to evaluate the between-batch repeatability of the present invention. The results are shown in Table 4 and Table 5. The coefficient of variation of both within-batch repeatability and between-batch repeatability is less than 10%, indicating good repeatability of the present invention.

[0062] Table 4 Within-batch repeatability

[0063]

[0064] Table 5 Between-batch repeatability

[0065]

[0066] In this specification, the various embodiments are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the various embodiments, reference may be made to each other.

[0067] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A primer set for detecting avian metapneumovirus based on the CRISPR-Cas13a system, characterized in that: The primer set includes a constant temperature amplification primer pair and a crRNA sequence; Furthermore, the nucleotide sequence of the isothermal amplification primer pair is shown as SEQ ID NO.3 and SEQ ID NO.5, and the nucleotide sequence of the crRNA sequence is shown as SEQ ID NO.

11.

2. A kit for detecting avian metapneumovirus based on the CRISPR-Cas13a system, characterized in that: The kit comprises the primer set according to claim 1.

3. The kit according to claim 2, characterized in that The kit also includes LwaCas13a protein, fluorescent RNA reporter molecule, T7 RNA polymerase, NTP mix, RNase inhibitor, and buffer.

4. The kit according to claim 3, characterized in that The sequence of the fluorescent RNA reporter molecule is 5'-FAM-UUUUUU-BHQ1-3', and the final concentration of the buffer solution is 30.6 mmol / L HEPES + 15.3 mmol / L MgCl2 aqueous solution.

5. Use of the primer set according to claim 1 or the kit according to any one of claims 2 to 4 in the preparation of an avian metapneumovirus detection product.

6. A method for detecting avian metapneumovirus based on the CRISPR-Cas13a system, characterized in that: The method is not for disease diagnosis purposes and comprises the following steps: S1: Extract RNA from the sample to be tested and reverse transcribe it into cDNA; S2: isothermal amplification of the cDNA to be tested, with the primers being the sequences shown in SEQ ID NO.3 and SEQ ID NO.5; S3: Using the CRISPR-Cas13a reaction system to cut the isothermal amplification product obtained in S2, the crRNA in the CRISPR / Cas13a system is the sequence shown in SEQ ID NO.11; S4: Result determination: the difference between the reaction endpoint fluorescence value and the starting fluorescence value of the test group and the difference between the reaction endpoint fluorescence value and the starting fluorescence value of the negative control group are subjected to biostatistical analysis. If there is a difference, the test result is positive, and if there is no difference, the test result is negative.

7. The method according to claim 6, characterized in that The isothermal amplification conditions described in S2 are 39° C. for 20 min.

8. The method according to claim 6, characterized in that The conditions for the CRISPR-Cas13a reaction described in S3 were 37°C for 40 min.

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