Primer composition for detecting canine enterovirus, kit thereof and LAMP (loop-mediated isothermal amplification) micro-fluidic chip detection method

Abstract

The invention relates to a primer composition for detecting canine enteroviruses, a kit of the primer composition and an LAMP (loop-mediated isothermal amplification) microfluidic chip detection method, the primer composition comprises LAMP primers with sequences as shown in SEQ ID No.1-SEQ ID No.39, and is used for detecting canine rotavirus, canine astrovirus, canine coronavirus, canine distemper virus, canine parvovirus, canine kobuvirus and canine norovirus. The LAMP method is optimized and constructed by taking positive plasmids or ssRNA of seven intestinal pathogen genomes of dogs as a template, a microfluidic chip detection disc is designed on the basis, the effectiveness of the method is verified through a clinical positive sample, and experimental results show that the method has the advantages of high specificity, high sensitivity, high sensitivity and the like. The LAMP micro-fluidic chip detection method constructed by the invention can simultaneously detect seven intestinal pathogens of dogs, is high in sensitivity, strong in specificity and short in reaction time, can be used for clinical rapid screening in primary pet hospitals, and can also be used for accurate diagnosis in professional laboratories.

Description

Technical Field

[0001] The invention relates to the technical field of animal virus detection, and in particular to a primer composition and a kit thereof for detecting canine enterovirus and a LAMP microfluidic chip detection method. Background Art

[0002] Canine enterovirus testing is an important process that helps detect viral infections in a timely manner and protect the health of canines. The current detection methods for canine enterovirus include: (1) virus isolation and culture: the collected sample is inoculated into a cell culture system suitable for the growth of canine enterovirus; if the sample contains virus, the virus will multiply in the cells and cause cytopathic effects. Further identification experiments, such as immunofluorescence and electron microscopy, are used to determine whether it is canine enterovirus. However, this method is time-consuming and has high requirements for experimental conditions and technology. In addition, some viruses may be difficult to culture in vitro. (2) Antigen or antibody detection: using the principle of antigen-antibody specific binding, the antigen component or antibody level of the virus in the sample is detected to determine whether the virus is infected, such as enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography. However, this method has limited sensitivity and poor specificity, and its detection results are greatly affected by the quality of the sample. (3) Nucleic acid detection: detecting the nucleic acid (DNA or RNA) of the virus to determine the presence of the virus, such as polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR). Although this method has the advantages of high sensitivity and strong specificity, it has high requirements for the operator's skills and instruments, making it difficult to popularize and apply in actual production at the grassroots level.

[0003] In 2002, Japanese researcher Notomi established a new in vitro nucleic acid detection technology named loop-mediated isothermal amplification (LAMP). This technology can achieve rapid and efficient nucleic acid fragment amplification by using 4-6 specific primers and a DNA polymerase with strand displacement activity. It can amplify 10 9The LAMP technology can achieve multiple copies of the target sequence and can achieve a sensitivity equivalent to that of real-time quantitative PCR technology. It is more convenient and specific than traditional PCR technology. The LAMP reaction is carried out under isothermal conditions throughout the process, and does not require repeated thermal denaturation. Only a thermally stable device is required to achieve continuous amplification, which greatly reduces the detection cost. With the development, LAMP technology has been widely used in the detection of pathogenic microorganisms. Jiang Ning et al. established a fast, simple, sensitive and accurate canine parvovirus (CPV) loop-mediated isothermal amplification (LAMP) visualization detection method. They designed specific primers based on the conservative region of the VP2 gene of canine parvovirus, and established a canine parvovirus LAMP visualization rapid detection method by optimizing the primer concentration, reaction temperature and reaction time. This method does not require special equipment, and the test results can be directly observed by the naked eye. It has the advantages of high efficiency, simplicity, good specificity and high sensitivity. It is suitable for on-site rapid diagnosis of canine parvovirus, but the above method is only for the detection of a single pathogen, and it cannot meet the clinical detection needs of multiple pathogens.

[0004] Microfluidic chips, also known as lab-on-a-chip (LOC), can integrate basic operating units such as sample processing, analysis, and detection involved in fields such as chemistry and biology onto a core of a few square centimeters, with the advantages of low reagent sample usage and fast analysis speed. The integrated detection mode based on centrifugal microfluidic chips can further enhance the integration and automation of nucleic acid detection, and significantly improve the convenience of nucleic acid detection. This type of method mainly drives and manipulates the flow of liquid on the chip by means of centrifugal force, and can achieve integrated high-throughput nucleic acid detection. Although microfluidic chips have been combined with the LAMP method to achieve the detection of multiple pathogens, it does not involve the detection of pathogens of multiple canine intestinal diseases, especially emerging viruses such as canine astrovirus, canine norovirus, and canine crest virus.

[0005] There are some shortcomings in the above-mentioned detection methods: (1) There are many pathogens that cause diarrhea in dogs clinically, and detection methods targeting only a single pathogen cannot meet the clinical detection needs; (2) The existing detection operations are relatively cumbersome and require certain professional laboratory skills, which is not conducive to the application and promotion of primary pet hospitals; (3) The sensitivity and specificity of the method still need to be further improved. Currently, there is an urgent need for a method that can screen multiple canine intestinal pathogens at one time to better meet the timeliness requirements of clinical diagnosis. Summary of the invention

[0006] In view of at least one deficiency in the prior art, the present invention utilizes the LAMP method and microfluidic chip technology to establish a set of rapid detection methods for seven pathogens that can cause canine intestinal diseases. Specifically, through the loop-mediated isothermal amplification fluorescence method and the layout design, process optimization and preparation of the microfluidic chip detection disk, a set of LAMP microfluidic chip detection methods are established for four common viruses that cause canine intestinal diseases: canine distemper virus, canine parvovirus, canine coronavirus, canine rotavirus, and three currently emerging viruses: canine astrovirus, canine norovirus, and canine crest virus, so as to meet the needs of professional laboratories and grassroots departments for rapid identification of these types of diseases.

[0007] To achieve the above object, the technical solution adopted by the present invention includes:

[0008] The first aspect of the present invention is to provide a primer composition for detecting canine enterovirus; wherein the canine enterovirus includes at least one of canine rotavirus, canine astrovirus, canine coronavirus, canine distemper virus, canine parvovirus, canine cristae virus, and canine norovirus.

[0009] Furthermore, the primer combination is designed for the most conserved regions of the VP6 gene of canine rotavirus, the Capsidprotein gene of canine astrovirus, the N gene of canine coronavirus, the N gene of canine distemper virus, the NS1 gene of canine parvovirus, the 3D gene of canine crest virus, and the ORF1 gene of canine norovirus.

[0010] Further, the primer composition includes at least one of the following LAMP primers:

[0011] LAMP primers for detecting canine rotavirus: sequences shown in SEQ ID No. 1 to SEQ ID No. 5;

[0012] LAMP primers for detecting canine astrovirus: sequences shown in SEQ ID No.6 to SEQ ID No.11;

[0013] LAMP primers for detecting canine coronavirus: sequences shown in SEQ ID No.12 to SEQ ID No.17;

[0014] LAMP primers for detecting canine distemper virus: sequences shown in SEQ ID No.18 to SEQ ID No.23;

[0015] LAMP primers for detecting canine parvovirus: sequences shown in SEQ ID No. 24 to SEQ ID No. 28;

[0016] LAMP primers for detecting canine crest virus: sequences shown in SEQ ID No. 29 to SEQ ID No. 34;

[0017] LAMP primers for detecting canine norovirus: sequences shown as SEQ ID No.35 to SEQ ID No.39.

[0018] In a specific embodiment, the primer composition consists of LAMP primers for detecting canine rotavirus, LAMP primers for detecting canine astrovirus, LAMP primers for detecting canine coronavirus, LAMP primers for detecting canine distemper virus, LAMP primers for detecting canine parvovirus, LAMP primers for detecting canine cristae virus, and LAMP primers for detecting canine norovirus.

[0019] The second aspect of the present invention is to provide a LAMP reaction system containing any one of the primer combinations described in the first aspect; wherein, in the LAMP reaction system, the FIP primer and the BIP primer are both 1.6 μM, the F3 primer and the B3 primer are both 0.2 μM, and the loop primers are both 0.8 μM.

[0020] Furthermore, the LAMP reaction system also includes a LAMP reaction solution; wherein, based on the total volume of the LAMP reaction system being 25 μL, the LAMP reaction solution includes 2.5 μL of 10x isothermalAmp Buffer, 1 to 2.2 mM dNTP, 4 to 10 mM MgSO4, 0 to 12.5 U of reverse transcriptase, 4 to 16 U of BstⅡPro DNA polymerase, 0.5 μL of 50x SYBR GREEN dye, 5 μL of nucleic acid and an appropriate amount of ddH2O.

[0021] Furthermore, based on the total volume of the LAMP reaction system of 25 μL: the LAMP reaction solution for detecting canine rotavirus, canine astrovirus, canine coronavirus, canine distemper virus, canine parvovirus, canine crest virus or canine norovirus includes 10x other Amp Buffer 2.5 μL, dNTP 1.8 mM, MgSO4 8 mM, reverse transcriptase 10 U, BstⅡ Pro DNA polymerase 12 U, 50x SYBR GREEN dye 0.5 μL, nucleic acid 5 μL and an appropriate amount of ddH2O.

[0022] The third aspect of the present invention is to provide a kit for detecting canine enterovirus; wherein the kit contains any primer combination described in the first aspect, or contains any LAMP reaction system described in the second aspect.

[0023] Furthermore, the kit also includes a microfluidic chip, a negative control substance, and a positive control substance.

[0024] Furthermore, the microfluidic chip adopts mold opening-injection molding technology to achieve later batch production. The microfluidic chip is a circular reaction chip composed of 4 reaction chambers, and there are 8 micro reaction chambers (amplification detection pools) under each reaction chamber. There is physical isolation between each micro reaction chamber (amplification detection pool), thereby eliminating the risk of cross contamination or interference between different reactions.

[0025] Furthermore, the microfluidic chip is provided with 4 independent reaction chambers, each reaction chamber comprises a sampling pool, a distribution pool, a capillary microvalve, and an amplification detection pool which are connected in sequence, and each reaction chamber has 8 amplification detection pools; among them, two reaction chambers are provided with a canine distemper virus detection hole, a canine parvovirus detection hole, a canine coronavirus detection hole, a canine rotavirus detection hole, a canine astrovirus detection hole, a canine norovirus detection hole and a canine cristal virus detection hole (the remaining hole is abandoned); one reaction chamber is provided with a canine distemper virus positive detection hole The positive control wells are canine parvovirus positive control wells, canine coronavirus positive control wells, canine rotavirus positive control wells, canine astrovirus positive control wells, canine norovirus positive control wells and canine crest virus positive control wells (the remaining well is discarded); one reaction chamber is set with canine distemper virus negative control wells, canine parvovirus negative control wells, canine coronavirus negative control wells, canine rotavirus negative control wells, canine astrovirus negative control wells, canine norovirus negative control wells and canine crest virus negative control wells (the remaining well is discarded).

[0026] Furthermore, a predetermined amount of primers are pre-packaged in the amplification detection pool of the microfluidic chip.

[0027] Furthermore, the primer packaging step includes: using a micro-spotting instrument to evenly distribute each primer in a specific position area on the microfluidic chip, adding a quantitative primer working solution to each reaction well, and drying the chip with the primer working solution at a constant temperature, ensuring that it is completely dried and then affixing a sealing film to seal the chip.

[0028] Furthermore, the negative control substance is ddH2O.

[0029] Furthermore, the positive control is a positive plasmid.

[0030] Furthermore, the positive plasmid is a plasmid containing the sequence shown as SEQ ID No.40.

[0031] Furthermore, the sequence shown in SEQ ID No.40 is a sequence formed by connecting the conserved sequences in canine rotavirus VP6 gene, canine astrovirus Capsidprotein gene, canine coronavirus N gene, canine distemper virus N gene, canine parvovirus NS1 gene, canine crest virus 3D gene, and canine norovirus ORF1 gene, and its preparation steps include: inserting the target DNA into a plasmid vector containing a T7 promoter, and then treating it with a restriction enzyme.

[0032] The fourth aspect of the present invention is to provide a LAMP microfluidic chip detection method for detecting canine enterovirus (i.e., a method for using any of the kits described in the third aspect), which uses any of the kits described in the third aspect, comprising the steps of:

[0033] S1. Collect samples to be tested and obtain nucleic acid extracts using a viral nucleic acid extraction kit;

[0034] S2, adding the pre-configured LAMP reaction liquid containing the nucleic acid extract to the injection pool of each reaction chamber in the microfluidic chip, packaging it and placing it in the detection device, and the liquid flows into the amplification detection pool under the action of centrifugal force to perform LAMP reaction, wherein a predetermined amount of primers are pre-packaged in the amplification detection pool;

[0035] S3, determining the detection result according to the fluorescent signals of each amplification detection pool collected in real time during the LAMP reaction;

[0036] Furthermore, in step S1, the sample to be detected is a stool sample. It is understandable that the sample may also be other forms of samples routinely collected in the art.

[0037] Furthermore, in step S2, 25 μL of LAMP reaction solution is added to the sample injection pool of each reaction chamber; further, the reaction procedure of the LAMP reaction is: transferring the sample to be tested to the detection hole of the microfluidic chip at 6000 rpm for 30 seconds; performing the reaction at 63-66°C (preferably 65°C) for 60 minutes, and collecting the fluorescence signal every 60 seconds.

[0038] Furthermore, in step S3, the criterion for determining the test result is: if an "S"-shaped amplification curve appears within the detection time, it can be determined as positive, otherwise it is negative.

[0039] Furthermore, in the microfluidic chip of the detection method, the minimum detection limits of the canine parvovirus detection module, the canine distemper virus detection module, the canine coronavirus detection module, the canine astrovirus detection module, the canine rotavirus detection module, the canine crest virus detection module, and the canine norovirus detection module are all 10 1 copies / uL.

[0040] The fifth aspect of the present invention is to provide the use of any of the kits described in the third aspect in detecting canine enterovirus, wherein the canine enterovirus includes canine rotavirus, canine astrovirus, canine coronavirus, canine distemper virus, canine parvovirus, canine cristae virus, and canine norovirus. The above detection can be used to screen related preventive and therapeutic drugs, and for epidemiological surveys of canine enterovirus.

[0041] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0042] In the microfluidic chip used in the present invention, one sample can undergo eight different reactions simultaneously in one reaction chamber, so a maximum of 32 different reactions can be performed on one chip at one time, which can meet the practical needs of "few samples and heavy screening" in the clinical diagnosis and treatment of pets. The present invention pre-embeds the LAMP detection reagent or the corresponding detection primer in the chip, and the operator only needs to add a certain amount of nucleic acid extract to the sample addition port of the reaction chamber of the microfluidic chip to complete the preliminary detection preparation, which is simple and convenient to operate. The present invention optimizes the various reaction parameters in the LAMP method and adopts the optimized reaction system to improve the amplification efficiency, minimum detection limit and specificity of the method.

[0043] The present invention realizes the clinical differential diagnosis of 7 canine intestinal pathogens at one time. The detection method is highly integrated and easy to operate, meeting the actual needs of primary pet hospitals. The method has high sensitivity, good specificity, and short reaction time. It can be used for rapid screening in primary pet hospitals and accurate diagnosis in professional laboratories. The above detection method also has the advantage of low cost. Both the instrument cost and the reagent cost are lower than the cost required for ordinary PCR and fluorescent quantitative PCR. Therefore, the detection method of the present invention can simultaneously and quickly realize the detection of 7 canine intestinal pathogens, which can be promoted and applied to the epidemiological survey and disease prevention and control of canine intestinal viruses, and has a broad scope of application and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0045] Figure 1 A schematic diagram of the manufacturing process of a microfluidic chip in one embodiment of the present invention;

[0046] Figure 2 The model diagram and the actual diagram of the microfluidic chip in one embodiment of the present invention are shown in FIG. 1 , wherein a: injection pool; b: distribution pool; c: amplification pool; d: capillary microvalve;

[0047] Figure 3 Schematic diagram of the layout of the microfluidic chip for real-time fluorescence isothermal amplification of seven intestinal pathogens of dogs in one embodiment of the present invention; wherein A1, B1: canine distemper virus detection holes; A2, B2: canine parvovirus detection holes; A3, B3: canine coronavirus detection holes; A4, B4: canine rotavirus detection holes; A5, B5: canine astrovirus detection holes; A6, B6: canine norovirus detection holes; A7, B7: canine crest virus detection holes; A8, B8: abandoned; C1, D1: canine distemper Thermovirus positive control well and negative control well; C2, D2: canine parvovirus positive control well and negative control well; C3, D3: canine coronavirus positive control well and negative control well; C4, D4: canine rotavirus positive control well and negative control well; C5, D5: canine astrovirus positive control well and negative control well; C6, D6: canine norovirus positive control well and negative control well; C7, D7: canine crest virus positive control well and negative control well; C8, D8: abandoned;

[0048] Figure 4 The amplification results of different reaction temperatures in the real-time fluorescent LAMP reaction in one embodiment of the present invention; wherein, Ⅰ to Ⅳ: 63°C, 64°C, 65°C, 66°C;

[0049] Figure 5 The amplification results of different MgSO4 concentrations in the real-time fluorescence LAMP reaction in one embodiment of the present invention; wherein, Ⅰ to Ⅳ: 4mM, 6mM, 8mM, 10mM;

[0050] Figure 6 The amplification results of different BstⅡPro DNA polymerase concentrations in the real-time fluorescent LAMP reaction in one embodiment of the present invention; wherein, Ⅰ to Ⅳ: 4U, 8U, 12U, 16U.

[0051] Figure 7 The amplification results of different dNTP concentrations in the real-time fluorescent LAMP reaction in one embodiment of the present invention; wherein, Ⅰ to Ⅳ: 1mM, 1.4mM, 1.8mM, 2.2mM;

[0052] Figure 8 The amplification results of different reverse transcriptase concentrations in the real-time fluorescent LAMP reaction in one embodiment of the present invention; wherein, Ⅰ to Ⅳ: 5U, 7.5U, 10U, 12.5U;

[0053] Fig. 9 Schematic diagram of the results of the sensitivity test of the canine parvovirus detection module of the microfluidic chip in one embodiment of the present invention; wherein A to D are positive plasmids respectively: 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 100 copies / μL;

[0054] Fig.10 Schematic diagram of the results of the sensitivity test of the canine distemper virus detection module of the microfluidic chip in one embodiment of the present invention; wherein A to D are ssRNA transcribed in vitro from plasmids: 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0 copies / μL;

[0055] Fig.11 Schematic diagram of the results of the sensitivity test of the canine coronavirus detection module of the microfluidic chip in one embodiment of the present invention; wherein A to D are ssRNA transcribed in vitro from plasmids: 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0 copies / μL;

[0056] Fig.12 Schematic diagram of the results of the sensitivity test of the canine astrovirus detection module of the microfluidic chip in one embodiment of the present invention; wherein A to D are ssRNA transcribed in vitro from plasmids: 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0 copies / μL;

[0057] Fig.13 Schematic diagram of the results of the sensitivity test of the canine rotavirus detection module of the microfluidic chip in one embodiment of the present invention; wherein A to D are ssRNA transcribed in vitro from plasmids: 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0 copies / μL;

[0058] Fig.14 Schematic diagram of the results of the sensitivity test of the canine crest virus detection module of the microfluidic chip in one embodiment of the present invention; wherein A to D are ssRNA transcribed in vitro from plasmids: 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0copies / μL;

[0059] Fig.15 Schematic diagram of the results of the sensitivity test of the canine norovirus detection module of the microfluidic chip in one embodiment of the present invention; wherein A to D are ssRNA transcribed in vitro from plasmids: 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0 copies / μL;

[0060] Fig.16 The figure is a schematic diagram of the results of the specificity test of the microfluidic chip canine parvovirus, canine distemper virus, canine coronavirus, canine astrovirus, canine rotavirus, canine cristomavirus, and canine norovirus detection module in one embodiment of the present invention; wherein A to G are canine parvovirus, canine distemper virus, canine coronavirus, canine astrovirus, canine rotavirus, canine cristomavirus, and canine norovirus, respectively;

[0061] Fig.17 This is a diagram of some actual test results of clinical sample testing using the LAMP microfluidic chip detection method in one embodiment of the present invention. DETAILED DESCRIPTION

[0062] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0063] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0064] In the following examples, the main reagents used: BstⅡPro DNA Polymerase Large Fragment, 10x isothermalAmp Buffer, HiScriptⅡReverse Transcriptase, dNTPs Mix, MgSO4 were purchased from Nanjing Norwegian Biotechnology Co., Ltd.; the viral nucleic acid extraction kit was purchased from Magen Biotech; the Ribo MAXTM LargeScale RNA Product Systems in vitro transcription kit was purchased from Promega Corporation of the United States; the positive plasmid was synthesized from Biotech Biotechnology (Shanghai) Co., Ltd.; One Step PrimeScript TM RT-PCR Kit (Perfect RealTime) was purchased from TaKaRa.

[0065]

[0066] In the following examples, ssRNA positive template preparation: (1) Preparation of positive plasmid: insert the target DNA into the plasmid vector containing T7 promoter, and then treat with restriction enzyme; the steps are as follows: first, use the restriction enzyme without recognition site in the inserted DNA fragment to linearize the plasmid, and then on the right side of the promoter region and downstream of the inserted DNA fragment; finally, after the reaction, Proteinase K treatment and purification reaction are carried out to obtain a positive plasmid containing the sequence shown in SEQ ID No.40. (2) Preparation of target DNA gene sequence: PCR amplification is performed using the positive plasmid as a template to obtain the target fragment, and the PCR product is recovered. (3) In vitro transcription: transcribe into ssRNA using Ribo MAXTM Large Scale RNA Product Systems in vitro transcription kit, measure the concentration and calculate the copy number, and store at -80℃ for future use.

[0067] The present invention is illustrated below by way of examples.

[0068] Example 1-LAMP primer design

[0069] The present embodiment is by querying the relevant sequence in NCBI Genbank, and the region with the highest homology in the viral genome is selected as the target gene of primer design by sequence alignment.Finally, the conservative sequence design primers in the N gene of canine distemper virus (MN296413.1), the NS1 gene of canine parvovirus (MH476590.1), the N gene of canine coronavirus (OQ623726.1), the VP6 gene of canine rotavirus (KP988017.1), the Capsid protein gene of canine astrovirus (ON733251.1), the ORF1 gene of canine norovirus (MK726262.1), and the 3D gene of canine crest virus (OP293748.1) are selected respectively.Conventional LAMP primers are designed using Primer Explorer V5.0 online software, and the primer set with the highest score is selected as the primer used in the present invention according to the software scoring.All the above primers are synthesized by Shanghai Saiheng Biotechnology Co., Ltd., and the effectiveness of the primers is evaluated after the primer synthesis.

[0070] This example compares the N gene of canine distemper virus, the NS1 gene of canine parvovirus, the N gene of canine coronavirus, the VP6 of canine rotavirus, the Capsid protein gene of canine astrovirus, the ORF1 gene of canine norovirus, and the 3D gene of canine crest virus in NCBI Genbank, finds the most conserved region, and designs primers with the help of Primer Explorer 5 online primer design software. The specific sequence information of the above primers is shown in the following table:

[0071] Table 1 - Primer information

[0072]

[0073]

[0074] Example 2-Optimization of LAMP reaction system

[0075] This embodiment uses virus positive plasmid or positive ssRNA for detection, and optimizes each reaction condition in the detection system through a single factor variation test. The initial selection of the LAMP reaction system (25 μL) is: FIP primer 1.6 μM, BIP primer 1.6 μM, F3 primer 0.2 μM, B3 primer 0.2 μM, loop primer 0.8 μM, 10x isothermalAmp Buffer 2.5 μL, dNTP 1-2.2 mM, MgSO4 4-10 mM, reverse transcriptase 0-12.5 U, BstⅡPro DNA polymerase 4-16 U, 50xSYBR GREEN dye 0.5 μL, nucleic acid (positive plasmid or positive ssRNA) 5 μL and an appropriate amount of ddH2O. The above primers are all designed in Example 1 as shown in Table 1. The judgment criteria for the detection results of the reaction system are: the appearance of an "S" type amplification curve within the detection time can be judged as positive, otherwise it is negative.

[0076] In this embodiment, different reaction temperatures (63°C, 64°C, 65°C, 66°C), Mg 2+ The LAMP reaction system was optimized by adjusting the concentration (4mM, 6mM, 8mM, 10mM), BstⅡPro DNA polymerase (4U, 8U, 12U, 16U), dNTP concentration (1mM, 1.4mM, 1.8mM, 2.2mM), and reverse transcriptase concentration (5U, 7.5U, 10U, 12.5U). In order to maximize the efficiency of real-time fluorescence LAMP amplification, the five important parameters of the real-time fluorescence LAMP reaction were optimized, and the condition that the earliest S-type positive amplification curve appeared was selected as the optimal reaction parameter of the detection system. The experimental results are shown in Figure 2. Figures 4 to 8 shown.

[0077] like Figures 4 to 8As shown in the figure, the optimal reaction parameters of real-time fluorescent LAMP for different pathogens are slightly different: the Tp value of real-time fluorescent LAMP is the lowest when the dNTP concentration is 1.8mM, the MgSO4 concentration is 8mM, the BstⅡPro DNA polymerase concentration is 12U, the reverse transcriptase concentration is 7.5U, and the reaction temperature is 65℃. The Tp value of canine astrovirus real-time fluorescent LAMP reaction system is the lowest when the reverse transcriptase concentration is 10U; the Tp value of canine norovirus real-time fluorescent LAMP reaction system is the lowest when the dNTP concentration is 1.4mM. The difference of canine parvovirus real-time fluorescent LAMP lies in two points: (1) Since canine parvovirus is a DNA virus, the reaction system does not involve the use of reverse transcriptase; (2) The Tp value of this method is the lowest when the MgSO4 concentration is 6mM.

[0078] However, for the convenience of subsequent experiments, the optimal reaction system of unified real-time fluorescence LAMP is 25 μL, which includes primers and LAMP reaction solution; wherein, in the LAMP reaction system, FIP and BIP primers are both 1.6 μM, F3 and B3 primers are both 0.2 μM, and loop primers are both 0.8 μM; in the LAMP reaction system, the LAMP reaction solution is: 10x isothermalAmpBuffer 2.5 μL, dNTP 1.8 mM, MgSO4 8 mM, reverse transcriptase 10 U, BstⅡPro DNA polymerase 12 U, 50x SYBRGREEN dye 0.5 μL, nucleic acid 5 μL and appropriate amount of ddH2O; the optimal reaction conditions are: 60 min at 65 °C, collecting fluorescence signals every 60 seconds, and finally heating to 90 °C to terminate the reaction.

[0079] Example 3 - Fabrication of microfluidic chip

[0080] This embodiment realizes multi-target LAMP amplification detection and performs qualitative and quantitative detection of the target. A microfluidic chip is combined with the LAMP reaction system constructed in Example 2 to perform simultaneous detection of canine distemper virus, canine parvovirus, canine coronavirus, canine rotavirus, canine astrovirus, canine norovirus, and canine cristae virus.

[0081] The production process of the above-mentioned microfluidic chip is as follows Figure 1 As shown, it uses precision mold opening / injection molding to complete the production of large quantities of chips. The structure and operation of the POCT optical disc microfluidic chip prepared by it are briefly described as follows:

[0082] (1) The microfluidic chip is a circular reaction chip composed of four reaction chambers. Under each reaction chamber, there are 8 micro reaction chambers (amplification detection pools). Each micro reaction chamber (amplification detection pool) is physically isolated, thereby eliminating the risk of cross contamination or interference between different reactions. Each of the above reaction chambers mainly includes four parts: a sample injection pool, a distribution pool, an amplification detection pool, and a capillary micro valve, wherein the sample injection pool, the distribution pool, the capillary micro valve, and the amplification detection pool are connected in sequence. Figure 2 As shown. a represents the injection pool: the nucleic acid extract enters the microfluidic chip from here and is used for subsequent testing; b represents the distribution pool: the distribution pool is used to evenly distribute the samples in the injection pool during the first round of centrifugation; c represents the amplification detection pool: the total volume is about 3μL (8 amplification detection pools / sample), and one chip simultaneously completes the detection of 2 samples; d represents the capillary microvalve: the capillary microvalve mainly uses the blocking effect of the capillary on the liquid to control the movement of the fluid in the chip.

[0083] (2) Primer embedding: Taking CPV as an example, first dilute the primers FIP / BIP, F3 / B3 and LB to 1.6μM, 0.2μM and 0.8μM respectively, and mix them in equal amounts. Use a micro-spotter to evenly spot 2μL of the primer mixture on the microfluidic chip for detecting CPV. Then place the chip in a 37℃ constant temperature drying oven for 30 minutes. After ensuring that it is completely dry, apply a sealing film to seal the chip. The primer embedding steps for other pathogens are the same as above.

[0084] (3) In order to detect seven canine enteroviruses, the reaction holes of the microfluidic chip were rationally arranged, as follows: Figure 3As shown: A1~A7 are respectively the canine distemper virus detection hole, canine parvovirus detection hole, canine coronavirus detection hole, canine rotavirus detection hole, canine astrovirus detection hole, canine norovirus detection hole, and canine cristal virus detection hole; B1~B7 are respectively the canine distemper virus detection hole, canine parvovirus detection hole, canine coronavirus detection hole, canine rotavirus detection hole, canine astrovirus detection hole, canine norovirus detection hole, and canine cristal virus detection hole; C1~C7 are respectively the canine distemper virus positive control hole, canine Positive control wells for parvovirus, positive control wells for canine coronavirus, positive control wells for canine rotavirus, positive control wells for canine astrovirus, positive control wells for canine norovirus, and positive control wells for canine crest virus; D1 to D7 are respectively negative control wells for canine distemper virus, negative control wells for canine parvovirus, negative control wells for canine coronavirus, negative control wells for canine rotavirus, negative control wells for canine astrovirus, negative control wells for canine norovirus, and negative control wells for canine crest virus; wherein A8, B8, C8, and D8 are abandoned. Among them, each detection well and the negative and positive control wells are pre-packaged with corresponding primers for detecting each virus, which use the collected sample as the detection sample, and each primer and its LAMP reaction system are respectively as shown in Example 1 and Implementation 2; corresponding primers are pre-packaged in all detection wells, the sample detection well uses the extracted sample nucleic acid as the detection sample, the positive control detection well uses the positive plasmid as the detection sample, and the negative control detection well uses ddH2O as the detection sample.

[0085] The microfluidic chip transfers the sample to be tested to the detection hole of the microfluidic chip at 6000rpm for 30s for LAMP reaction. The detection steps include: collecting the sample to be tested, obtaining nucleic acid extracts using a viral nucleic acid extraction kit; adding a pre-configured LAMP reaction solution containing nucleic acid extracts (10x isothermalAmp Buffer 2.5μL, dNTP1.8mM, MgSO4 8mM, reverse transcriptase 10U, BstⅡPro DNA polymerase 12U, 50x SYBR 0.5 μL of GREEN dye, 5 μL of nucleic acid and an appropriate amount of ddH2O) are added to the sample injection pool of each reaction chamber in the microfluidic chip in 25 μL, and the sample is placed in the detection device after packaging. The liquid flows into the amplification detection pool under the action of centrifugal force to carry out LAMP reaction (the reaction conditions are as described in Example 2), wherein the primers are pre-packaged in the amplification detection pool; the detection result is determined according to the fluorescent signal of each amplification detection pool collected in real time during the LAMP reaction, and the determination standard of the detection result is: if an "S"-shaped amplification curve appears within the detection time, it can be determined as positive, otherwise it is negative.

[0086] Example 4-LAMP microfluidic chip detection system reaction sensitivity test

[0087] In this example, the positive plasmid or the ssRNA transcribed from the positive plasmid was diluted in a gradient manner (103 copies / μL~10 0 copies / μL) were used as templates to carry out a sensitivity test of the microfluidic chip method, which used the LAMP reaction system of Example 2 and the microfluidic chip of Example 3.

[0088] The positive plasmid and the ssRNA transcribed in vitro by the plasmid were serially diluted to 10 3 copies / μL~10 0 copies / μL, and used this as a template to add 5uL of the corresponding detection module of the microfluidic chip. The lowest template concentration that resulted in an S-shaped positive amplification curve was used as the minimum detection limit of the LAMP microfluidic chip detection method. The results are shown in Figures 9 to 15 shown.

[0089] The above experimental results show that Fig. 9 As shown in Figure 2, the minimum detection limit of canine parvovirus detection module in the microfluidic chip is 10 1 copies / μL; Fig.10 As shown in Figure 2, the minimum detection limit of the canine distemper virus detection module in the microfluidic chip is 10 1 copies / μL; Fig.11 As shown in Figure 2, the minimum detection limit of the canine coronavirus detection module in the microfluidic chip is 10 1 copies / μL; Fig.12 As shown in Figure 2, the lowest detection limit of canine astrovirus detection module in the microfluidic chip is 10 1 copies / μL; Fig.13 As shown in Figure 2, the lowest detection limit of canine rotavirus detection module in the microfluidic chip is 10 1 copies / μL; Fig.14 As shown in Figure 2, the lowest detection limit of the canine crest virus detection module in the microfluidic chip is 10 1 copies / μL; Fig.15 The lowest detection limit of canine norovirus detection module in the microfluidic chip is 10 1 copies / μL.

[0090] Example 5-LAMP microfluidic chip detection system reaction specificity test

[0091] In this example, nucleic acids of positive samples of canine rotavirus, canine astrovirus, canine coronavirus, canine distemper virus, canine parvovirus, canine crest virus, and canine norovirus collected clinically were used as templates; among them, sick dogs with intestinal symptoms were selected, and sterile cotton swabs were inserted into the anus, rotated 2 to 3 times to scrape feces, and then placed in a centrifuge tube containing an appropriate amount of PBS, shaken and mixed, and stored at -80°C for later use. Sample nucleic acid was extracted using the MAGEN virus nucleic acid extraction kit.

[0092] The LAMP reaction system of Example 2 and the microfluidic chip of Example 3 were used for detection. The sample nucleic acid was added to the prepared LAMP reaction system, a total of 25uL, and added to the corresponding detection modules of the microfluidic chip. The specificity of the method was evaluated based on whether other viral nucleic acids could be detected. The results are as follows: Fig.16 shown.

[0093] It can be seen from the above experimental results that the amplification reaction will only occur when the positive nucleic acid of the corresponding detection module is used as a template for detection, and there is no peak in the other samples. This shows that the LAMP microfluidic chip detection method has strong amplification specificity.

[0094] Example 6 - Validation of clinical samples

[0095] In this example, canine anal swabs were collected from different pet hospitals and canine quarantine centers in Shanghai for nucleic acid extraction. The LAMP microfluidic chip detection method (using the LAMP reaction system of Example 2 and the microfluidic chip of Example 3) and fluorescent quantitative PCR were used for detection, and the detection results of the two methods were compared to calculate the coincidence rate of the two methods.

[0096] Source of clinical samples: Clinical canine anal swab samples were collected from different pet hospitals and canine quarantine centers in Shanghai at different times; the sample collection process is as follows: select sick dogs with clinical intestinal symptoms, insert sterile cotton swabs into the anus, rotate 2 to 3 times to scrape the feces, and then put them into a centrifuge tube containing an appropriate amount of PBS and shake to mix, and store at -80℃ for later use. Use the MAGEN virus nucleic acid extraction kit to extract sample nucleic acid. Add the sample nucleic acid to the prepared LAMP reaction system for a total of 25uL, and then add the corresponding detection modules of the microfluidic chip for LAMP amplification detection, and then judge the test results. Some of the test results are as follows Fig.17 shown.

[0097] Canine anal swab samples collected from various pet hospitals and quarantine sites in Shanghai were tested for 7 types of canine enteroviruses and compared with the positive coincidence rate of the same fluorescence quantitative PCR method. The results are shown in the table below.

[0098] Table 2 - Real-time fluorescence LAMP clinical sample verification

[0099]

[0100] It can be seen from the above results that the LAMP fluorescence detection method established by the present invention has a high coincidence rate with the real-time fluorescence quantitative PCR method. The coincidence rate of positive samples of canine distemper virus, canine astrovirus, and canine rotavirus reached 100%, and the coincidence rates of canine parvovirus, canine coronavirus, canine crest virus, and canine norovirus reached 97.65%, 90.48%, 94.44%, and 90.00%, respectively.

[0101] It can be seen from the above embodiments that in the early stage of the present invention, the positive plasmids or ssRNAs of the genomes of seven canine intestinal pathogens were used as templates to optimize and construct the LAMP method, and on this basis, a microfluidic chip detection disk was designed and developed, and finally the effectiveness of the method was verified by clinical positive samples. The experimental results all show that the LAMP microfluidic chip detection method constructed by the present invention can simultaneously detect seven canine intestinal pathogens, and has high sensitivity, strong specificity and short reaction time. It can be used for rapid clinical screening in primary pet hospitals, as well as for accurate diagnosis in professional laboratories, and can be applied to epidemiological surveys and disease prevention and control of canine enterovirus.

[0102] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer composition for detecting canine enterovirus, characterized in that: The canine enterovirus comprises at least one of canine rotavirus, canine astrovirus, canine coronavirus, canine distemper virus, canine parvovirus, canine cristae virus, and canine norovirus; wherein the primer composition comprises at least one of the following LAMP primers: LAMP primers for detecting canine rotavirus: sequences shown in SEQ ID No. 1 to SEQ ID No. 5; LAMP primers for detecting canine astrovirus: sequences shown in SEQ ID No.6 to SEQ ID No.11; LAMP primers for detecting canine coronavirus: sequences shown in SEQ ID No.12 to SEQ ID No.17; LAMP primers for detecting canine distemper virus: sequences shown in SEQ ID No.18 to SEQ ID No.23; LAMP primers for detecting canine parvovirus: sequences shown in SEQ ID No. 24 to SEQ ID No. 28; LAMP primers for detecting canine crest virus: sequences shown in SEQ ID No. 29 to SEQ ID No. 34; LAMP primers for detecting canine norovirus: sequences shown as SEQ ID No.35 to SEQ ID No.

39.

2. A LAMP reaction system comprising the primer combination according to claim 1, characterized in that: In the LAMP reaction system, the concentrations of the FIP primer and the BIP primer were both 1.6 μM, the concentrations of the F3 primer and the B3 primer were both 0.2 μM, and the concentrations of the loop primers were both 0.8 μM.

3. The LAMP reaction system according to claim 2, characterized in that: The LAMP reaction system also includes a LAMP reaction solution; wherein, based on the total volume of the LAMP reaction system being 25 μL: The LAMP reaction solution used to detect canine rotavirus, canine astrovirus, canine coronavirus, canine distemper virus, canine parvovirus, canine crest virus or canine norovirus includes 10x isothermalAmp Buffer 2.5μL, dNTP 1.8mM, MgSO48mM, reverse transcriptase 10U, BstⅡPro DNA polymerase 12U, 50x SYBR GREEN dye 0.5μL, nucleic acid 5μL and an appropriate amount of ddH2O.

4. A kit for detecting canine enterovirus, characterized in that: The kit contains the primer composition according to claim 1, or contains the LAMP reaction system according to any one of claims 3 to 4.

5. The kit according to claim 4, characterized in that The kit also includes a microfluidic chip, a negative control substance, and a positive control substance.

6. The kit according to claim 5, characterized in that The microfluidic chip is provided with 4 independent reaction chambers, each reaction chamber comprises a sampling pool, a distribution pool, a capillary microvalve, and an amplification detection pool which are connected in sequence, and each reaction chamber has 8 amplification detection pools; wherein, two reaction chambers are provided with canine distemper virus detection holes, canine parvovirus detection holes, canine coronavirus detection holes, canine rotavirus detection holes, canine astrovirus detection holes, canine norovirus detection holes, and canine cristae virus detection holes; one reaction chamber is provided with a corresponding positive control hole; and one reaction chamber is provided with a corresponding negative control hole.

7. The kit according to claim 5, characterized in that A predetermined amount of primers is pre-packaged in the amplification detection pool of the microfluidic chip.

8. A LAMP microfluidic chip detection method for detecting canine enterovirus, characterized in that: The LAMP microfluidic chip detection method adopts the kit described in any one of claims 4 to 7, comprising the steps of: S1. Collect samples to be tested and obtain nucleic acid extracts using a viral nucleic acid extraction kit; S2, adding the pre-configured LAMP reaction liquid containing the nucleic acid extract to the injection pool of each reaction chamber in the microfluidic chip, packaging it and placing it in the detection device, and the liquid flows into the amplification detection pool under the action of centrifugal force to perform LAMP reaction, wherein a predetermined amount of primers are pre-packaged in the amplification detection pool; S3. Determine the detection result according to the fluorescent signal of each amplification detection pool collected in real time by the LAMP reaction.

9. The LAMP microfluidic chip detection method according to claim 8, characterized in that: In step S1, the sample to be detected is a stool sample; In step S2, 25 μL of LAMP reaction solution is added to the sample injection pool of each reaction chamber; the reaction procedure of the LAMP reaction is: transferring the sample to be tested to the detection hole of the microfluidic chip at 6000 rpm for 30 seconds; performing at 65° C. for 60 minutes, and collecting the fluorescence signal every 60 seconds; In step S3, the judgment standard of the test result is: if an "S"-shaped amplification curve appears within the detection time, it can be judged as positive, otherwise it is negative.

10. Use of the kit according to any one of claims 4 to 7 in detecting canine enterovirus, characterized in that: The canine enterovirus includes canine rotavirus, canine astrovirus, canine coronavirus, canine distemper virus, canine parvovirus, canine crest virus, and canine norovirus.

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