RAA primer, kit and method for detecting feline herpes virus type I

Abstract

The invention belongs to the technical field of biological detection. The invention provides RAA primers, a kit and a method for detecting feline herpes virus type I. The nucleotide sequences of the RAA primer pair are as shown in SEQ ID NO. 1 and SEQ ID NO. 2. The composition comprises a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas13a (CRISPR / Cas13a) system for detecting the feline type I herpes virus and a lateral flow chromatography test strip matched with the CRISPR / Cas13a system, wherein the CRISPR / Cas13a system for detecting the feline type I herpes virus comprises the RAA primer pair. The invention provides a feline herpesvirus type I detection technology based on RAA-CRISPR / Cas13a. The technology has the advantages of high sensitivity, strong specificity, simplicity in operation and visualization. In addition, the invention also has the advantages of short detection time, controllable temperature, capability of completing detection at human body temperature and suitability for field detection, and has potential prospects of popularization and application.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to RAA primers, a kit and a method for detecting feline herpesvirus type I. Background Art

[0002] Feline upper respiratory tract disease is one of the common diseases of cats. The pathogens that cause cat upper respiratory tract diseases mainly include feline herpesvirus, feline calicivirus, mycoplasma, chlamydia and some bacteria. Among them, feline herpesvirus is one of the most important pathogens causing cat upper respiratory tract diseases. At present, only one serotype of feline herpesvirus has been confirmed, namely feline herpesvirus type-1 (FHV-1). FHV-1 is a double-stranded DNA virus belonging to the family Herpesviridae, the subfamily Alphaherpesvirinae, and the genus Varicellavirus. It is susceptible to all cats, and it mainly infects the conjunctiva and upper respiratory tract of susceptible cats through direct contact and proliferates. FHV-1 can exist in the neuronal cells of infected cats, making them latent carriers for life, so clinically recovered cats are the main carriers of FHV-1. FHV-1 undergoes periodic viral reactivation in the body of recovered cats, especially after stress in recovered cats. According to statistics, more than 90% of cats are FHV-1 positive, about 80% of cats are latently infected, and 45% of cats carry and excrete the virus throughout their lives. Moreover, based on clinical symptoms alone, it is difficult to distinguish FHV-1 from other pathogens that cause upper respiratory tract diseases in cats. Therefore, it is urgent to establish an efficient detection method for the detection of FHV-1 in order to more accurately treat sick animals.

[0003] Since nucleic acid testing can target specific segments of the genome and accurately detect viral DNA in samples, it is widely used in the detection of FHV-1. However, current detection methods either have problems of insufficient sensitivity or low detection convenience. For example, invention patent CN 116622909 A discloses a constant temperature amplification detection reagent and detection method for feline herpesvirus type I. Although this method combines nucleic acid test strip technology and has the advantage of simple operation, its sensitivity is only 25 copies / rxn; invention patent CN 116377131A discloses a fluorescent quantitative primer and probe combination for diagnosing feline parvovirus and feline herpesvirus type I (FPV, FHV-1). Although its sensitivity reaches 10 copies, this technology has high requirements for equipment; invention patent CN 116083645 A discloses a primer set, a kit and an application for simultaneously detecting feline panleukopenia virus, feline calicivirus and feline herpesvirus type I. It combines nanotechnology with PCR technology, and the PCR reaction can quickly reach the target temperature, which improves the amplification efficiency. However, its detection sensitivity for FHV-1 is only 1.78×10 2For example, the invention patent CN 114940989 A discloses a primer-probe combination, a kit and a preparation method for detecting feline herpes virus and feline calicivirus, although the sensitivity obtained is 10 3 Copies / mL, but it uses the fluorescence method, which has high requirements for equipment; similarly, invention patent CN 113862401 A discloses a LAMP primer set, a fluorescence visualization rapid detection kit and a method for detecting feline herpes virus type I, and the obtained sensitivity reaches 8 copies / reaction, but it also has high requirements for detection equipment.

[0004] Through the above-mentioned prior art reports, combined with the general understanding of FHV-1 detection by those skilled in the art, it is not difficult to know that the existing detection technology for FHV-1 is difficult to take into account both the convenience of detection and high sensitivity of detection, making it difficult for easy-to-implement detection technology to obtain high sensitivity, while detection technology with high sensitivity is not easy to promote and use.

[0005] Therefore, there is an urgent need for a FHV-1 detection technology with high sensitivity and easy implementation in the art. Summary of the invention

[0006] In view of the defects of the prior art, the object of the present invention is to provide a FHV-1 detection scheme with high sensitivity, high specificity and simple operation advantages. Specifically, a primer, a kit and a corresponding detection method of the RAA-CRISPR / Cas13a system for detecting feline herpesvirus type I with the above advantages are provided.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] A RAA primer pair of a CRISPR / Cas13a system for detecting feline herpesvirus type I, wherein the nucleotide sequence of the RAA primer pair is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0009] A composition for detecting feline herpesvirus type 1, the composition comprising a CRISPR / Cas13a system for detecting feline herpesvirus type 1 and a lateral flow chromatography test strip matched with the system, wherein the CRISPR / Cas13a system for detecting feline herpesvirus type 1 comprises the RAA primer pair described in claim 1.

[0010] Preferably, the composition further comprises LwCas13a protein, CrRNA, and Reporter-RNA; the nucleotide sequence of the CrRNA is shown in SEQ ID NO.3, and the target sequence of the CrRNA is shown in SEQ ID NO.4.

[0011] Preferably, the composition further comprises T7 reverse transcriptase.

[0012] Preferably, the Reporter-RNA consists of 20 U, and its two ends are labeled with FAM and biotin, respectively.

[0013] A kit for detecting feline herpesvirus type I, comprising a CRISPR / Cas13a system for detecting feline herpesvirus type I and a lateral flow chromatography test strip supporting the system; the CRISPR / Cas13a system for detecting feline herpesvirus type I comprises the RAA primer pair described in claim 1 and the LwCas13a protein, CrRNA, Reporter-RNA, and T7 reverse transcriptase described in claims 2 and 3, and also comprises rNTP Mix, RNase Inhibitor, HEPES buffer, MgCl2 solution, and DEPC water.

[0014] Preferably, the CRISPR / Cas13a system for detecting feline herpesvirus type I consists of the following components: 5 μL of RAA reaction product, 4.2 μL of rNTP Mix, 2 μL of RNase Inhibitor, 2 μL of LwCas13a protein, 1.2 μL of T7 reverse transcriptase, 0.6 μL of MgCl2 solution, 1 μL of HEPES buffer, 3 μL of CrRNA, 5 μL of Reporter-RNA, and 26 μL of DEPC water; the RAA reaction product is obtained by amplifying the sample nucleic acid using the RAA primers described in claim 1.

[0015] Application of the above RAA primer pair, composition or kit in the preparation of a product for detecting feline herpesvirus type I.

[0016] A method for detecting feline herpesvirus type I, which is a method for non-disease diagnosis purposes, comprises the steps of: (1) extracting sample nucleic acid, amplifying with the aforementioned primer pair to obtain a RAA reaction product; and (2) performing a reaction using the aforementioned kit, and dropping the reaction product onto a lateral flow chromatography test strip to observe the detection result.

[0017] Preferably, in step (1), the amplification temperature is 37° C. and the amplification time is 25 minutes; in step (2), after incubation at 37° C. for 20 minutes, 50 μL of the reaction product is dropped onto a lateral flow chromatography test strip, and the test result is observed within 3 minutes.

[0018] Beneficial effects of the present invention:

[0019] The present invention provides a feline herpes virus type I detection technology based on RAA-CRISPR / Cas13a, which has the advantages of high sensitivity, strong specificity, simple operation and visualization. In addition, the present invention also has the advantages of short detection time, controllable temperature, and can be completed at human body temperature and is suitable for on-site detection, and has potential prospects for promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a working principle diagram of the detection technology of the present invention;

[0021] Figure 2 This is a diagram of the LWcas13a protein preparation and activity identification results;

[0022] Figure 3 This is the result of random combination screening of primer pairs identified by RAA agarose gel electrophoresis;

[0023] Figure 4 Results of random combination screening of primer pairs for RAA-CRISPR / Cas13a nucleic acid test strip identification;

[0024] Figure 5 This is the result diagram of optimization of RAA amplification temperature;

[0025] Figure 6 This is the optimization result diagram of RAA amplification time;

[0026] Figure 7 This is a screen result diagram of the working concentration of Cas13a protein;

[0027] Figure 8 This is a graph showing the results of the CRISPR trans-cleavage incubation temperature test;

[0028] Fig. 9 This is a graph showing the results of the CRISPR trans-cleavage incubation time experiment;

[0029] Fig.10 This is a graph showing the results of the detection sensitivity evaluation of the present invention, with the positive plasmid as a reference;

[0030] Fig.11 This is a graph showing the results of the detection specificity evaluation of the present invention;

[0031] Fig.12 and Fig.13 This is a graph showing the results of the clinical sample detection capability evaluation of the present invention. DETAILED DESCRIPTION

[0032] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technical personnel in this field based on the above invention content still fall within the scope of protection of the present invention.

[0033] Example 1

[0034] (1) LWCas13a protein preparation and activity identification

[0035] The LWCas13a protein expression plasmid was expressed in E. coli Rosetta (DE3) in a pilot test, purified, and then screened for high-yield expression strains. A 20L biofermenter was used to induce the expression for 16 hours at 18°C. After purification by nickel column, the protein eluted by 100mmol / L and 200mmol / L imidazole was similar in size to the target protein LwCas13a (150kD). SDS-PAGE was used to identify the protein. Figure 2 As shown in part A, LwCas13a was successfully purified and successfully cleaved by SUMO. The cleaved protein was purified by ion exchange (500mmol / LNaCl) to obtain LwCas13a. After SUMO cleavage of this part of the protein, the protein band was located at around 130kD and the protein became smaller, indicating that this part of the protein was successfully cleaved by SUMO and the protein purified in the previous step was the target protein. The cleaved protein was further purified by ion exchange (500mmol / LNaCl) to obtain LwCas13a. In order to further confirm that the purified protein is the target protein, anti-His antibody was used for Western blot identification. As shown Figure 2 As shown in part B of the figure, a protein band was detected between 130 and 175 kD, and the size was consistent with the expected value, indicating that LwCas13a was obtained. However, no protein band was detected by anti-His antibody after SUMO digestion. Figure 2 Obvious protein bands can be seen in the SDS-PAGE electrophoresis shown in part C of FIG. It can be seen that the His tag was successfully removed after the final purified protein was cleaved by SUMO. Then the fluorescence method was used to identify the activity of LwCas13a protein, such as Figure 2 As shown in part D, compared with the negative control without LwCas13a protein, after adding LwCas13a protein, the fluorescence signal has been rising within 60 minutes, indicating that LwCas13a protein has a strong function of cleaving fluorescent reporter RNA, and the RNA cleavage activity of LwCas13a protein can only be activated by Matched crRNA (such as Figure 2As shown in the E part of the figure, the RNA shearing effect of LwCas13a protein increases linearly with the increase of the working concentration of LwCas13a protein (as shown in the E part of the figure). Figure 2 As shown in part F of the figure). As described in the review, we successfully obtained the LwCas13a protein, and the protein has specific RNA cleavage activity.

[0036] (2) Design and screening of RAA primers

[0037] The positive plasmid reference was used as a template. The positive reference plasmid was synthesized by Beijing Qingke Biotechnology Co., Ltd. The plasmid vector was PUC57, the cloning method was seamless cloning, and the preserved strain was Escherichia coli top10. The primers designed in Table 1 were used in different combinations (F1\R1, F2\R1, F2\R3, F3\R1, F1\R2, F1\R3, F2\R2, F3\R2, F3\R3) to screen out the primer pair with the best amplification efficiency. The three pairs of designed RAA primers were randomly combined in pairs for RAA amplification and purification, and verified by 2% nucleic acid gel electrophoresis. The results are as follows Figure 3 As shown in the figure, the amplification results of the four primer pairs F1\R1, F2\R1, F2\R3, and F3\R1 were good, and the positions were consistent with the expected products. The amplification effects of other primer pairs were poor, with primer dimers or non-specific amplification.

[0038] Using low-concentration virus separation supernatant as a template, F1\R1, F2\R1, F2\R3, and F3\R1 were used for RAA amplification, and the RAA reaction product was added to the CRISPR / Cas13a system for reaction. The reaction product was then dropped onto the test strip detection well. The results were as follows: Figure 4 10 -4 The CT of the supernatant of the isolated strain diluted by 10 times was about 30, and all four primer pairs detected it; -5 10 times diluted virus culture supernatant (CT is about 34) and -6 In the virus culture supernatant diluted 100 times (CT was about 37), no primer pairs except F1\R1 were detected. The results showed that F1\R1 (also recorded as SEQ ID NO.1 and SEQ ID NO.2) had the best amplification efficiency.

[0039] The sequence information of F1, F2, F3, R1, R2, R3 and other primers and oligonucleotides in the present invention is shown in Table 1. In the table, FHV-F1 is abbreviated as F1, and the abbreviations of other sequences are the same.

[0040] Table 1

[0041]

[0042] (3) Optimization of RAA amplification temperature and time

[0043] Take 30 μL of DNA template for RAA amplification for 30 min, and take 5 μL of amplified product for CRISPR reaction for 30 min. Select 37°C, 38°C, and 39°C to evaluate the optimal temperature for RAA amplification. Figure 5 The results show that RAA recombinase has good amplification efficiency between 37 and 39°C, so the optimal temperature for RAA amplification is 37°C. RAA amplification reacts for 15 minutes, 20 minutes, and 25 minutes at the optimal temperature. RAA amplification mainly depends on magnesium acetate and the energy supply system in the reaction system. Since the RAA system contains polyethylene glycol (PEG), the entire reaction is relatively viscous. When the reaction time is 20 minutes, the entire amplification reaction may not be completed. In order to achieve the best amplification efficiency, it is necessary to extend the reaction time as much as possible to consume the energy in the system, such as Figure 6 The experimental results show that the repeatability and sensitivity of RAA detection are best when the amplification is carried out at 37°C for 25 minutes. Therefore, the optimal reaction temperature and time for RAA amplification are incubation at 37°C for 25 minutes.

[0044] (4) Screening of Cas13a protein working concentration

[0045] The working concentration of Cas13a protein is related to the trans-cleavage efficiency of CRISPR / Cas13a, so four different gradient concentrations of Cas13a protein were set at 22.5nmol / L, 45nmol / L, 90nmol / L, and 180nmol / L. -6 , 10 -7 , 10 -8 The three different concentrations of positive plasmids were evaluated to screen out the optimal working concentration of Cas13a protein. Figure 7 As shown, when the concentration of Cas13a protein increases from 22.5nmol / L to 45nmol / L, the trans-cleavage efficiency of CRISPR / Cas13a increases. However, when the concentration of Cas13a protein continues to increase, the trans-cleavage efficiency of CRISPR / Cas13a decreases. Therefore, the optimal working concentration of Cas13a protein is 45nmol / L.

[0046] (5) CRISPR trans-cleavage incubation temperature and time

[0047] The reaction temperature of Cas13a protein is usually between 35 and 42°C. 37°C, 38°C, 39°C, and 45°C were selected to evaluate the optimal temperature of Cas13a protein reaction. Figure 8As shown in Figure 2, it was found that the activity of lwCas13a was best between 37 and 39 °C, and 37 °C was subsequently selected as the optimal reaction temperature. CRISPR trans-cleavage was incubated at the optimal temperature for 20 min, 25 min, and 30 min, respectively. Fig. 9 The results show that Cas13a protein can stably trans-cleave the target sequence within 20 to 30 minutes, and 20 minutes is subsequently selected as the CRISPR incubation time. Therefore, the optimal incubation temperature and time for CRISPR trans-cleavage are 37°C and 20 minutes.

[0048] (6) RAA amplification system

[0049] Amplify according to RAA optimized parameters, amplify at 37℃ for 25min, take 5μL RAA amplification product and add it to a new EP tube. The RAA amplification system is as shown in Table 2:

[0050] Table 2

[0051]

[0052] (7) CRISPR / Cas13a reaction system

[0053] CRISPR trans-cleavage was performed according to the optimized parameters of CRISPR / Cas13a, incubated at 37°C for 20 min, 50 μL of the reaction product was dripped onto the lateral flow chromatography test strip, and the test results were observed within 3 min. The CRISPR / Cas13a reaction system is shown in Table 3.

[0054] Table 3

[0055]

[0056] (8) Sensitivity evaluation

[0057] Under the optimal reaction conditions, the synthesized FHV-1 positive reference was quantified by a nucleic acid microspectrophotometer, and the original reference concentration was calculated to be 1.0 × 10 8 copies / μL, and the detection sensitivity was evaluated after 10-fold gradient dilution of the plasmid. Fig.10 As shown, the detection limit of RAA-CRISPR / Cas13a is 0.1 copies / μL. Since the amount of template added in this experimental system is 10 μL, the sensitivity of this method is 1 copy / reaction.

[0058] (9) Specificity assessment

[0059] The specificity of the test was evaluated by using the culture supernatant of feline herpesvirus (FHV), feline calicivirus (FCV), feline panleukopenia virus (FPV), canine distemper virus (CDV), canine parvovirus (CPV), Tritrichomonas, and Babesia canis isolated from clinical strains or parasites of Zhejiang Agricultural and Forestry University and then extracting nucleic acid using the magnetic bead method. Fig.11 As shown, except for FHV which showed a positive result, the other samples were all negative, and RAA-CRISPR / Cas13a had good specificity in detecting FHV-1.

[0060] (10) Evaluation of the detection capability of actual clinical samples

[0061] The culture supernatant of the FHV-1 strain isolated from Zhejiang Agricultural and Forestry University was used. -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Gradient dilution, use magnetic beads to extract nucleic acid and then fluorescent quantitative PCR analysis (the average of three results is taken). Fig.12 The results showed that except for the negative control, the supernatants of the five gradients of clinical strain cultures were all positive, which was consistent with the results of fluorescent quantitative PCR, and the detection effect reached the critical value of PCR detection.

[0062] 24 clinical swab samples from central animal hospitals or catteries in different regions (Chongqing, Hangzhou, Ningbo, Shanghai, Chengdu, and Beijing) were extracted with magnetic beads to evaluate the detection capability of clinical actual samples. Fig.13 As shown, except for the negative control, all 24 clinical swab samples were positive (24 / 24), which was consistent with the clinical qPCR results. RAA / CRISPR-cas13a can be used for actual clinical detection of FHV-1.

Claims

1. A RAA primer pair for a CRISPR / Cas13a system for detecting feline herpesvirus type I, characterized in that The nucleotide sequences of the RAA primer pair are shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. A composition for detecting feline herpesvirus type I, characterized in that: The composition comprises a CRISPR / Cas13a system for detecting feline herpesvirus type 1 and a lateral flow chromatography test strip matched with the system, and the CRISPR / Cas13a system for detecting feline herpesvirus type 1 comprises the RAA primer pair described in claim 1.

3. The composition according to claim 2, characterized in that The composition also includes LwCas13a protein, CrRNA, and Reporter-RNA; the nucleotide sequence of the CrRNA is shown in SEQ ID NO.3, and the target sequence of the CrRNA is shown in SEQ ID NO.

4.

4. The composition according to claim 3, characterized in that The composition also includes T7 reverse transcriptase.

5. The composition according to claim 4, characterized in that The reporter-RNA consists of 20 U, and both ends are labeled with FAM and biotin respectively.

6. A kit for detecting feline herpesvirus type I, characterized in that: It comprises a CRISPR / Cas13a system for detecting feline herpesvirus type I and a lateral flow chromatography test strip matched with the system; the CRISPR / Cas13a system for detecting feline herpesvirus type I comprises the RAA primer pair described in claim 1 and the LwCas13a protein, CrRNA, Reporter-RNA, and T7 reverse transcriptase described in claims 2 and 3, and also comprises rNTP Mix, RNase Inhibitor, HEPES buffer, MgCl2 solution, and DEPC water.

7. The kit according to claim 6, characterized in that The CRISPR / Cas13a system for detecting feline herpesvirus type I is composed of the following components: 5 μL of RAA reaction product, 4.2 μL of rNTP Mix, 2 μL of RNaseInhibitor, 2 μL of LwCas13a protein, 1.2 μL of T7 reverse transcriptase, 0.6 μL of MgCl2 solution, 1 μL of HEPES buffer, 3 μL of CrRNA, 5 μL of Reporter-RNA, and 26 μL of DEPC water; the RAA reaction product is obtained by amplifying the sample nucleic acid using the RAA primer described in claim 1.

8. Use of the RAA primer pair according to claim 1, the composition according to any one of claims 2 to 5, or the kit according to claim 6 or 7 in the preparation of a product for detecting feline herpesvirus type 1.

9. A method for detecting feline herpesvirus type I, characterized in that: The detection method is a method for non-disease diagnosis purposes, comprising the steps of: (1) extracting sample nucleic acid, amplifying with the primer pair described in claim 1 to obtain a RAA reaction product; (2) reacting with the kit described in claim 6 or 7, and dropping the reaction product onto a lateral flow chromatography test strip to observe the detection result.

10. The method according to claim 9, characterized in that In step (1), the amplification temperature is 37° C. and the amplification time is 25 minutes. In step (2), after incubation at 37° C. for 20 minutes, 50 μL of the reaction product is dropped onto the lateral flow chromatography test strip and the test result is observed within 3 minutes.

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