Visual detection method for canine distemper virus based on RPA-CRISPR / Cas13d

Abstract

The invention discloses a visual detection method of a canine distemper virus based on RPA-CRISPR / Cas13d, and belongs to the technical field of molecular biology diagnosis. Through the two-step design of combining EsCas13d protein and RPA isothermal amplification, remarkable technical breakthrough is realized, on one hand, the detection sensitivity reaches 5cp / mu L, and meanwhile, cross reaction of various common viruses such as canine parvovirus and coronavirus can be effectively eliminated; on the other hand, visual interpretation is achieved through 470 nm ultraviolet fluorescence or a lateral chromatography test strip, the whole-process detection only needs 60 minutes, the clinical sample detection positive rate is remarkably higher than that of qPCR, the omission ratio is lower, and the method is not only suitable for ultrasensitive screening in the early stage of canine infection, but also capable of detecting CDV of endangered animals such as pandas and red pandas, and has broad application prospects. A rapid, portable and cost-effective POCT solution is provided for grass-roots pet hospitals, farms and wild animal protection sites, and the method has important practical significance in improving the early prevention and control capability of canine distemper epidemic situations, reducing the economic loss of breeding and protecting endangered species.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology diagnosis, and particularly relates to a visual detection method for canine distemper virus based on RPA-CRISPR / Cas13d. BACKGROUND

[0002] Canine distemper (CD) is an acute, highly contagious disease caused by canine distemper virus (CDV), mainly harming dogs, especially puppies. The disease can be transmitted through the respiratory tract, digestive tract and eye-nose secretions, and often leads to high fever, respiratory inflammation, digestive disorders and severe nervous system symptoms such as convulsions and paralysis. Canine distemper has a very high mortality rate, and even surviving individuals often leave permanent neurological damage, causing serious economic losses and social impact to the dog industry, and is one of the most important and dangerous diseases in canine epidemic prevention. It is worth noting that the host range of CDV has expanded to cats, civets and non-human primates, and has been proven to be able to cross-species infect giant pandas and even cause death, so canine distemper is not only one of the most dangerous diseases in canine epidemic prevention, but also an important infectious disease threatening the health of endangered species. Therefore, it is of great significance to establish a rapid, sensitive and on-site real-time deployment diagnostic method for the early detection and prevention and control of CDV.

[0003] CRISPR / Cas-based diagnostic technology has become an important breakthrough in the field of point-of-care testing (POCT) in recent years due to its rapid detection, high specificity and simple reaction conditions. According to the principle of action and application scene, CRISPR-Dx mainly has two strategies: one is not pre-amplification, directly using CRISPR / Cas protein to detect pathogenic nucleic acid, this method has slightly lower sensitivity than qPCR, but has the advantages of simple operation and is suitable for on-site real-time detection; the other is to combine CRISPR-Dx with isothermal amplification technology, although it increases the operation steps, but significantly improves the detection sensitivity, and is more suitable for early detection and monitoring of infection.

[0004] Most of the current CRISPR-Dx platforms use Cas12 and Cas13 family proteins as detection elements. It is worth noting that in the Cas12a-based single-tube reaction, the target DNA competes with the isothermal amplification for the binding of the target DNA, leading to the degradation of the DNA template, thereby affecting the detection efficiency of the one-step method. In addition, the dependence of Cas12a on PAM sequence also limits the design flexibility of crRNA, limiting its application in highly mutated viruses. In contrast, Cas13 family proteins specifically recognize single-stranded RNA and do not cause a decrease in the sensitivity of the one-step method due to competition with isothermal amplification for the template. However, the requirement of Cas13a for PFS also limits the design of crRNA, and the Cas13a protein is relatively large and difficult to express. Cas13d is a newly discovered VI-D type RNA-targeting CRISPR effector protein, which has a molecular weight of 190-300 amino acids less than Cas12 family proteins and Cas13a-Cas13c subtypes, making it easier to purify and more suitable for subsequent industrial production. In addition, Cas13d does not require PAM and PFS, and the crRNA design is more flexible in detection. The current research on CRISPR diagnostic technology based on Cas13d is still limited, but the existing research has shown that Cas13d has excellent ability in field detection. SUMMARY

[0005] The application establishes a two-step canine distemper virus (CDV) detection platform based on RPA-CRISPR / EsCas13d. The method first uses recombinase-mediated isothermal amplification (RPA) to amplify the target nucleic acid, and then combines EsCas13d protein and specific crRNA in an independent detection reaction for recognition and signal amplification. Compared with the direct detection method without amplification, the operation steps of the method of the application are increased, but the detection sensitivity is significantly improved, and low copy level viral nucleic acid can be detected, which is suitable for early diagnosis and monitoring scene.

[0006] The application provides a crRNA for detecting canine distemper virus based on a CRISPR / Cas13d system, wherein the sequence of the crRNA molecule is shown as SEQ ID NO. 8.

[0007] The application also provides a reagent for detecting canine distemper virus based on a CRISPR / Cas13d system, wherein the reagent contains the above-mentioned crRNA molecule.

[0008] The application also provides a kit for detecting canine distemper virus based on a CRISPR / Cas13d system, wherein the kit contains the above-mentioned crRNA molecule or reagent.

[0009] Preferably, the kit further comprises a Cas13d protein, a fluorescent RNA reporter probe, and an upstream primer SEQ ID NO. 2 and a downstream primer SEQ ID NO. 6 for recombinase polymerase nucleic acid amplification.

[0010] More preferably, the fluorescent RNA reporter probe is 5'-6-FAM-UUUUUU-BHQ-1-3'.

[0011] More preferably, the kit further comprises a lateral flow test strip.

[0012] More preferably, the fluorescent RNA reporter probe is 5'-6-FAM-UUUUUU-Biotin-3'.

[0013] The application also provides the use of the above-mentioned crRNA molecule or reagent in the preparation of a canine distemper virus detection product.

[0014] More preferably, the product is a kit.

[0015] Compared with the prior art, the application has the following beneficial effects: The application realizes a significant breakthrough in technology through the two-step method design of EsCas13d protein combined with RPA isothermal amplification. On the one hand, the application makes the detection sensitivity reach 5 cp / μL, while effectively excluding the cross-reaction of canine parvovirus, coronavirus and other common viruses. On the other hand, the method realizes visual interpretation through 470 nm ultraviolet fluorescence or a lateral flow test strip, and the whole detection only takes 60 minutes, and the positive rate of clinical sample detection is significantly higher than that of qPCR, and the missed detection rate is lower. It is not only suitable for the ultra-sensitive screening of early canine infection, but also can detect endangered animal sources CDV such as giant pandas and red pandas, and provides a rapid, portable and high-performance POCT solution for primary pet hospitals, breeding farms and wild animal protection sites. It has important practical significance for improving the early prevention and control ability of canine distemper epidemic, reducing breeding economic losses and protecting endangered species. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 1 is the screening result of the RPA primer in Example 1, wherein A: RPA primer screening gel electrophoresis map; B: RPA primer screening fluorescence value heat map.

[0017] Figure 2A Figure 1 is the screening result of the RPA primer in Example 1, wherein A: RPA primer screening gel electrophoresis map; B: RPA primer screening fluorescence value heat map.

[0018] Figure 2B Figure 1 is the screening result of the RPA primer in Example 1, wherein A: RPA primer screening gel electrophoresis map; B: RPA primer screening fluorescence value heat map.

[0019] Figure 3Sensitivity detection results of EsCas13d in Example 1, in which NC is a negative control.

[0020] Figure 4 Specificity detection results of EsCas13d in Example 1, in which NC is a negative control. DETAILED DESCRIPTION

[0021] Example 1 1. Materials and methods 1.1 Reagents The main reagents used in this example are shown in Table 1.

[0022] Table 1 Main reagents used in this example

[0023] 1.2 Nucleic acid extraction Canine parvovirus (CPV), canine rotavirus (CRV), influenza A virus (IAV), feline herpesvirus type 1 (FHV-1), feline panleukopenia virus (FPV), and feline calicivirus (FCV) positive nucleic acids were provided by the Biotechnology and Pharmaceutical Research and Development and Application Laboratory of Henan City Construction College to verify the specificity of the method. Viral DNA or RNA was extracted according to the instructions of the FastPure Viral DNA / RNA Mini Kit, and cDNA was transcribed and synthesized using PrimeScript RT Master Mix. The extracted nucleic acids and cDNA were stored at -80 ℃ for later use. The detection performance of the clinical samples used in this application was evaluated using the commercial qPCR kit from Jianglai Biological Co., Ltd. as the gold standard. The CDV strain from giant panda was preserved at the Military Veterinary Research Institute of the Military Medical Science Academy.

[0024] 1.3 RPA primer and crRNA design and synthesis With CDV N gene as the target, CDV N genes of different genomes were downloaded from GenBank database, and the highly conserved region was screened out. Three RPA upstream primers and three downstream primers were designed and synthesized according to the highly conserved region, which were combined to form nine primer pairs for screening the best primer combination (Table 2). The 5' end of the RPA upstream primer was introduced with T7 promoter sequence (SEQ ID NO. 1: 5'-GAAATTAATACGACTCACTATAGGG-3') to facilitate subsequent in vitro transcription (Table 2). Three crRNAs were designed in the region amplified by the best primer pair, and the most suitable crRNA was screened out (Table 2). All the nucleotide sequences in Table 2 were synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd. RPA amplification was performed according to the RPA amplification kit instructions. After incubation at 37 ℃ for 20 min, the best primer group was screened by agarose gel electrophoresis.

[0025] Table 2 Nucleotide sequences used in this study

[0026] 1.4 RPA and crRNA screening RPA amplification was performed according to the RPA amplification kit instructions. After incubation at 37 ℃ for 20 min, the best primer group was screened by agarose gel electrophoresis.

[0027] EsCas13d 2x reaction buffer was prepared, and the specific components were as follows: 40 mM HEPES, pH 7.1, 100 mM KCl, 10 mM MgCl2, 10% glycerol. The 20 μL activity verification reaction system was as follows: 10 μL 2x reaction buffer, 100 nM EsCas13d, 100 nM crRNA, 10 5 cp / μL of target RNA, RNase inhibitor 0.5 U / μL, FQ-ssRNA reporter 1 μM. The most suitable crRNA was screened by fluorescence curve.

[0028] 1.5 Establishment of RPA-EsCas13d detection method First, isothermal amplification was performed using recombinase polymerase amplification (RPA), and then detection was performed using a CRISPR / EsCas13d-based system and a single-stranded RNA (ssRNA) fluorescent reporter molecule. In the RPA reaction, in addition to the standard components, Invitrogen Superscript IV (SSIV) reverse transcriptase and murine ribonuclease inhibitor were added, both at a final concentration of 2 U / μL. After adding viral nucleic acids and 14 mM magnesium acetate, the reaction mixture was incubated at 37°C in a metal bath for 30 minutes (see TWISTA MP® BASIC instructions for details). The detailed reaction system components are shown in Table 3. After amplification, 1 μL of RPA product was mixed with 19 μL of detection master mix to establish the final detection system. The system includes 1x Cas13d buffer, 50 nM EsCas13d protein, 100 nM crRNA, 1.5 U / μL T7 RNA polymerase, 1 mM rNTP mixture, 1 μM fluorescent reporter molecule, 2 U / μL murine ribonuclease inhibitor, and 0.05 U / μL ribonuclease H. The final 20 μL reaction was incubated at 37°C for 30 minutes in a QuantStudio™ 1 PLUS real-time PCR system.

[0029] Table 3 RPA reaction system

[0030] 1.6 Specificity and sensitivity of RPA-EsCas13d To achieve visual detection, visual observation under 470 nm ultraviolet light irradiation was used. FAM-BHQ labeled probe (Probe1) was used for qPCR instrument and visual fluorescence reading, and FAM-biotin poly U reporter (Probe2) was used for lateral flow test strip visualization. Gradient dilution of positive samples with known viral load (determined by qPCR and quantified) was used to evaluate the sensitivity of the detection.

[0031] 1.7 Clinical sample detection To evaluate the application effect of the EsCas13d platform in clinical sample detection, 30 clinical samples suspected of CDV infection were collected, of which 23 clinical samples were from dogs, 6 clinical samples were from red pandas, and 1 cell sample was a CDV infection positive sample from a giant panda. All samples were stored and transported under low temperature conditions after collection, and then detected using the established RPA-EsCas13d platform, and compared with the qPCR method. The positive detection rate and other information are shown in Table 4.

[0032] 2 Results 2.1 RPA primer and crRNA screening First, nine RPA primer pairs targeting the N gene region were screened. Agarose gel electrophoresis and fluorescence signal intensity showed that primer pair 1F / 2R had the highest amplification efficiency. Figure 1 Subsequently, three different crRNAs were designed based on the conserved sequence of the N gene and evaluated using the complete EsCas13d detection system. Among them, crRNA1 showed the strongest fluorescence signal, therefore crRNA1 was selected for subsequent experiments. Figure 2A , 2B ).

[0033] 2.4 Visualization, sensitivity, and specificity of the EsCas13d reaction In both in-tube fluorescence observation under 470 nm UV irradiation and LFA mode, the detection sensitivity of this platform is 5 cp / μL ( Figure 3 Using the giant panda-derived CDV strain as the target and other common pet virus nucleic acids as controls, no cross-reactivity was observed, indicating that this method has good specificity. Figure 4 ).

[0034] 2.5 Clinical Sample Testing To systematically evaluate the clinical application efficacy of the one-step EsCas13d detection method, this study selected 30 suspected CDV clinical samples for testing and analysis. The positive rate and other summary information are shown in Table 4. The results showed that RPA-EsCas13d detected 10 positive samples, while the commercial qPCR kit from Jianglai Biotechnology, used strictly according to the instructions, only detected 8 positive samples. For the two samples that were positive for RPA-EsCas13d but negative for qPCR, after being seeded with Vero-SLAM cells and passaged for three consecutive generations, the results of retesting with the same qPCR kit both turned positive, further validating the accuracy of the RPA-EsCas13d detection method and indicating that this method has superior sensitivity to qPCR for clinical sample detection.

[0035] Table 4 Summary of Clinical Sample Assessment Results

[0036] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A crRNA for detecting canine distemper virus based on a CRISPR / Cas13d system, characterized in that, The sequence of the crRNA molecule is shown in SEQ ID NO.

8.

2. A reagent for detecting canine distemper virus based on the CRISPR / Cas13d system, characterized in that, The reagent contains the crRNA molecule as described in claim 1.

3. A kit for detecting canine distemper virus based on the CRISPR / Cas13d system, characterized in that, The kit contains the crRNA molecule as described in claim 1 or the reagent as described in claim 2.

4. The reagent kit according to claim 3, characterized in that, The kit also contains Cas13d protein, fluorescent RNA reporter probe, and upstream primer SEQ ID NO.2 and downstream primer SEQ ID NO.6 for recombinase polymerase nucleic acid amplification.

5. The reagent kit according to claim 4, characterized in that, The fluorescent RNA reporter probe is 5'-6-FAM-UUUUUU-BHQ-1-3'.

6. The reagent kit according to claim 4, characterized in that, The kit also contains lateral chromatography test strips.

7. The reagent kit according to claim 6, characterized in that, The fluorescent RNA reporter probe is 5'-6-FAM-UUUUUU-Biotin-3'.

8. Use of the crRNA molecule of claim 1 or the reagent of claim 2 in the preparation of canine distemper virus detection products.

9. The use according to claim 8, characterized in that, The product in question is a reagent kit.

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