RPA-PAND detection kit and detection method for canine parvovirus and canine adenovirus

Abstract

The invention provides an RPA-PAND detection kit and detection method for canine parvovirus and canine adenovirus, and belongs to the technical field of canine virus detection.The kit comprises PfAgo protein and a gDNA and MB combination used for detecting canine parvovirus MVC, canine parvovirus CPV and canine adenovirus CAV. The RPA-PAND rapid nucleic acid detection method for detecting canine parvoviruses MVC and CPV and canine adenovirus CAV is successfully established on the basis of PfAgo protein mediated target detection and in combination with the RPA technology, and the method has the advantages of high sensitivity, high specificity, low cost, relatively low position targeting limitation, simple and convenient reaction system, no aerosol pollution, capability of performing multi-channel nucleic acid detection and the like; and a new reagent and a new way are provided for clinical diagnosis and epidemiological monitoring of viruses MVC, CPV and CAV.

Description

Technical Field

[0001] This invention relates to the field of canine virus detection technology, specifically to an RPA-PAND detection kit and method for canine parvovirus and canine adenovirus. Background Technology

[0002] Canine bocavirus (MCV, also known as CPV-1), canine parvovirus (CPV, referring to CPV-2 and its variants in this article), and canine adenovirus (CAV) are the main pathogens causing infectious diseases in dogs, seriously endangering their health. Canine bocavirus disease belongs to the genus *Covaviridae* of the family Parvoviridae. It was first isolated from the feces of healthy dogs in 1970. It is a non-enveloped, single-stranded DNA virus that can infect domestic dogs of all ages, causing respiratory distress, diarrheal enteritis, and even embryonic death in newborn puppies. Canine parvovirus belongs to the genus *Parvoviridae* of the family Parvoviridae. It was first isolated from the feces of dogs suffering from enteritis in 1978. It is a non-enveloped, single-stranded, negative-sense DNA virus that causes severe enteritis in young dogs and myocarditis in newborn puppies. The nucleocapsid protein of CPV and feline parvovirus (FPV) shares 98% amino acid sequence homology. Due to this high homology and the severe prevalence of canine diseases in recent years, CPV is considered a novel host variant of FPV. Canine adenovirus is the most pathogenic and structurally well-understood animal virus among the mammalian adenoviruses. CAV-2 is one of the two serotypes of canine adenovirus and has an antigenic correlation with CAV-1 (which causes canine infectious hepatitis), but it mainly causes canine infectious laryngotracheitis and enteritis, especially affecting puppies significantly. These three viruses occupy an important position in canine infectious diseases, and their prevalence and mutations pose a continuous threat to canine health, requiring prevention and control through accurate testing and effective vaccines.

[0003] The clinical diagnostic criteria for canine bocavirus, canine parvovirus, and canine adenovirus are primarily based on medical history, exposure history, symptoms, and various examinations, including complete blood count, serological tests, and pathological examinations. Detection methods for canine bocavirus and canine parvovirus mainly include rapid diagnostic strips, immunofluorescence assays, PCR, and antigen detection. Detection methods for canine adenovirus primarily involve PCR and virus isolation and culture. Traditional detection methods are complex, time-consuming, and require specific conditions, making them unsuitable for early diagnosis. Multiplex fluorescent RT-PCR cannot simultaneously detect all three viruses.

[0004] With the establishment of viral nucleic acid diagnostic methods represented by the CRISPR / Cas system, traditional clinical molecular diagnostic techniques have entered a new stage characterized by low cost, high sensitivity, and high specificity. However, while the development of CRISPR / Cas detection technology has achieved high sensitivity and specificity, it still suffers from limitations such as Cas effector protein off-target effects, high gRNA synthesis costs, dependence on PAM sequences, and complex multiplex detection systems. Currently, novel diagnostic methods based on the endonuclease Argonaute protein have also been established. This method achieves specific cleavage of DNA or RNA under the guidance of gDNA. Using the endonuclease Argonaute protein for typing and detecting the above three viruses is simpler, faster, and less expensive. Summary of the Invention

[0005] In view of this, the present invention aims to utilize based on Pf Ago's nucleic acid testing method PAND ( Pf By combining the Ago-mediated Nucleic Acid Detection (PAND) technology platform with recombinase polymerase amplification (RPA) technology, a rapid RPA-PAND nucleic acid detection system for canine parvovirus and canine adenovirus was established.

[0006] In a first aspect, the present invention provides a detection kit for canine parvovirus and canine adenovirus, comprising: Pf Ago protein and gDNA and MB used to detect canine parvovirus MVC, CPV, and canine adenovirus CAV; among which, The sequence of MVC-MB is shown in SEQ ID NO.2, and the sequences of MVC-gt, MVC-gf, and MVC-gr are shown in SEQ ID NO.3~5, respectively. The sequence of CPV-MB is shown in SEQ ID NO.7, and the sequences of CPV-gt, CPV-gf, and CPV-gr are shown in SEQ ID NO.8~10, respectively. The sequence of CAV-MB is shown in SEQ ID NO.12, and the sequences of CAV-gt, CAV-gf, and CAV-gr are shown in SEQ ID NO.13~15, respectively.

[0007] Preferably, the above-mentioned canine parvovirus and canine adenovirus detection kits further include the following RPA primers: The sequence is MVC-RPA-F / R as shown in SEQ ID NO.16 and SEQ ID NO.17; The sequence is CPV-RPA-F / R as shown in SEQ ID NO.18 and SEQ ID NO.19; The sequence is CAV-RPA-F / R as shown in SEQ ID NO.20 and SEQ ID NO.21.

[0008] Preferably, the MB carries a fluorescent group, and the fluorescent group is selected from FAM, CY5, ROX, etc.

[0009] In a first aspect, the present invention provides the application of the above-mentioned canine parvovirus and canine adenovirus detection kit in the detection of canine parvovirus MVC, CPV and / or canine adenovirus CAV, the application method comprising the following steps: S1. Extract DNA from the sample to be tested; S2. Using the DNA sample obtained in S1 as the detection target, and employing the canine parvovirus and canine adenovirus detection kit described in claim 1, a detection method containing... Pf The reaction was performed using a PAND detection system for Ago protein, gDNA, and MB.

[0010] Preferably, step S1 specifically involves: extracting DNA from the sample to be tested, using the obtained DNA as a template for amplification with RPA primers to obtain an RPA product; wherein the RPA product contains gDNA and the target region for MB detection. In some embodiments of the present invention, the RPA primers include MVC-RPA-F / R with sequences as shown in SEQ ID NO. 16-17, CPV-RPA-F / R with sequences as shown in SEQ ID NO. 18-19, and CAV-RPA-F / R with sequences as shown in SEQ ID NO. 20-21.

[0011] More preferably, the amplification conditions using RPA primers are: reaction at 37°C for 10-20 min.

[0012] Preferably, the reaction conditions in step S2 are: reaction at 95°C for 30 min.

[0013] Preferably, the MB carries a fluorescent group, so that the type of virus in the sample can be determined based on the different fluorescent groups carried by the MB of different viruses during detection.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) High social value. This invention is based on Pf Ago protein-mediated target detection has successfully established a rapid nucleic acid detection method (including PAND and RPA-PAND methods) for canine parvovirus MVC, CPV and canine adenovirus CAV. This method has advantages such as speed, high sensitivity, high specificity, low cost, low location targeting limitation, simple reaction system, no aerosol contamination, and the ability to perform multi-channel nucleic acid detection. It can handle complex samples and is suitable for clinical applications.

[0015] (2) Strong core competitiveness. Compared with clinical diagnostic standards, traditional virus isolation and culture, RT-PCR / qRT-PCR nucleic acid detection and serological detection, the present invention is an ideal pathogen detection method that meets the requirements of POCT (Point-of-Care Testing, i.e., rapid, sensitive, specific, low equipment requirements and economical). The RPA-PAND method of the present invention can reach 0.01-0.1 copy / μL, and has a higher degree of freedom in screening targets.

[0016] (3) Good potential benefits. Compared with conventional virus detection technologies and equipment, the present invention can save labor costs and solvent costs; RPA amplification and PAND detection only need to be carried out under two temperature conditions (37℃ and 95℃ are preferred), which greatly reduces the requirements for equipment and personnel operation. Attached Figure Description

[0017] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0018] Figure 1 This is a schematic diagram illustrating the detection of canine parvovirus MVC, CPV, and canine adenovirus CAV based on RPA-PAND in this invention. Figure 2 Used in the examples Pf Figure showing the prokaryotic expression and purification results of Ago protein; Figure 3 Used in the examples Pf Figure showing the results of endonuclease activity identification of Ago protein; Figure 4 This refers to the specific regions corresponding to the gDNA and MB used in Example 1 for canine parvovirus MVC, CPV, and canine adenovirus CAV; Figure 5 The images show the screening results of gDNA and MB used for canine parvovirus MVC(a), CPV(b), and canine adenovirus CAV(b) in Example 1. The left image in ac shows the screening of gn and molecular beacon MB, and the right image shows the screening of three gDNAs. Figure 6 The TBE-PAGE images of canine parvovirus MVC(a), CPV(b) and canine adenovirus CAV(b) detected by the PAND method in Example 1 are shown. Figure 7The results of agarose gel assay for RPA primer amplification fragments of canine parvovirus MVC(a), CPV(b) and canine adenovirus CAV(b) in Example 2 are shown. M is DL 2000 DNA maker. Figure 8 The results show the sensitivity of the PAND method detected in Example 4; Figure 9 The results show the sensitivity of the RPA / PCR-PAND method detected in Example 4; Figure 10 The results of agarose gel assay for the amplification products of various canine viruses in Example 5 are shown below. M is DL 2000 DNA maker. Figure 11 This is the specificity result of the RPA-PAND method detected in Example 5. Detailed Implementation

[0019] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art; the terminology used herein is for the purpose of describing particular implementation only and is not intended to limit the invention.

[0021] Canine parvoviruses (MVC), canine parvovirus (CPV), and canine adenovirus (CAV) all play important roles in canine health management. Although their pathogenicity, transmission methods, and control strategies differ, they all pose varying degrees of threat to canine health and play a crucial role in veterinary clinical practice and the prevention and control of canine infectious diseases. MVC, a relatively mild parvovirus, primarily affects puppies and immunocompromised individuals, potentially causing mild gastrointestinal or respiratory symptoms. However, in pregnant dogs, it can cause reproductive disorders such as fetal resorption or abortion, thus requiring special attention in the management of breeding populations. Although MVC has low pathogenicity and is generally not included in core vaccines, it still needs to be controlled through environmental management and biosecurity measures in specific situations (such as outbreaks in kennels or breeding farms). In contrast, CPV is significantly more virulent and infectious, making it one of the most deadly viruses in dogs, especially posing a significant threat to puppies. CPV directly attacks intestinal epithelial cells and the immune system, leading to severe hemorrhagic enteritis, leukopenia, and high mortality. Infection in unvaccinated dog populations can cause large-scale outbreaks. The continuous emergence of CPV variants (such as CPV-2a, 2b, and 2c) challenges the cross-protection of vaccines. Therefore, regular immunization and strict disinfection measures (such as the use of sodium hypochlorite) are the core means of prevention and control. The importance of CAV-2 is mainly reflected in respiratory disease prevention and control and cross-protection against vaccines. As one of the common pathogens of canine infectious tracheobronchitis ("kennel cough"), CAV-2 often synergizes with other pathogens (such as canine parainfluenza virus and Bordetella bronchiseptica), leading to respiratory symptoms such as cough and pneumonia. Furthermore, the CAV-2 vaccine can induce cross-immunity against CAV-1 (canine infectious hepatitis virus), thereby avoiding the "blue eye" side effect that may be caused by the CAV-1 vaccine. Therefore, in modern canine immunization programs, CAV-2 has replaced CAV-1 as a safer vaccine option. Overall, these three viruses each have their own focus in canine disease control: MVC requires vigilance in breeding management, CPV is a key focus for the prevention and control of fatal infectious diseases, while CAV-2 indirectly maintains canine health through respiratory protection and vaccination strategies. Scientific vaccination (such as the DHPP combined vaccine covering both CPV and CAV-2), environmental disinfection, and monitoring of high-risk groups are key measures to reduce their harm.

[0022] To achieve low-cost, high-sensitivity, rapid, and simultaneous detection of canine parvovirus MVC, canine parvovirus CPV, and canine adenovirus CAV, this invention provides an RPA-PAND detection kit and method for canine parvovirus and canine adenovirus, providing a reliable tool for canine disease prevention and control.

[0023] In a first aspect, embodiments of the present invention provide an RPA-PAND detection kit for canine parvovirus and canine adenovirus, comprising at least the following: Pf Ago protein; The gDNA and MB combination used for specific detection of canine parvovirus MVC, CPV and canine adenovirus CAV, wherein MVC-MB is shown in SEQ ID NO.2, MVC-gt, MVC-gf and MVC-gr are shown in SEQ ID NO.3~5 respectively, CPV-MB is shown in SEQ ID NO.7, CPV-gt, CPV-gf and CPV-gr are shown in SEQ ID NO.8~10 respectively, CAV-MB is shown in SEQ ID NO.12, and CAV-gt, CAV-gf and CAV-gr are shown in SEQ ID NO.13~15 respectively.

[0024] Furthermore, the above kit also includes RPA primers, and primers MVC-RPA-F / R are shown in SEQ ID NO.16~17, primers CPV-RPA-F / R are shown in SEQ ID NO.18~19, and primers CAV-RPA-F / R are shown in SEQ ID NO.20~21.

[0025] In the above kit, Pf Ago proteins can be naturally occurring proteins isolated from organisms or recombinant proteins with the same function prepared using biotechnology. When preparing recombinant proteins, a tag protein for expression promotion or purification can be added to the C-terminus or N-terminus, as in the embodiments of this invention. Pf The Ago protein was modified by adding a His-tagged protein, and the amino acid sequence of the resulting recombinant protein is shown in SEQ ID NO.22.

[0026] Secondly, based on the above-mentioned kit, this invention provides an RPA-PAND detection method for canine parvovirus and canine adenovirus, referring to... Figure 1 The method includes the following steps: (1) Extract DNA from the sample to be tested (such as a clinical serum sample); (2) The DNA extracted in step (1) was amplified using RPA primers to obtain the RPA amplification product (i.e., Figure 1 (Target DNA in the middle) (3) Construct a structure containing RPA amplification products, Pf The Ago protein, gDNA, and MB are reacted in a PAND detection system; when the MB carries a fluorescent group, the presence and type of the target virus in the sample can be directly determined by the collected fluorescence signal.

[0027] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0028] In the following examples, the 5' end of gDNA was phosphorylated using the system shown in Table 1: Table 1. Reaction system for phosphorylated gDNA

[0029] Used in the examples Pf Ago proteins are prepared and their nuclease activity is verified through the following process: Pf Preparation of Ago protein: Construction of pET-23a- Pf Ago recombinant plasmid, pET-23a- Pf Ago recombinant plasmid was transformed into E. coli BL21(DE3) strain; after induction of expression with 0.5 mM IPTG at 18℃ for 16 h, the bacterial cell pellet was collected and used... Pf Resuspend the cells in apolysis buffer (20 mM Tris-HCl, 200 mM NaCl, 0.2 mM MnCl2) and sonicate to lyse the bacteria; centrifuge at 12000 rpm for 10 min, collect the supernatant, and treat it at 70°C to remove contaminating proteins. Then, incubate it with a Ni-NTA column containing different concentrations of imidazole. Pf Gradient elution with ago lysis buffer; ultrafiltration concentration of protein solution containing 50-300 mM imidazole, followed by buffer replacement. Pf The gel was then subjected to 10% SDS-PAGE gel electrophoresis in an ago elution buffer (20 mM Tris-HCl, 300 mM NaCl, 0.5 mM MnCl2) to verify the results. Figure 2 The SDS-PAGE diagram shown illustrates Pf The prokaryotic expression and purification results of Ago protein showed that the obtained protein was consistent with the expected protein size of 91.4 kDa, and the protein concentration was 1.875 mg / ml after ultrafiltration concentration and liquid replacement.

[0030] Identification of endonuclease activity: Design and synthesize two complementary sequences containing the target gene fragment, three gDNA sequences targeting the target gene site, and the corresponding molecular beacon MB (specifically as follows). Figure 3(As shown in Figure A); phosphorylated gDNA is added to the detection system (gDNA and...) Pf The ago molar ratio was 1:10), and the reaction was carried out at 95℃ for 30 min. A sample was then added to 2 × TBE-PAGE loading, incubated at 95℃ for 5 min, and then subjected to 20% TBE-PAGE electrophoresis. The results are as follows: Figure 3 As shown in Figure B, the single-stranded DNA is 52 nt in size. Pf The Ago digestion reaction produced corresponding cleavage product sizes: gt cleavage products were 24 nt and 28 nt, GF cleavage products were 40 nt and 12 nt, and gr cleavage products were 40 nt and 12 nt, consistent with the expected cleavage results. This indicates that the purified... Pf Ago proteins have the activity of cleaving DNA from position 10 to 11 at the 5' end under the action of phosphorylated gDNA.

[0031] Example 1 This example provides a PAND kit for detecting canine parvovirus and canine adenovirus, including... Pf Ago proteins, as well as gDNA and MB used to detect viral MVC, CPV, and CAV-2.

[0032] First, based on the gene sequences of canine parvovirus MVC, canine parvovirus CPV, and canine adenovirus CAV provided by GeneBank, multiple sequence alignment was performed in MEGA 11, and phylogenetic analysis was conducted. Based on the phylogenetic analysis, multiple sequence alignment was performed on the viral MVC, CPV, and CAV-2 sequences to identify conserved regions in the viral genome that could be used for gDNA and MB design. Positive plasmids for each virus were then constructed based on these conserved regions, denoted as MVC P, CPV P, and CAV P, respectively.

[0033] Then, specific fragments in conserved regions were screened, corresponding gDNA and MB were designed, and the results were shown in Table 2. Pf The Ago restriction enzyme digestion assay was used for screening, and TBE-PAGE electrophoresis was used for verification. Three groups of gDNA and MB corresponding to different viruses were finally obtained, as shown in Table 3. The specific regions corresponding to these gDNA and MB are as follows: Figure 4 As shown, and the corresponding TBE-PAGE results are as follows. Figure 5 As shown.

[0034] Table 2 Pf Ago enzyme digestion detection system

[0035] Table 3 shows the gDNA and MB used for detecting various viruses.

[0036] In Table 3, gMB is a single-stranded fragment complementary to MB. gMB binds to PfAgo protein to form a complex and cleaves MB. Further, based on MB in Table 3, molecular beacons carrying FAM groups were synthesized, and a PAND detection system was constructed (Table 2). After 20% TBE-PAGE electrophoresis, gel electrophoresis verified that in the wavelength range of 495-518 nm, 28 nt FAM-MB (with 6 bases added before and after 16 nt MB to form a hairpin structure) was detected. Pf Ago enzyme cleavage into 16 nt beacon products, after staining with SYBR Gold nucleic acid dye in the dark for 5 min, was verified to be effective. Pf Under Ago active cleavage, the amplification products of canine parvovirus and canine adenovirus undergo changes. The test results are as follows: Figure 6 As shown, the gDNA and MB provided in this example can be used to establish Pf Ago-mediated targeted detection system for canine parvovirus and canine adenovirus.

[0037] Example 2 Based on Example 1, this example provides an RPA-PAND kit for the detection of canine parvovirus and canine adenovirus. The kit also includes RPA primers for the detection of canine parvovirus and canine adenovirus, as shown in Table 4.

[0038] Table 4. RPA primer information for canine parvovirus and canine adenovirus.

[0039] Based on the design principles of primers used in RPA amplification reactions, targeting three viruses... Figure 4 Multiple sets of RPA upstream and downstream primers can be designed for each of the specific fragments shown. For any virus, RPA amplification experiments were performed using different primer sets. Specifically, 29.4 μL of Abuffer was added to each dry powder reaction tube, and after thorough mixing, 2 μL of upstream / downstream primers (10 μM), nucleic acid template, and ddH2O were added sequentially. Finally, 2.5 μL of Bbuffer was added and thoroughly mixed to start the amplification reaction (see Table 5). The mixture was incubated at 37℃ for 30 min, and an equal volume of amplification product was added to a 6 × Loading buffer metal bath and incubated at 56℃ for 5 min. Detection was then performed using 3% agarose gel electrophoresis. Finally, primer combinations with high sensitivity at low copy numbers were determined.

[0040] Table 5 RPA amplification reaction system

[0041] Meanwhile, PAND detection was performed on RPA amplification products of different combinations (same as in Example 1), and the fluorescence signal intensity was compared by detecting the fluorescence signal using an enzyme-linked immunosorbent assay (ELISA) reader. Finally, RPA primer pairs that can produce high fluorescence signal values ​​in the detection methods of canine parvovirus and canine adenovirus using RPA amplification combined with PAND detection were obtained, as shown in Table 4. Figure 7 The results of agarose gel assays of the amplification products obtained using the primers in Table 4 are shown.

[0042] Example 3 Based on the detection kit in Example 2, this example establishes an RPA-PAND detection method for canine parvovirus and canine adenovirus, including the following steps: (1) RPA amplification.

[0043] The RPA product was obtained by amplifying the sample using RPA primers, with the preferred amplification conditions being: reaction at 37℃ for 10-20 min.

[0044] (2) Synthesize MB carrying fluorescent groups (such as FAM), establish the PAND detection system as shown in Table 6 and carry out the reaction. The preferred reaction conditions are: 95℃ for 30 min; after the reaction is completed, perform 20% TBE-PAGE electrophoresis and stain with SYBRGold nucleic acid dye in the dark for 5 min to verify the enzyme digestion results.

[0045] Table 6 PAND Reaction System

[0046] It is understandable that in step (2), the MB of each virus carries different fluorescent groups in order to distinguish and identify canine parvovirus MVC, CPV and canine adenovirus CAV while detecting changes in fluorescence signals.

[0047] Example 4 Based on Example 3, this example conducts sensitivity tests on the PAND method and the RPA-PAND method. Compared with the RPA-PAND method, the PAND method uses conventional PCR amplification reaction instead of RPA amplification.

[0048] (1) Minimum Detectable Concentration (MDC) of PAND method.

[0049] Positive plasmids for canine parvovirus MVC, CPV, and canine adenovirus CAV were amplified by PCR (same as in Example 1). The amplified products were purified (using an Omega gel extraction kit, and the purified products were measured using a NanoDrop 8000 high-throughput spectrophotometer) and serially diluted to 1, 2, 3, 4, 5, 10, 15, 20, 25, and 30 nM. Equal volumes were added to the PAND detection system, and the reaction was carried out at 95°C for 30 min. The limit of detection was determined based on the fluorescence value measured by the microplate reader.

[0050] The results are as follows Figure 8 As shown, the minimum sensitivity of MVC can reach 5 nM, the minimum sensitivity of CPV can reach 3 nM, and the minimum sensitivity of CAV can reach 3 nM.

[0051] (2) The lowest detection limit of the RPA-PAND method.

[0052] Using positive plasmids of canine parvovirus MVC, CPV, and canine adenovirus CAV as targets, plasmid concentrations were measured using NanoDrop 8000. The concentrations were calculated according to the formula: (copy number (copies / μL) = [6.02 × 10⁻⁶)). 23 [× plasmid concentration (ng / μL)] / [DNA length (bp) × 660 × 10] 9 ]) Calculate and dilute the copy number by a factor of 10, specifically to a dilution of 1.66 × 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 aM. RPA amplification was performed using RPA primers (incubated at 37℃ for 10 min), and then an equal volume was added to the PAND detection system and incubated at 95℃ for 30 min to establish the lower limit of detection for different viruses. Additionally, PCR was performed on samples of different concentrations, and after amplification, an equal volume was added to the PAND detection system and incubated at 95℃ for 30 min. The PCR amplification system included 0.3 μL template, 0.3 μL each of the front and rear primers (R / F), 5 μL Taq enzyme, and 4.1 μL ddH2O. The PCR amplification process was as follows: 95℃ for 3 min; 95℃ for 15 s, 60℃ for 15 s, 72℃ for 8 s, 25-30 cycles; 72℃ for 5 min. The entire PCR amplification process took approximately one hour.

[0053] The results are as follows Figure 9As shown, after RPA or PCR amplification, the MDC of canine parvovirus MVC, CPV, and canine adenovirus CAV was significantly increased, reaching 1.66 × 10⁻⁶. -2 -1.66 × 10 -1 aM (0.01-0.1 copy / μL), where CPV is 1.66×10 -1 aM, while MVC and CAV reach 1.66×10 -2 aM. However, compared to PCR amplification, RPA-PAND detection takes 45 minutes less time.

[0054] Example 5 In this example, non-canine parvovirus and canine adenovirus were used as controls to perform a specificity test of the RPA-PAND detection method. The specific procedure is as follows: (1) For canine parvovirus MVC, CPV, and canine adenovirus CAV, the corresponding DNA fragments were amplified using the RPA primers shown in Table 4; additionally, DNA fragments of HBoV1-NP1, B19-VP1, LuIII-VP, and EV71-2A were amplified (randomly selected from conserved regions of each virus). The agarose gel analysis results of each amplification product are shown below. Figure 10 As shown.

[0055] (2) Referring to Table 6, construct a PAND reaction system containing canine parvovirus MVC, CPV, and canine adenovirus CAV; and follow the instructions in Table 6. Figure 11 As shown, different DNA fragments or combinations from step (1) are added to the PAND reaction system for reaction (at 95°C for 30 min).

[0056] The results are as follows Figure 11 As shown, when only RPA amplification products that can be specifically targeted by gDNA are present, the fluorescence signal in the reaction is enhanced, while no difference in fluorescence signal is observed in the reaction detection systems for non-canine parvovirus and canine adenovirus gene products. This indicates that the present invention has successfully established a detection system capable of detecting RPA amplification products that can be specifically targeted by gDNA. Pf Ago-mediated PAND typing detection method for MVC, CPV, and CAV, with high specificity.

[0057] In summary, the PAND detection kit and method provided by this invention can be successfully used for the typing and detection of canine parvovirus MVC, CPV, and canine adenovirus CAV, and have the following main technical advantages: (1) Compared with the detection platform based on the CRISPR-Cas system, the PAND method can use low-cost guide DNA to screen a wider range of target regions without being limited by the PAM sequence; (2) Compared with PCR amplification, the time required for RPA-PAND detection is reduced by 45 min; (3) The minimum detectable concentration of the PAND method without RPA amplification reaction is 3-5 nM; after RPA or PCR amplification, the MDC is greatly increased to 1.66 × 10⁻⁶. -2 - 1.66 × 10 -1 aM, where CPV is 1.66 × 10 -1 aM, while MVC and CAV reach 1.66×10 -2 aM.

[0058] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A detection kit for canine parvovirus and canine adenovirus, characterized in that, include Pf Ago protein and gDNA and MB combinations used for detecting canine parvovirus MVC, CPV, and canine adenovirus CAV; among which, MVC-MB is shown in SEQ ID NO.2, and MVC-gt, MVC-gf, and MVC-gr are shown in SEQ ID NO.3~5 respectively; CPV-MB is shown in SEQ ID NO.7, and CPV-gt, CPV-gf, and CPV-gr are shown in SEQ ID NO.8~10 respectively; CAV-MB is shown in SEQ ID NO.12, and CAV-gt, CAV-gf, and CAV-gr are shown in SEQ ID NO.13~15 respectively.

2. The canine parvovirus and canine adenovirus detection kit according to claim 1, characterized in that, It also includes the following RPA primers: The sequence is MVC-RPA-F / R as shown in SEQ ID NO.16 and SEQ ID NO.17; The sequence is CPV-RPA-F / R as shown in SEQ ID NO.18 and SEQ ID NO.19; The sequence is CAV-RPA-F / R as shown in SEQ ID NO.20 and SEQ ID NO.

21.

3. The canine parvovirus and canine adenovirus detection kit according to claim 1, characterized in that, The MB carries a fluorescent group, which is selected from FAM, CY5, and ROX.

4. The application of the canine parvovirus and canine adenovirus detection kit as described in claim 1 in the detection of canine parvovirus MVC, CPV and / or canine adenovirus CAV for non-diagnostic and non-therapeutic purposes.

5. A method for detecting canine parvovirus and canine adenovirus for non-diagnostic and non-therapeutic purposes, characterized in that, Includes the following steps: S1. Extract DNA from the sample to be tested; S2. Using the DNA sample obtained in S1 as the detection target, and employing the canine parvovirus and canine adenovirus detection kit described in claim 1, a detection method containing... Pf The reaction was performed using a PAND detection system for Ago protein, gDNA, and MB.

6. The method for detecting canine parvovirus and canine adenovirus according to claim 5, characterized in that, Step S1 specifically involves: extracting DNA from the sample to be tested, using the obtained DNA as a template to amplify it through RPA primers to obtain RPA products; wherein the RPA products contain gDNA and the target region for MB detection.

7. The method for detecting canine parvovirus and canine adenovirus according to claim 6, characterized in that, The RPA primers are MVC-RPA-F / R with sequences as shown in SEQ ID NO.16-17, CPV-RPA-F / R with sequences as shown in SEQ ID NO.18-19, and CAV-RPA-F / R with sequences as shown in SEQ ID NO.20-21.

8. The method for detecting canine parvovirus and canine adenovirus according to claim 6 or 7, characterized in that, The conditions for amplification using RPA primers are: reaction at 37℃ for 10-20 min.

9. The method for detecting canine parvovirus and canine adenovirus according to claim 5, characterized in that, The reaction conditions described in step S2 are: reaction at 95°C for 30 min.

10. The method for detecting canine parvovirus and canine adenovirus according to claim 5, characterized in that, The MB carries a fluorescent group, and the type of virus in the sample to be tested can be determined based on the different fluorescent groups carried by the MB of different viruses.

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