LAMP (loop-mediated isothermal amplification) primer group, micro-fluidic chip, kit and method for rapidly detecting five canine tick pathogens

Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) primer group, a micro-fluidic chip, a kit and a method for rapidly detecting five canine tick pathogens, and belongs to the technical field of biology. The LAMP primer composition comprises a primer group used for detecting canine liver cluster worms, a primer group used for detecting canine babesia canis, a primer group used for detecting canine babesia gibsoni, a primer group used for detecting canine Ehrlick and a primer group used for detecting anaplasma planus. The LAMP freeze-dried microspheres contain one primer group in the primer composition, the LAMP freeze-dried microspheres are contained in different reaction cavities of the micro-fluidic chip, and the kit comprises the micro-fluidic chip. The method comprises the following steps: carrying out loop-mediated isothermal amplification reaction on a to-be-detected sample by adopting the micro-fluidic chip or the kit, and determining the canine tick pathogen infection condition of the to-be-detected sample according to a generated fluorescence curve. According to the invention, simultaneous rapid detection of five canine tick pathogens can be realized.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a LAMP primer set, a microfluidic chip, a kit and a method for rapidly detecting five canine tick pathogens. Background Art

[0002] With the increasing pressure of modern life and the improvement of living standards, more and more pets have become people's life partners. Among them, pet dogs are very popular and suitable for family breeding. Pet dogs like to stay in areas where feces gather, such as grasslands, and are easily bitten by ticks (also known as dog beans or ticks), which can cause tick disease. Common tick-borne blood protozoan diseases include babesiosis, ehrlichiosis, anaplasmosis and hepatozoonosis. The main infectious pathogens of these canine blood infections are canine hepatozoons ( Hepatozoon canis ), Babesia canis ( Babesia canis ), Babesia canis ( Babesia gibsoni )、Erica canis( Ehrlichia canis )、Flat pulpless body( Anaplasma platys ). The above-mentioned tick-borne blood protozoan diseases are highly contagious and have a high incidence rate. After being infected with blood protozoan diseases, dogs' resistance is reduced and they may be infected with other viral diseases. Therefore, the simultaneous detection of these canine pathogens is conducive to obtaining rapid diagnostic results, which is crucial to preventing the spread of the disease.

[0003] At present, the main methods for detecting tick-borne blood protozoan diseases include blood smear microscopy, immunoassay and molecular biology methods. Among them, blood smear microscopy is to identify species from a morphological perspective, but it is very difficult to identify specific insect species, and the quality of smear preparation and staining will also affect the detection limit and accuracy of the experimental results. The immunoassay detects the target protein through the specific binding of antigen and antibody. The immunoassay detection reagent mainly detects the antibodies produced after infection by the pathogen or directly detects the specific antigen of the pathogen through a certain method. This detection method has a long window period and has limited effect on early screening of the disease. At the same time, its sensitivity and detection specificity are relatively low, which is easy to produce misjudgment of the results. Molecular biological methods include PCR, real-time fluorescence quantitative PCR (Quantitative Real-time PCR), etc. Among them, the PCR method mainly amplifies the specific gene of the target pathogen, detects the fragment length, or adds fluorescent substances to the reaction to achieve real-time detection of pathogens. However, this method has high requirements for equipment and site, requires professional operators, and has cumbersome operation steps. If disinfection and ventilation measures are not taken, it is easy to cause nucleic acid contamination, thereby affecting the accuracy of the test results.

[0004] Loop-mediated isothermal amplification (LAMP) is a new in vitro isothermal amplification technology for specific nucleic acid fragments. It is characterized by designing 4-6 specific primers for the 6 regions of the target gene, and performing constant temperature amplification at 60-65°C under the action of chain displacement DNA polymerase. The results can be determined within 15-30 minutes. It has the characteristics of simple operation, strong specificity, easy product detection, and low equipment requirements.

[0005] Microfluidics integrates basic operating units such as sample preparation, biological and chemical reactions, separation, and detection involved in the fields of biology and chemistry into chips to complete different biological or chemical reaction processes and analyze their products. It has the characteristics of "sample in, result out". Among them, the most commonly used method in microfluidics is centrifugal microfluidics, which can manipulate liquids on a submillimeter scale by rotating centrifugal microfluidics chips. Compared with the traditional method of extracting nucleic acids manually or using nucleic acid extractors, the use of centrifugal microfluidics to extract nucleic acids can complete all reaction processes in the chip chambers and pipes, effectively preventing the possibility of leakage of amplified products or samples, greatly reducing the possibility of nucleic acid contamination, and at the same time, it has the characteristics of good liquid flow controllability, fast analysis speed, and minimal consumption of samples and reagents, which can also significantly reduce the space requirements of experimental sites. Therefore, the automatic completion of nucleic acid analysis processes such as amplification and detection on microfluidic chips through microfluidics has become an economical and rapid molecular diagnostic method.

[0006] In summary, based on the current status of canine tick-borne hematoprotozoan infection, how to combine loop-mediated isothermal amplification technology with microfluidics technology to seek a detection method that can simultaneously and rapidly detect five canine tick pathogens with high detection sensitivity and specificity has become an urgent problem to be solved. Summary of the invention

[0007] In order to overcome the shortcomings of the prior art, one of the objects of the present invention is to provide a LAMP primer set for rapid detection of five canine tick pathogens, which are used to detect Hepatozoon canis, Babesia canis, Babesia gibbsii, Ehrlichia canis and Anaplasma flatulum.

[0008] To solve the above problems, the technical solution adopted by the present invention is as follows: A LAMP primer composition for simultaneously and rapidly detecting five canine tick pathogens, comprising a primer set for detecting Hepatozoon canis, a primer set for detecting Babesia canis, a primer set for detecting Babesia gibbsii, a primer set for detecting Ehrlichia canis, and a primer set for detecting Anaplasma planum; wherein, The primer set for detecting Hepatozoon canis includes: outer primers Hepca-F3 and Hepca-B3, whose nucleotide sequences are shown in SEQ ID NOs. 1 and 2, respectively; inner primers Hepca-FIP and Hepca-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 3 and 4, respectively; and loop primer Hepca-LB, whose nucleotide sequence is shown in SEQ ID NO. 5; The primer set for detecting Babesia canis includes: outer primers Babeca-F3 and Babeca-B3, whose nucleotide sequences are shown in SEQ ID NOs. 6 and 7, respectively; inner primers Babeca-FIP and Babeca-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 8 and 9, respectively; loop primers Babeca-LF and Babeca-BF, whose nucleotide sequences are shown in SEQ ID NOs. 10 and 11, respectively; The primer set for detecting Babesia gibbsii of dogs comprises: outer primers Babegi-F3 and Babegi-B3, whose nucleotide sequences are shown in SEQ ID NOs. 12 and 13, respectively; inner primers Babegi-FIP and Babegi-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 14 and 15, respectively; and loop primer Babegi-LB, whose nucleotide sequence is shown in SEQ ID NO. 16; The primer set for detecting Ehrlichia canis includes: outer primers Ehrca-F3 and Ehrca-B3, whose nucleotide sequences are shown in SEQ ID NOs. 17 and 18, respectively; inner primers Ehrca-FIP and Ehrca-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 19 and 20, respectively; and loop primer Ehrca-LF, whose nucleotide sequence is shown in SEQ ID NO. 21; The primer set for detecting flat anaplasma includes: outer primers AnaP-F3 and AnaP-B3, whose nucleotide sequences are shown in SEQ ID NOs. 22 and 23, respectively; inner primers AnaP-FIP and AnaP-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 24 and 25, respectively; and loop primer AnaP-LB, whose nucleotide sequence is shown in SEQ ID NO. 26.

[0009] As a preferred embodiment of the present invention, the primer set for detecting Hepatozoon canis is designed for the conservative region of the 18S rRNA gene sequence of Hepatozoon canis; the primer set for detecting Babesia canis is designed for the conservative region of the 18S rRNA gene sequence of Babesia canis; the primer set for detecting Babesia gibbsii is designed for the conservative region of the 18S RNA gene sequence of Babesia gibbsii; the primer set for detecting Ehrlichia canis is designed for the conservative region of the 16S rRNA gene sequence of Ehrlichia canis; the primer set for detecting Anaplasma planum is designed for Anaplasma planum gltA Design of conserved regions of gene sequences.

[0010] The second object of the present invention is to provide a LAMP freeze-dried microsphere for rapid simultaneous detection of five canine tick pathogens, wherein the LAMP freeze-dried microsphere contains a primer set in the primer composition as described above.

[0011] As a preferred embodiment of the present invention, the LAMP freeze-dried microspheres include 4X LAMP Buffer, Bst DNA polymerase, MgSO 4 , 5XSYTO9 fluorescent dye, 25XLAMP Primer Mix, mannitol, trehalose, bovine serum albumin and PEG20000; the 25XLAMP Primer Mix is ​​a primer mix prepared by mixing any one of the primer sets of Hepatozoon canis, the primer set of Babesia canis, the primer set of Babesia gibbsii, the primer set of Ehrlichia canis, the primer set of Anaplasma flatulum and the primer set of the internal reference gene (ACTB gene) of canine at a concentration 10 times the final concentration of the primers when used; the final concentration of the inner primers FIP / BIP in each of the primer sets is 0.8-1.6 μM, the final concentration of the loop primers LF / LB is 0.2-0.4 μM, and the final concentration of the outer primers F3 / B3 is 0.1-0.2 μM.

[0012] A third object of the present invention is to provide a microfluidic chip for simultaneously and rapidly detecting five canine tick pathogens, wherein different reaction chambers of the microfluidic chip contain the above-mentioned LAMP freeze-dried microspheres.

[0013] As a preferred embodiment of the present invention, the microfluidic chip is provided with a reagent tank, a sample processing tank, a nucleic acid extraction area, a nucleic acid dilution area, a quantitative filling area and a reaction detection area which are connected to each other; the nucleic acid extraction area and the quantitative filling area are respectively connected to a waste liquid tank through a microfluidic channel; the nucleic acid dilution area, the quantitative filling area and the waste liquid tank are respectively connected to the atmosphere through a microfluidic channel, and the reaction chamber of the reaction detection area has a transparent area for fluorescence detection.

[0014] As a preferred embodiment of the present invention, the reagent tank includes a first cleaning liquid tank, a second cleaning liquid tank, an elution liquid tank and a dilution liquid tank, each of the reagent tanks is provided with an opening on the same centrifugal radius, and the opening is filled with paraffin.

[0015] A fourth object of the present invention is to provide a kit for simultaneously and rapidly detecting five canine tick pathogens, the kit comprising the microfluidic chip as described above.

[0016] A fifth object of the present invention is to provide a method for simultaneously and rapidly detecting five canine tick pathogens for non-disease diagnosis purposes, the method comprising performing a loop-mediated isothermal amplification reaction on a test sample using the microfluidic chip as described above or the kit as described above, and determining the canine tick pathogen infection status of the test sample based on the generated fluorescence curve.

[0017] As a preferred embodiment of the present invention, the method specifically comprises the following steps: S1. Add 100-200 μL of dog blood to the mixture of lysate and proteinase K, mix well and add to the sample processing tank of the microfluidic chip, heat at 54-58°C for 10-15 minutes, and centrifuge at 2500-3000 rpm for 60-90 seconds to allow the lysed sample to pass through the nucleic acid extraction column in the nucleic acid extraction area, and then the waste liquid enters the waste liquid area; S2, using laser to irradiate the paraffin in the opening of the first cleaning liquid tank to melt it and release the first cleaning liquid, centrifuging at 2500-3000 rpm for 60-90 seconds, and the first cleaning liquid enters the waste liquid area through the nucleic acid extraction column; using laser to irradiate the paraffin in the opening of the second cleaning liquid tank to melt it and release the second cleaning liquid, centrifuging at 2500-3000 rpm for 120-180 seconds, and the second cleaning liquid enters the waste liquid area through the nucleic acid extraction column to complete the nucleic acid purification; S3, using laser to irradiate the paraffin in the opening of the eluent tank to melt it and release the eluent, centrifuging at 2500-3000rpm for 30-60s, so that the eluent elutes the nucleic acid adsorbed on the nucleic acid extraction column and enters the nucleic acid extraction area under the action of centrifugation; using laser to irradiate the paraffin in the opening of the diluent tank to melt it and release the diluent, centrifuging at 2500-3000rpm for 30-60s, so that the diluted nucleic acid sample liquid enters the reaction chamber in the reaction detection area; S4. The nucleic acid sample solution enters the reaction chamber to re-dissolve the preset LAMP freeze-dried microspheres to form a LAMP reaction system. The LAMP reaction system is amplified at a constant temperature of 62-67°C. As the reaction proceeds, the fluorescent signal is used to determine whether the sample contains canine tick pathogens.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The primer combination provided by the present invention has strong specificity, and the time required for LAMP amplification is short, which further shortens the detection time and simplifies the operation. The kit made by the primer combination can quickly and accurately detect whether the sample contains five canine tick pathogens, including hepatozoon canis, babesia canis, babesia gibbsii, errichosis canis, and flat anaplasma. The present invention prepares LAMP freeze-dried microspheres by using the primer set of each pathogen and the LAMP fluorescence detection reagent. The LAMP freeze-dried microspheres can be stored at room temperature, which can greatly reduce the transportation and storage costs of the reagents. At the same time, the present invention combines the LAMP freeze-dried microspheres for detecting five pathogens with a microfluidic chip based on the LAMP technology, and can achieve the purpose of rapid detection of five common tick-transmitted pathogens of dogs simultaneously by single addition of samples. No human intervention is required for the whole process, and the whole detection process only takes 1 hour, with high detection sensitivity and specificity, which can well meet the market demand for rapid detection of pet pathogens, and provide strong technical support for early diagnosis and monitoring of canine pathogen infection, and can also effectively prevent the possibility of cross-species transmission.

[0019] The detection method of the present invention requires simple equipment, occupies a small space, is highly automated, does not require professional operators and analysts, and the entire detection process can be completed in an ordinary laboratory. Based on the above characteristics, the present invention can be used in ordinary laboratories without professional nucleic acid detection laboratories, pet clinics with poor basic conditions, etc., and has obvious advantages in reducing sample transportation costs, reducing detection time and detection costs, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a fluorescence intensity diagram of the detection sensitivity test of the present invention; wherein the sample plasmid concentration of 1a is 10 6 copies / mL, the sample plasmid concentration of 1b is 10 5 copies / mL, the sample plasmid concentration of 1c is 10 4 copies / mL, and the sample plasmid concentration for 1 day was 10 3 copies / mL, the sample plasmid concentration of 1e is 10 2 copies / mL.

[0021] Figure 2 This is a fluorescence intensity diagram of the detection specificity test of the present invention; wherein, the sample 2a contains Hepatozoon canis DNA, the sample 2b contains Babesia canis DNA, the sample 2c contains Babesia gibbsii DNA, the sample 2d contains Ehrlichia canis DNA, and the sample 2e contains Anaplasma flatulum DNA.

[0022] Figure 3This is a fluorescence intensity diagram of the detection specificity test of the present invention; wherein, the sample 3a contains bovine Babesia DNA, the sample 3b contains Escherichia coli DNA, the sample 3c contains Staphylococcus aureus DNA, the sample 3d contains Toxoplasma gondii DNA, the sample 3e contains canine parvovirus DNA, and the sample 3f contains canine distemper virus DNA.

[0023] Figure 4 This is a fluorescence intensity diagram of samples No. 1 to No. 12 in the positive sample detection test of the present invention. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with specific implementation modes.

[0025] 1. Design and sequence information of LAMP primers The present invention is directed against Babesia canis (including Babesia canis canis , Babesia canis vogeli and Babesia rossi ) were downloaded and compared, and the conserved sequence of Babesia canis was selected. The LAMP specific primers were designed using the LAMP online primer design software (Primer Explorer V5). Babesia gibsoni ) of the 18S RNA gene sequence, Ehrlichia canis ( Ehrlichia canis , E. canis ) of the 16S rRNA gene sequence, Hepatozoon canis ( Hepatozoon canis ) of the conserved region of the 18S rRNA gene sequence, the flat anaplasma ( Anaplasma platys )of gltA LAMP-specific primers were designed in the conserved region of the gene sequence. Each set of LAMP-specific primers contained two outer primers (F3 and B3), two inner primers (FIP and BIP) and one or two loop primers (LF / LB).

[0026] Specifically, the primer set for detecting Hepatozoon canis includes: outer primers Hepca-F3 and Hepca-B3, whose nucleotide sequences are shown in SEQ ID NOs. 1 and 2, respectively; inner primers Hepca-FIP and Hepca-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 3 and 4, respectively; loop primer Hepca-LB, whose nucleotide sequence is shown in SEQ ID NO. 5; The primer set for detecting Babesia canis includes: outer primers Babeca-F3 and Babeca-B3, whose nucleotide sequences are shown in SEQ ID NOs. 6 and 7, respectively; inner primers Babeca-FIP and Babeca-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 8 and 9, respectively; loop primers Babeca-LF and Babeca-BF, whose nucleotide sequences are shown in SEQ ID NOs. 10 and 11, respectively; The primer set for detecting Babesia gibbsii of dogs comprises: outer primers Babegi-F3 and Babegi-B3, whose nucleotide sequences are shown in SEQ ID NOs. 12 and 13, respectively; inner primers Babegi-FIP and Babegi-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 14 and 15, respectively; and loop primer Babegi-LB, whose nucleotide sequence is shown in SEQ ID NO. 16; The primer set for detecting Ehrlichia canis includes: outer primers Ehrca-F3 and Ehrca-B3, whose nucleotide sequences are shown in SEQ ID NOs. 17 and 18, respectively; inner primers Ehrca-FIP and Ehrca-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 19 and 20, respectively; and loop primer Ehrca-LF, whose nucleotide sequence is shown in SEQ ID NO. 21; The primer set for detecting flat anaplasma includes: outer primers AnaP-F3 and AnaP-B3, whose nucleotide sequences are shown in SEQ ID NOs. 22 and 23, respectively; inner primers AnaP-FIP and AnaP-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 24 and 25, respectively; and loop primer AnaP-LB, whose nucleotide sequence is shown in SEQ ID NO. 26.

[0027] The LAMP primer set sequences for each of the above pathogens are shown in Table 1 below.

[0028] Table 1 Sequence information of LAMP primers for five tick-transmitted pathogens in dogs

[0029] The final concentrations of the primers for each pathogen in the LAMP reaction system are: 0.8-1.6 μM for the inner primer FIP / BIP, 0.2-0.4 μM for the loop primer LF / LB, and 0.1-0.2 μM for the outer primer F3 / B3. In the actual reaction, for ease of operation, 5-6 primers for each pathogen can be prepared into a 25X Primer Mix for use.

[0030] 2. Composition and formulation of LAMP freeze-dried microspheres LAMP freeze-dried microspheres are prepared by freeze-drying the LAMP reaction solution.

[0031] The LAMP reaction solution contains 4X LAMP Buffer, Bst DNA polymerase, MgSO 4 , 5XSYTO9 fluorescent dye, 25XLAMP Primer Mix, mannitol, trehalose, bovine serum albumin and PEG20000. Among them, 25XLAMP Primer Mix refers to the primer mix of 5-6 LAMP primers of Hepatozoon canis, Babesia canis, Babesia gibbsii, Ehrlichia canis, Anaplasma flatulum and the internal reference gene (ACTB gene) of canine, at 10 times the concentration. 4X LAMP Buffer contains 2-8mM dNTPs, 60-100mM Tris HCl, 30-50mM (NH 4 ) 2 SO 4 , 50-300mM KCl, 0.2%-0.5% (volume percentage) Tween-20, 1-4M betaine. MgSO 4 The final concentration of is 4-10mM, the concentration of 5XSYTO 9 is 10-50μM. The amount of Bst DNA polymerase used in each LAMP reaction is 4-16U. The mass concentration of trehalose is 5%-15%, the mass concentration of mannitol is 2%-10%, the mass concentration of bovine serum albumin is 0.5-3%, and the mass concentration of PEG2000 is 2%-10%.

[0032] The LAMP reaction solution is freeze-dried to obtain freeze-dried microspheres and pre-placed in the reaction chamber of the microfluidic chip. Different reaction chambers can be pre-placed with LAMP freeze-dried microspheres containing different pathogen primer sets, and the function of microfluidics can be used to achieve the purpose of rapid detection of five pathogens at the same time. At the same time, LAMP freeze-dried microspheres containing canine internal reference gene-specific primer sets can also be pre-placed in the reaction chamber of the microfluidic chip as an indicator for evaluating DNA quality and LAMP amplification detection capabilities.

[0033] The preparation method of the above-mentioned LAMP freeze-dried microspheres for detecting each pathogen is as follows: after mixing the components of the above-mentioned LAMP reaction solution according to a proportion, using a ball dropper to drop into microspheres with a volume of 10-20 μL, and then placing the microspheres in a freeze dryer for freeze drying. The following Table 2 shows the freeze drying procedure.

[0034] Table 2 Freeze-drying procedure of freeze-dried microspheres

[0035] 3. Microfluidic Chips and Kits The microfluidic chip includes a covering layer and a chip body stacked from top to bottom, and the covering layer is adapted to the chip body. Among them, the covering layer can be a cover plate or a covering film, preferably a covering film, and the covering film is used to seal the chip body, which can achieve rapid closure and low cost, and the covering film is a pressure-sensitive film or a heat-sensitive film. Specifically, the chip body includes a reagent tank, a sample processing tank, a nucleic acid extraction area, a nucleic acid dilution area, a quantitative subpackaging area and a reaction detection area connected in sequence through a microchannel, and the nucleic acid extraction area and the quantitative subpackaging area are respectively connected to a waste liquid tank through a microchannel; the sample lysis area, the nucleic acid extraction area, the nucleic acid dilution area, the quantitative subpackaging area and the waste liquid tank are respectively connected to the atmosphere through a microchannel. The reaction chamber of the reaction detection area has a transparent area for fluorescence detection.

[0036] Specifically, the sample and lysate are added to the sample processing tank through the sample addition hole, and the sample and lysate are mixed under the action of centrifugal force. The heating plate installed on the upper and lower surfaces of the microfluidic chip provides a uniform heat source for heating and lysis, thereby releasing the nucleic acid. The heating time and temperature in the above lysis process can be precisely controlled by an external control device.

[0037] Specifically, the nucleic acid extraction area includes a mixing buffer pool and a nucleic acid extraction column arranged from top to bottom in sequence, thereby ensuring that the flow path of the reagent under the action of centrifugal force is unidirectional flow to avoid cross contamination. The reagent tank and the sample processing tank are respectively connected to the mixing buffer pool through microchannels, the mixing buffer pool is connected to the nucleic acid extraction column, and the nucleic acid extraction column is respectively connected to the nucleic acid dilution area and the waste liquid tank through microchannels. Further, the reagent tank includes a first cleaning liquid tank, a second cleaning liquid tank, an elution liquid tank and a dilution liquid tank, each reagent tank is provided with an opening on the same centrifugal radius, and the opening is filled with paraffin to seal the reagent. When used, the paraffin can be melted by laser irradiation and the reagent is released by centrifugation. More specifically, the nucleic acid extraction column includes an extraction chamber with a cylindrical structure and a pressure ring sealed and installed in the extraction chamber, and a small hole connected to the extraction chamber is opened on the bottom wall of the extraction chamber for liquid outflow. An extraction membrane is provided on the bottom wall of the extraction chamber, and the extraction membrane is preferably a silicone mold. The pressure ring is against the extraction membrane and is used to limit the position of the extraction membrane in the flow direction of the liquid, ensuring that the extraction membrane is stable and the fluid flow path is controlled. The nucleic acid extraction column is embedded in the microfluidic chip and combined with the microfluidic channel, and the centrifugal force is used to achieve automatic flow and separation of the liquid, avoiding sample loss and external interference, ensuring high extraction efficiency and stable nucleic acid purity.

[0038] Specifically, the nucleic acid dilution area includes a dilution pool connected to the dilution liquid tank through a microchannel, and the dilution pool is connected to the atmosphere through the microchannel. The dilution pool is connected to the nucleic acid extraction area and the quantitative filling area through the microchannel. The dilution liquid enters the dilution pool under the action of centrifugal force to dilute the eluted nucleic acid, and the diluted nucleic acid sample liquid to be tested enters the quantitative filling area under the drive of centrifugal force.

[0039] Specifically, the quantitative filling area includes a liquid channel and a plurality of quantitative tanks of equal volume arranged at intervals. The liquid channel is connected to the nucleic acid dilution area and the waste liquid tank through microfluidics respectively. Each quantitative tank is connected to the liquid channel respectively, and the nucleic acid sample liquid to be tested is driven into the plurality of quantitative tanks of equal volume by centrifugal force.

[0040] Specifically, the reaction detection area includes a plurality of reaction chambers arranged one by one with the quantitative grooves, and the reaction chamber is connected to the quantitative groove through a microchannel, and the sample enters the reaction chamber from the quantitative groove. In order from left to right, the reaction chamber is pre-installed with LAMP freeze-dried microspheres and fluorescent probes containing primers of canine hepatozoon, canine Babesia, canine Babesia gibbsii, canine Ericella, flat anaplasma and canine internal reference genes. After the sample enters the reaction chamber, the LAMP reagent freeze-dried microspheres can be quickly and automatically redissolved to ensure reaction sensitivity and specificity. In order to avoid evaporation of liquid in the reaction well and to avoid aerosol diffusion of the amplification product, a paraffin tank is provided above the reaction chamber, and the paraffin tank is filled with paraffin. A blocking tank connected to the paraffin tank is provided on the microchannel between the reaction chamber and the quantitative groove. After being heated to a certain temperature, the paraffin melts and flows into the blocking tank above the reaction chamber, and the outlet of the reaction chamber can be sealed.

[0041] The kit provided by the present invention comprises the above-mentioned microfluidic chip.

[0042] 4. Method for rapid simultaneous detection of five canine tick pathogens for non-disease diagnostic purposes The method comprises using the above-mentioned microfluidic chip or kit to perform a loop-mediated isothermal amplification reaction on the sample to be tested, and determining the canine tick pathogen infection status of the sample to be tested based on the generated fluorescence curve. The specific steps are as follows: S1. Add 100-200 μL of dog blood to the mixture of lysate and proteinase K, mix well and add to the sample processing tank of the microfluidic chip, then use the extraction program of the instrument to start the DNA extraction process, heat the sample processing tank at 54-58°C for 10-15 minutes, and then centrifuge at 2500-3000 rpm for 60-90 seconds to make the lysed sample pass through the nucleic acid extraction column in the nucleic acid extraction area, and then the waste liquid enters the waste liquid area; S2, using laser to irradiate the paraffin in the opening of the first cleaning liquid tank to melt it and release the first cleaning liquid, centrifuging at 250-3000 rpm for 60-90 seconds, and the first cleaning liquid enters the waste liquid area through the nucleic acid extraction column; using laser to irradiate the paraffin in the opening of the second cleaning liquid tank to melt it and release the second cleaning liquid, centrifuging at 2500-3000 rpm for 120-180 seconds, and the second cleaning liquid enters the waste liquid area through the nucleic acid extraction column to complete the nucleic acid purification; S3, using laser to irradiate the paraffin in the opening of the eluent tank to melt and release the eluent, centrifuging at 2500-3000rpm for 30-60s, so that the eluent elutes the nucleic acid adsorbed on the nucleic acid extraction column and enters the nucleic acid extraction area under the action of centrifugation. In order to reduce the inhibition of DNA on subsequent reactions, the eluent needs to be diluted a certain multiple. This step is achieved by releasing the diluent into the nucleic acid dilution area and mixing it with the eluent obtained in the previous step. Specifically, laser is used to irradiate the paraffin in the opening of the diluent tank to melt and release the diluent, centrifuging at 2500-3000rpm for 30-60s, so that the diluted nucleic acid sample liquid enters the reaction chamber in the reaction detection area; S4. The nucleic acid sample solution enters the reaction chamber to dissolve the preset LAMP freeze-dried microspheres to form a LAMP reaction system. The LAMP reaction system performs constant temperature amplification at 62-67°C. As the reaction proceeds, the fluorescence signal is used to determine whether the sample contains dog tick pathogens: if an S-type amplification curve appears in the internal reference reaction well within 30 minutes, and an obvious S-type curve or an obvious increase in fluorescence signal appears in the corresponding pathogen detection well within 30 minutes, it can be determined that the sample contains the pathogen; if an S-type amplification curve appears in the internal reference reaction well within 30 minutes, but no obvious S-type curve or no obvious increase in fluorescence signal appears in the corresponding pathogen detection well within 30 minutes, it can be determined that the sample does not contain the pathogen; if no S-type amplification curve appears in the internal reference reaction well within 30 minutes, it means that the DNA extraction has failed or the sample is unqualified, and it is necessary to re-extract the DNA or re-sample and repeat the test.

[0043] 5. Detection sensitivity test In order to test the detection sensitivity of the kit, five plasmids, including Hepatozoon canis, Babesia canis, Babesia gibbsii, Ehrlichia canis and Anaplasma planum, were mixed into dog blood samples to make the concentration of each plasmid in the sample reach 10 6 copies / mL, 10 5 copies / mL, 10 4 copies / mL, 10 3 copies / mL, 10 2 The samples containing different concentrations of plasmids were added to the microfluidic chip, and the five canine tick pathogens and internal reference genes were detected according to the above operation steps. The test results are shown in Tables 3-4 and Figure 1 .

[0044] Among them, the specific composition of the kit and LAMP freeze-dried microspheres used for the detection sensitivity test is as follows: The volume of the first cleaning solution contained in the first cleaning solution tank is 250 μL; the volume of the second cleaning solution contained in the second cleaning solution tank is 250 μL; the volume of the eluting solution contained in the eluting solution tank is 150 μL; and the volume of the diluting solution contained in the diluting solution tank is 250 μL.

[0045] The composition of the LAMP reaction solution is as follows: MgSO 4 The final concentration of 5XSYTO 9 is 6mM, and the concentration of 5XSYTO 9 is 50μM. The amount of Bst DNA polymerase used in each LAMP reaction is 10U. The mass concentration of trehalose is 10%, the mass concentration of mannitol is 5%, the mass concentration of bovine serum albumin is 0.5%, and the mass concentration of PEG2000 is 2%; 4X LAMP Buffer contains 5mM dNTPs, 60mM Tris HCl, 50mM (NH 4 ) 2 SO 4 , 100mM KCl, 0.2% (volume percentage) Tween-20, 2M betaine; the primer group concentration in each 25XLAMP Primer Mix containing any one of the primer sets of Hepatoma canis primer set, Babesia canis primer set, Babesia gibbsii primer set, Ehrlichia canis primer set, Anaplasma flatulum primer set and canine internal reference gene (ACTB gene) primer set is 10 times the final concentration of the primer set when used, and the final concentration of each primer in each primer set in the LAMP reaction system is: 0.8μM for inner primer FIP / BIP, 0.2μM for loop primer LF / LB, and 0.1μM for outer primer F3 / B3.

[0046] The LAMP freeze-dried microspheres are prepared by freeze-drying the above LAMP reaction solution.

[0047] The extraction membrane in the nucleic acid extraction column is a silica gel membrane with a pore size of 0.7 μm.

[0048] Table 3 Test results of kit detection sensitivity

[0049] Table 4 Summary of the results of the kit detection sensitivity

[0050] Note: “+” in the table indicates that the test result is positive and the corresponding pathogen is detected; “-” indicates that the test result is negative and no pathogen is detected.

[0051] Table 3~4 and Figure 1The results showed that the kit of the present invention combined with the microfluidic chip can simultaneously realize the rapid detection of five pathogens, and the detection sensitivity of five tick-transmitted pathogens of dogs can reach 1000 copies / mL.

[0052] 6. Detection Specificity Test In order to confirm whether the detection kit for the five tick-transmitted pathogens of dogs, Hepatozoon canis, Babesia canis, Babesia gibbsii, Ehrlichia canis and Anaplasma planum, can only specifically detect the corresponding pathogens and have no cross-reaction to other pathogens, DNA of Hepatozoon canis, Babesia canis, Babesia gibbsii, Ehrlichia canis and Anaplasma planum, as well as DNA of Babesia bovis ( Babesia bovis ), Escherichia coli ( Escherichia coli , Staphylococcus aureus ( Staphylococcus aureus )、Toxoplasma gondii ( Toxoplasma gondii ), canine parvovirus ( Canine parvovirus )、Canine distemper virus( Canine parvovirus ) DNA, the concentration of the above DNA was 15ng / μL, the DNA of the above pathogens was used as the detection template and added into the detection kits of five pathogens, namely, Hepatozoon canis, Babesia canis, Babesia gibbsii, Ehrlichia canis and Anaplasma flatulum. The kit was completely consistent with the kit used for the detection sensitivity test. The test results are shown in Tables 5-6 and Figure 2~3 shown.

[0053] Table 5 Kit detection specificity results

[0054] Note: Since the target was not detected in the samples containing DNA of Babesia bovis, Escherichia coli, Staphylococcus aureus, Toxoplasma gondii, canine parvovirus, and canine distemper virus, their results are not included in Table 5.

[0055] Table 6 Summary of kit specificity results

[0056] Table 5-6 and Figures 2-3 The results showed that the kit of the present invention has good detection specificity, can detect the target pathogens, and has no non-specific amplification of other common canine pathogens.

[0057] VII. Detection of Positive Samples In order to test the detection of the kit for clinical samples, 12 whole blood samples that tested positive by qPCR were collected. 100 μL of each sample was mixed with lysis buffer and proteinase K and added to the microfluidic chip for detection. The kit was completely consistent with the kit used for the sensitivity test. The qPCR test results of the 12 samples and the test results of the kit of the present invention are shown in Tables 7 to 8 and Figure 4 shown.

[0058] Table 7 Test results of the kit for positive samples Well number project Sample name aisle Target Ct 1 Dog LAMP No. 1 FAM Hepca 12.64 2 Dog LAMP No. 1 FAM Babeca NoC 3 Dog LAMP No. 1 FAM Babegi NoC 4 Dog LAMP No. 1 FAM Ehrca NoC 5 Dog LAMP No. 1 FAM Anap NoC 6 Dog LAMP No. 1 FAM ACTB NoC 1 Dog LAMP No. 2 FAM Hepca 15.81 2 Dog LAMP No. 2 FAM Babeca NoC 3 Dog LAMP No. 2 FAM Babegi NoC 4 Dog LAMP No. 2 FAM Ehrca NoC 5 Dog LAMP No. 2 FAM Anap NoC 6 Dog LAMP No. 2 FAM ACTB 8.93 1 Dog LAMP No.3 FAM Hepca 21.34 2 Dog LAMP No.3 FAM Babeca NoC 3 Dog LAMP No.3 FAM Babegi NoC 4 Dog LAMP No.3 FAM Ehrca NoC 5 Dog LAMP No.3 FAM Anap NoC 6 Dog LAMP No.3 FAM ACTB 10.25 1 Dog LAMP No. 4 FAM Hepca NoC 2 Dog LAMP No. 4 FAM Babeca 19.38 3 Dog LAMP No. 4 FAM Babegi NoC 4 Dog LAMP No. 4 FAM Ehrca NoC 5 Dog LAMP No. 4 FAM Anap NoC 6 Dog LAMP No. 4 FAM ACTB 9.64 1 Dog LAMP No. 5 FAM Hepca NoC 2 Dog LAMP No. 5 FAM Babeca 22.66 3 Dog LAMP No. 5 FAM Babegi NoC 4 Dog LAMP No. 5 FAM Ehrca NoC 5 Dog LAMP No. 5 FAM Anap NoC 6 Dog LAMP No. 5 FAM ACTB 10.18 1 Dog LAMP No. 6 FAM Hepca NoC 2 Dog LAMP No. 6 FAM Babeca NoCt 3 Dog LAMP No. 6 FAM Babegi 17.39 4 Dog LAMP No. 6 FAM Ehrca NoCt 5 Dog LAMP No. 6 FAM Anap NoCt 6 Dog LAMP No. 6 FAM ACTB 9.98 1 Dog LAMP No. 7 FAM Hepca NoCt 2 Dog LAMP No. 7 FAM Babeca NoCt 3 Dog LAMP No. 7 FAM Babegi 25.38 4 Dog LAMP No. 7 FAM Ehrca NoCt 5 Dog LAMP No. 7 FAM Anap NoCt 6 Dog LAMP No. 7 FAM ACTB 10.33 1 Dog LAMP No. 8 FAM Hepca NoCt 2 Dog LAMP No. 8 FAM Babeca NoCt 3 Dog LAMP No. 8 FAM Babegi NoCt 4 Dog LAMP No. 8 FAM Ehrca 18.87 5 Dog LAMP No. 8 FAM Anap NoCt 6 Dog LAMP No. 8 FAM ACTB 9.37 1 Dog LAMP No. 9 FAM Hepca NoCt 2 Dog LAMP No. 9 FAM Babeca NoCt 3 Dog LAMP No. 9 FAM Babegi NoCt 4 Dog LAMP No. 9 FAM Ehrca 23.6 5 Dog LAMP No. 9 FAM Anap NoCt 6 Dog LAMP No. 9 FAM ACTB 10.79 1 Dog LAMP No. 10 FAM Hepca NoCt 2 Dog LAMP No. 10 FAM Babeca NoCt 3 Dog LAMP No. 10 FAM Babegi NoCt 4 Dog LAMP No. 10 FAM Ehrca 21.77 5 Dog LAMP No. 10 FAM Anap NoCt 6 Dog LAMP No. 10 FAM ACTB 10.66 1 Dog LAMP No. 11 FAM Hepca NoCt 2 Dog LAMP No. 11 FAM Babeca NoCt 3 Dog LAMP No. 11 FAM Babegi NoCt 4 Dog LAMP No. 11 FAM Ehrca NoCt 5 Dog LAMP No. 11 FAM Anap 16.91 6 Dog LAMP No. 11 FAM ACTB 9.93 1 Dog LAMP No. 12 FAM Hepca NoCt 2 Dog LAMP No. 12 FAM Babeca NoCt 3 Dog LAMP No. 12 FAM Babegi NoCt 4 Dog LAMP No. 12 FAM Ehrca NoCt 5 Dog LAMP No. 12 FAM Anap 22.37 6 Dog LAMP No. 12 FAM ACTB 10.32 Table 8 Detection results of the kit on positive samples

[0059] From Tables 7~8 and Figure 4 The results show that, through the detection of 12 clinical positive samples, it is shown that the kit of the present invention combined with the microfluidic chip can effectively detect the target pathogens, and the detection results are consistent with those of qPCR.

[0060] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A LAMP primer composition for rapid simultaneous detection of five canine tick pathogens, characterized in that: It includes a primer set for detecting Hepatozoon canis, a primer set for detecting Babesia canis, a primer set for detecting Babesia gibbsii, a primer set for detecting Ehrlichia canis, and a primer set for detecting Anaplasma planus; wherein, The primer set for detecting Hepatozoon canis includes: outer primers Hepca-F3 and Hepca-B3, whose nucleotide sequences are shown in SEQ ID NOs. 1 and 2, respectively; inner primers Hepca-FIP and Hepca-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 3 and 4, respectively; and loop primer Hepca-LB, whose nucleotide sequence is shown in SEQ ID NO. 5; The primer set for detecting Babesia canis includes: outer primers Babeca-F3 and Babeca-B3, whose nucleotide sequences are shown in SEQ ID NOs. 6 and 7, respectively; inner primers Babeca-FIP and Babeca-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 8 and 9, respectively; loop primers Babeca-LF and Babeca-BF, whose nucleotide sequences are shown in SEQ ID NOs. 10 and 11, respectively; The primer set for detecting Babesia gibbsii of dogs comprises: outer primers Babegi-F3 and Babegi-B3, whose nucleotide sequences are shown in SEQ ID NOs. 12 and 13, respectively; inner primers Babegi-FIP and Babegi-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 14 and 15, respectively; and loop primer Babegi-LB, whose nucleotide sequence is shown in SEQ ID NO. 16; The primer set for detecting Ehrlichia canis includes: outer primers Ehrca-F3 and Ehrca-B3, whose nucleotide sequences are shown in SEQ ID NOs. 17 and 18, respectively; inner primers Ehrca-FIP and Ehrca-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 19 and 20, respectively; and loop primer Ehrca-LF, whose nucleotide sequence is shown in SEQ ID NO. 21; The primer set for detecting flat anaplasma includes: outer primers AnaP-F3 and AnaP-B3, whose nucleotide sequences are shown in SEQ ID NOs. 22 and 23, respectively; inner primers AnaP-FIP and AnaP-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 24 and 25, respectively; and loop primer AnaP-LB, whose nucleotide sequence is shown in SEQ ID NO.

26.

2. The LAMP primer composition for simultaneous and rapid detection of five canine tick pathogens according to claim 1, characterized in that: The primer set for detecting Hepatozoon canis is designed for the conservative region of the 18S rRNA gene sequence of Hepatozoon canis; the primer set for detecting Babesia canis is designed for the conservative region of the 18S rRNA gene sequence of Babesia canis; the primer set for detecting Babesia gibbsii is designed for the conservative region of the 18S RNA gene sequence of Babesia gibbsii; the primer set for detecting Ehrlichia canis is designed for the conservative region of the 16S rRNA gene sequence of Ehrlichia canis; the primer set for detecting Anaplasma planum is designed for Anaplasma planum gltA Design of conserved regions of gene sequences.

3. A LAMP freeze-dried microsphere for rapid simultaneous detection of five canine tick pathogens, characterized in that: The LAMP freeze-dried microspheres contain a primer set in the primer composition according to claim 1 or 2.

4. The LAMP freeze-dried microspheres for simultaneous rapid detection of five canine tick pathogens according to claim 3, characterized in that: The LAMP freeze-dried microspheres include 4X LAMP Buffer, Bst DNA polymerase, MgSO4, 5XSYTO9 fluorescent dye, 25XLAMP Primer Mix, mannitol, trehalose, bovine serum albumin and PEG20000; the 25XLAMP Primer Mix is ​​a primer mix prepared by mixing any one of a primer set of Hepatozoon canis, a primer set of Babesia canis, a primer set of Babesia gibbsii, a primer set of Ehrlichia canis, a primer set of Anaplasma flatulum and a primer set of an internal reference gene of a dog at a concentration 10 times of the final concentration of the primers when used; in each of the primer sets, the final concentration of the inner primers FIP / BIP is 0.8-1.6 μM, the final concentration of the loop primers LF / LB is 0.2-0.4 μM, and the final concentration of the outer primers F3 / B3 is 0.1-0.2 μM.

5. A microfluidic chip for rapid and simultaneous detection of five canine tick pathogens, characterized in that: Different reaction chambers of the microfluidic chip contain the LAMP freeze-dried microspheres as described in any one of claims 3 to 4.

6. The microfluidic chip for rapid simultaneous detection of five canine tick pathogens according to claim 5, characterized in that: The microfluidic chip is provided with a reagent tank, a sample processing tank, a nucleic acid extraction area, a nucleic acid dilution area, a quantitative subpackaging area and a reaction detection area which are connected to each other. The nucleic acid extraction area and the quantitative subpackaging area are respectively connected to a waste liquid tank through a microfluidic channel; the nucleic acid dilution area, the quantitative subpackaging area and the waste liquid tank are respectively connected to the atmosphere through the microfluidic channel, and the reaction chamber of the reaction detection area has a transparent area for fluorescence detection.

7. The microfluidic chip for rapid simultaneous detection of five canine tick pathogens according to claim 6, characterized in that: The reagent tank comprises a first cleaning liquid tank, a second cleaning liquid tank, an eluting liquid tank and a diluting liquid tank. Each of the reagent tanks is provided with an opening on the same centrifugal radius, and the opening is filled with paraffin.

8. A kit for rapid simultaneous detection of five canine tick pathogens, characterized in that: The kit comprises the microfluidic chip as described in any one of claims 5 to 7.

9. A method for simultaneously and rapidly detecting five canine tick pathogens for non-disease diagnosis purposes, characterized in that: The method comprises using the microfluidic chip according to any one of claims 5 to 7 or the kit according to claim 13 to perform a loop-mediated isothermal amplification reaction on the sample to be tested, and determining the canine tick pathogen infection status of the sample to be tested based on the generated fluorescence curve.

10. The method for simultaneously and rapidly detecting five canine tick pathogens for non-disease diagnosis purposes according to claim 9, characterized in that: The following steps are involved: S1. Add 100-200 μL of dog blood to the mixture of lysate and proteinase K, mix well and add to the sample processing tank of the microfluidic chip, heat at 54-58°C for 10-15 minutes, and centrifuge at 2500-3000 rpm for 60-90 seconds to allow the lysed sample to pass through the nucleic acid extraction column in the nucleic acid extraction area, and then the waste liquid enters the waste liquid area; S2, using laser to irradiate the paraffin in the opening of the first cleaning liquid tank to melt it and release the first cleaning liquid, centrifuging at 2500-3000 rpm for 60-90 seconds, and the first cleaning liquid enters the waste liquid area through the nucleic acid extraction column; using laser to irradiate the paraffin in the opening of the second cleaning liquid tank to melt it and release the second cleaning liquid, centrifuging at 2500-3000 rpm for 120-180 seconds, and the second cleaning liquid enters the waste liquid area through the nucleic acid extraction column to complete the nucleic acid purification; S3, using laser to irradiate the paraffin in the opening of the eluent tank to melt it and release the eluent, centrifuging at 2500-3000rpm for 30-60s, so that the eluent elutes the nucleic acid adsorbed on the nucleic acid extraction column and enters the nucleic acid extraction area under the action of centrifugation; using laser to irradiate the paraffin in the opening of the diluent tank to melt it and release the diluent, centrifuging at 2500-3000rpm for 30-60s, so that the diluted nucleic acid sample liquid enters the reaction chamber in the reaction detection area; S4. The nucleic acid sample solution enters the reaction chamber to re-dissolve the preset LAMP freeze-dried microspheres to form a LAMP reaction system. The LAMP reaction system is amplified at a constant temperature of 62-67°C. As the reaction proceeds, the fluorescent signal is used to determine whether the sample contains canine tick pathogens.

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