A crisper-cas13 system for detecting fadv-4 and a kit and method thereof

Abstract

The application discloses a guide RNA probe, the nucleic acid sequence of which is shown in SEQ ID No. 1 or SEQ ID No. 2. The application discloses application of the guide RNA probe in preparation of a product for detecting fowl adenovirus serotype 4. The application discloses a CRISPR-Cas13 system for detecting fowl adenovirus serotype 4, which comprises a Cas13a nuclease, a guide RNA probe as claimed in claim 1, and an FB-RNA reporter molecule. The application discloses a kit, which comprises a RAA kit and the CRISPR-Cas13 system for detecting fowl adenovirus serotype 4. The RAA kit comprises a primer pair for fowl adenovirus serotype 4. The application discloses a use method of the kit.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection of avian adenovirus serotype 4, and particularly relates to a CRISPR-Cas13 system for detecting FAdV-4, a kit and a method thereof. BACKGROUND

[0002] Avian adenovirus serotype 4 (FAdV-4) is the pathogen of hepatitis pericardial effusion syndrome (HHS). It was first discovered in Ankarain Pakistan in 1987, and then found in Mexico, Peru, Chile, India, South Korea, Japan and other regions. Since 2015, FAdV-4 has begun to spread in China.

[0003] FAdV-4 mainly infects 3 to 6-week-old broilers, but there are also reports of egg-laying chicken infection. In addition, there are 12 serotypes of FAdVs (FAdV-1 to FAdV-11 and FAdV-8b), and FAdV-4 infection is easily confused with other serotype infections, making its diagnosis, prevention and control more difficult. There is no effective vaccine or treatment method for FAdV-4. Therefore, it is of great significance to establish a rapid and accurate on-site diagnostic method for effective prevention and control of the disease.

[0004] Currently, the detection method of FAdV-4 is still relatively traditional, mainly relying on virus isolation culture or serological detection. These methods have the disadvantages of low specificity, easy false positives, etc. The nucleic acid detection method of FAdV-4 also has many limitations, such as long time-consuming of PCR; qPCR requires expensive equipment; the primer design of loop-mediated isothermal amplification technology (LAMP) is complex, and non-specific amplification is easy to occur, causing false positives. Therefore, a new technical method is needed to solve the above problems.

[0005] Hexon is the main structural protein of FAdV, which contains major genus and subgenus specific epitopes and minor species specific epitopes. Hexon is currently recognized as the standard for diagnosing FAdV serotypes, which provides a basis for developing a rapid diagnostic method for different avian adenovirus serotype infections.

[0006] Research has found that after CRISPR / Cas13a cuts the target RNA under the guidance of specific crRNA, it can activate its accompanying cutting activity to cut the surrounding non-specific RNA. Based on this principle, specific RNA reporter probes can be added to the reaction system to achieve specific detection of target molecules. This method has the advantages of convenience, rapidness, sensitivity, specificity, etc., and provides a new idea for the development of nucleic acid-based pathogen detection technology. SUMMARY

[0007] Based on the technical problems existing in the background art, in order to establish a simple FAdV-4 detection method, the applicant establishes a detection system combining a lateral flow test strip based on CRISPR / Cas13a, which provides technical support for timely detection of FAdV-4.

[0008] One of the purposes of the present application is to provide a guide RNA probe, FAdV-4probe1 or FAdV-4probe3, the nucleic acid sequence of FAdV-4probe1 is shown in SEQ ID No. 1, and the nucleic acid sequence of FAdV-4probe3 is shown in SEQ ID No. 2.

[0009] Preferably, the target gene of the guide RNA probe is the Hexon gene of avian adenovirus serotype 4.

[0010] The second purpose of the present application is to provide the use of the above-mentioned guide RNA probe in the preparation of a product for detecting avian adenovirus serotype 4.

[0011] Preferably, the product is any one of a drug, a reagent, a kit, etc.

[0012] The third purpose of the present application is to provide a CRISPR-Cas13 system for detecting avian adenovirus serotype 4, comprising: a Cas13a nuclease, the above-mentioned guide RNA probe, and an FB-RNA reporter molecule; the above-mentioned guide RNA probe is directed against the Hexon gene of avian adenovirus serotype 4, and the Cas13a nuclease activates the enzyme activity after recognizing the target gene of the guide RNA probe, and cuts the FB-RNA reporter molecule to release a detection signal.

[0013] Preferably, the Cas13a nuclease is an LwCas13a nuclease.

[0014] Preferably, it further comprises: an RNases inhibitor, a ribonucleic acid, and a T7 polymerase.

[0015] The fourth purpose of the present application is to provide a primer pair for avian adenovirus serotype 4, comprising any one of the following groups:

[0016] (1) FAdV-4primer1-F and FAdV-4primer1-R, the nucleic acid sequences of which are shown in SEQ ID No. 3 and SEQ ID No. 4;

[0017] (2) FAdV-4primer3-F and FAdV-4primer3-R, the nucleic acid sequences of which are shown in SEQ ID No. 5 and SEQ ID No. 6.

[0018] The fifth object of the present application is to provide a kit comprising: a RAA kit, and the CRISPR-Cas13 system for detecting avian adenovirus serotype 4.

[0019] Preferably, the RAA kit comprises: the primer pair for avian adenovirus serotype 4.

[0020] The fifth object of the present application is to provide a method for using the kit, comprising the following steps:

[0021] (1) using the RAA kit to amplify the Hexon gene with the sample DNA as a template;

[0022] (2) using the CRISPR-Cas13 system for detecting avian adenovirus serotype 4 to specifically recognize the Hexon gene, detecting the detection signal to determine whether the sample to be tested contains avian adenovirus serotype 4.

[0023] The present application uses the guide RNA of the CRISPR / Cas13 system of the gene editing system, which can specifically and accurately recognize the Hexon gene in avian adenovirus serotype 4 (FAdV-4) and start the Cas13 enzyme activity, cut off the special reporter molecule, and the reporter molecule can appear a band on the test strip. By the difference of the band position, it is easy to confirm the presence or absence of the target sequence, so as to quickly infer whether there is FAdV-4 in the sample.

[0024] The detection method of the present application has high sensitivity, and the minimum detection limit can reach 10 copies of virus per microliter. Compared with the qRT-PCR technology, the method is simple and easy to operate, and is expected to improve the sensitivity and reduce the false positive rate, thereby providing a new method for rapid and accurate detection of FAdV-4. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The results of the combination of 1-7 gene editing system detection.

[0026] Figure 2 The results of FAdV-4 primer1+probe1 sensitivity detection.

[0027] Figure 3 The results of FAdV-4 primer3+probe3 sensitivity detection.

[0028] Figure 4 The results of the first optimization of FAdV-4 primer3+probe3 sensitivity detection.

[0029] Figure 5 The results of the second optimization of FAdV-4 primer3+probe3 sensitivity detection.

[0030] Figure 6 The second optimization of the detection results for FAdV-4 primer 3 + probe 3 sensitivity.

[0031] Figure 7 The detection results for FAdV-4 primer 3 + probe 3 specificity.

[0032] In the above figures, ZL represents a plasmid, and NTC represents a negative control. DETAILED DESCRIPTION

[0033] The technical solutions of the present application will be described in detail below through specific examples.

[0034] Example 1: Method for preparing and detecting avian adenovirus serotype 4

[0035] The overall process is as follows: the specific sequence fragments obtained are used to design isothermal amplification primers and specific probes. The double-stranded DNA probe prepared is prepared into RNA by in vitro transcription. Isothermal amplification reaction is carried out using a plasmid as a template, and Q-sep100 is used for amplification product detection. The qualified primer combination is selected into the detection experiment. The constant temperature amplification product is used as a template for enzyme cutting reaction using Cas13a nuclease and prepared specific RNA probe, and the reaction product is diluted and loaded on a lateral flow test strip, and the strip change of the test strip is observed.

[0036] 1.1 Primer and probe design

[0037] For FAdV-4 Hexon gene, NCBI Primer BLAST is used for primer design, using default parameters, amplicon size is (100-300 nt), primer annealing temperature is 54-67℃, and primer size is 30-35 nt. Seven pairs of primers are selected for DNA sequence synthesis, as follows:

[0038]

[0039]

[0040] The probe design principles are as follows: 1. The target region size is 28 bases, and the reverse complementary base sequence is designed according to the target region; 2. The flanking sequence at the 3' end of the target region cannot contain base sequence G, and other bases are allowed; 3. It cannot overlap with the RPA primer sequence.

[0041] The guide RNA probe sequence is as follows:

[0042]

[0043] According to WIPO ST.26 standard requirements, the above sequence will be changed to "T" instead of "U" in the sequence listing.

[0044] 1.2 Probe preparation

[0045] The DNA sequence of the probe is synthesized, and then the guide RNA probe (crRNA probe) is transcribed in vitro. First, pay attention to the 5' end accessory T7 promoter sequence when designing the probe. Two pieces of DNA are annealed to form double-stranded DNA with a final concentration of 10 μM, and then transcribed in vitro using the HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs).

[0046] 1.3 Isothermal amplification

[0047] The nucleic acid amplification uses a recombinase-aided amplification (RAA) one-step method to amplify the target DNA. At 37°C, the sample DNA template can be used to amplify the target gene fragment in 30 minutes. Generally, a single amplification reaction system includes primers (100-500 pmol), amplification buffer + enzyme Mix (25 μL), MgOAc solution (3 μL), nucleic acid template (1 μL), and nucleic acid-free water (supplemented to 50 μL).

[0048] 1.4 Targeted detection

[0049] LwCas13a nuclease (45 nM), crRNA probe (22.5 nM, transcribed in vitro in step 1.2), FB-RNA reporter molecule (125 nM), RNase inhibitor (0.25 μL), ATP (1 mM), GTP (1 mM), UTP (1 mM), CTP (1 mM), and T7 polymerase (0.4 μL) are used to supplement the detection Buffer to 9 μL, and 1 μL of the amplification product in step 1.3 is added, and incubated at 37°C for 40 minutes. Insert the lateral flow test strip into the detection product and place it for 3-5 minutes, and observe the results.

[0050] The FB-RNA reporter molecule is as follows:

[0051] Name Sequence (5' to 3') FB-RNA Reporter / 56-FAM / mArArUrGrGrCmAmArArUrGrGrCmA / 3Bio /

[0052] Example 2

[0053] 2.1 Detection system test

[0054] The detection system verification template plasmid was input at a copy number of 2 x 10 8copies / μL; RAA time: 30 min, test strip detection results as shown in Figure 1 .

[0055] In the verification of the gene editing detection system, 7 primer probe combinations were tested, and only primer1+probe1 combination and primer3+probe3 combination passed the verification. In all combination system verifications, the corresponding NTC was not detected, and the experimental results were reliable.

[0056] 2.2 Sensitivity test

[0057] The primer1+probe1 combination and primer3+probe3 combination were tested for sensitivity using the test strip method. Experimental conditions: RAA amplification system primer dosage 240 pM; amplification time 40 min; DNA template: plasmid copy number 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10, 1 copies / μL; template input amount: 1 μL.

[0058] The FAdV-4 primer1+probe1 sensitivity test is shown in Figure 2 . The results show that the FAdV-4 primer1+probe1 primer and probe combination: the test strip method can detect the lower limit of plasmid copy number of 10 6 copies / μL.

[0059] The FAdV-4 primer3+probe3 sensitivity test is shown in Figure 3 . The results show that the FAdV-4 primer3+probe3 primer and probe combination: the test strip method can detect the lower limit of plasmid copy number of 100 copies / μL.

[0060] 2.3 primer3+probe3 sensitivity optimization

[0061] 2.3.1 First sensitivity optimization test on primer3+probe3

[0062] Experimental conditions: RAA amplification system primer dosage 240 pM; amplification time 40 min; DNA template: plasmid copy number 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 102 , 10 copies / μL.

[0063] Optimization condition: RAA template input amount was optimized to 5 μL, and cas detection system template input amount was optimized to 5 μL (before, RAA template input amount and cas detection system template input amount were both 1 μL).

[0064] The detection results are shown in Figure 4 .

[0065] 2.3.2 Second sensitivity optimization test on primer3 + probe3

[0066] Experimental conditions: RAA amplification system primer amount was 240 pM; amplification time was 40 min; DNA template: plasmid copy number was 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 copies / μL.

[0067] Optimization condition: RAA template input amount was optimized to 5 μL (before, RAA template input amount was all 1 μL).

[0068] The detection results are shown in Figure 5 and Figure 6 .

[0069] Through the sensitivity optimization of FAdV-4 primer3 + probe3 (as shown in Figures 4-6 ), the results showed that the primer and probe combination of FAdV-4 primer3 + probe3 could detect the lower limit of plasmid copy number of 10 copies / μL.

[0070] The above results showed that the plasmid was diluted to 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10, 1 copies / μL, respectively, and the sensitivity of two pairs of primers and corresponding probes (primer1 + probe1 and primer3 + probe3) was tested by using test strip method, wherein the combination of primer3 + probe3 had the highest sensitivity, and the detection lower limit could reach 10 copies / μL; the detection lower limit of primer1 + probe1 combination could reach 10 6 copies / μL, and the sensitivity was poor.

[0071] Note:

[0072] The plasmid used in the application: the vector is 2692 bp, the target fragment is 2800 bp, and the total length of the plasmid is 5492 bp; Qubit

[0073] If the concentration of the detection plasmid 1 is 10 9 ng / μL, the copy number is about (9.12×10 11 )×10 9 / 5492=1.18×10 10 ≈2×10 10 copies / μL.

[0074] Example 3 Specific detection

[0075] 3.1 Specific detection of sample by Primer 3+probe 3 combination

[0076] The FAdV-4 strain, avian infectious laryngotracheitis virus (ILTV), Marek's disease virus (MDV), infectious bursal disease virus (IBDV), and infectious bronchitis virus (IBV) used for specific detection were isolated and preserved by the laboratory.

[0077] Avian influenza H9 subtype vaccine (AIV-H9) and Newcastle disease virus (NDV) attenuated vaccine were purchased from Shandong Lvdou Biotechnology Co., Ltd., and were used for extracting viral nucleic acid.

[0078] FAdV-1, FAdV-8b, FAdV-9, and FAdV-10 were donated by Professor Ye Jianqiang of Yangzhou University.

[0079] The specific detection results are shown in Figure 7 The results show that FAdV-4 primer 3+probe 3 has strong specificity and can be distinguished from other viruses and other subtypes of FAdV.

[0080] Example 4 Clinical sample detection

[0081] 20 samples of liver grinding liquid collected from diseased chicks in some areas of Anhui were detected by CRISPR-Cas13a lateral flow test strip detection method and fluorescence quantitative PCR method respectively, and the detection results were compared, as follows:

[0082]

[0083] The above results show that each sample test strip shows a positive band and the negative result is established, and the coincidence degree with the fluorescence quantitative PCR detection result is 100%; it is proved that the method of the application is effective and can be applied for on-site diagnosis.

[0084] The above description is only the preferred embodiment of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can make equivalent substitutions or changes according to the technical solution and the inventive concept of the present application within the technical range disclosed by the present application, which should be covered in the protection scope of the present application.

Claims

1. A probe-primer pair combination against avian adenovirus serotype 4, characterized in that, Comprising: a guide RNA probe and a primer pair; the nucleic acid sequence of the guide RNA probe is shown as SEQ ID No. 2; the primer pair is FAdV-4 primer3-F and FAdV-4 primer3-R, and the nucleic acid sequences thereof are shown as SEQ ID No. 5 and SEQ ID No. 6, respectively.

2. The set of probe primer pairs according to claim 1, wherein the target gene of the guide RNA probe is the Hexon gene of avian adenovirus serotype 4.

3. Use of the probe primer pair combination of claim 1 in the preparation of a product for detecting avian adenovirus serotype 4.

4. A CRISPR-Cas13 system for detecting avian adenovirus serotype 4, characterized in that, Comprising: a Cas13a nuclease, the probe primer pair combination of claim 1, an FB-RNA reporter molecule; the Cas13a nuclease activates the enzyme activity after recognizing the target gene by the guide RNA probe, and cuts the FB-RNA reporter molecule to release the detection signal.

5. The CRISPR-Cas13 system for detecting avian adenovirus serotype 4 according to claim 4, characterized in that, the Cas13a nuclease is a LwCas13a nuclease.

6. The CRISPR-Cas13 system for detecting avian adenovirus serotype 4 according to claim 4, characterized in that, Further comprising: an RNases inhibitor, a ribonucleic acid, and a T7 polymerase.

7. A kit characterized in that, Comprising: a RAA kit, and a CRISPR-Cas13 system for detecting avian adenovirus serotype 4 according to any one of claims 4-6.

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