Application of primer pairs and probes in the preparation of kits for detecting canine insect-borne pathogenic microorganisms

Abstract

The present invention relates to the field of detecting canine insect-borne pathogenic microorganisms, and more specifically, to the use of a primer pair and a probe in preparing a kit for detecting canine insect-borne pathogenic microorganisms. The primer pair is shown in SEQ ID NOs: 3 and 4, and the probe is shown in SEQ ID NOs: 5 and 6. The kit includes the primer pair and the probe. The primer pair and probe provided by the present invention amplify specific sequences and enable fluorescence quantitative detection, thereby improving detection sensitivity and specificity.

Description

Technical Field

[0001] The present invention relates to the field of detection of canine insect-borne pathogenic microorganisms, and in particular to the application of a primer pair and a probe in preparing a kit for detecting canine insect-borne pathogenic microorganisms. Background Art

[0002] The main canine insect-borne pathogenic microorganisms are canine ear mites ( Otodectescynotis Otodendron spp. is a non-burrowing, monospecific mite that parasitizes the skin of the vertical and horizontal ear canals. It has a wide host range and is found worldwide, infecting both canine and feline species. There have also been reports of ruminant infections, posing a zoonotic risk. Animals carrying the mites are the primary source of infection. Transmission can occur through direct contact or indirect contact via utensils and objects. Infection is particularly common during lactation, when puppies and cats frequently come into contact with their mothers. Following infection, infected animals experience symptoms such as pruritus and external otitis, and are at increased risk of developing cerumen tumors, which can reduce their quality of life and cause economic losses. Microscopy is currently the gold standard for clinical diagnosis of otodendron spp. A cotton swab is used to collect external ear canal secretions from infected animals. The specimen is then placed on a glass slide and cleared with a few drops of 10% KOH solution. Microscopic examination is performed. Confirmation is confirmed by the presence of one or more mites or eggs, with a sensitivity of approximately 57%. This method is subject to numerous subjective factors, and the expertise and technical skills of clinical laboratories vary widely, which can lead to missed or misdiagnoses, compromising prevention and treatment. Molecular biology methods are widely used in the diagnosis of clinical parasitic diseases, offering high sensitivity and specificity. They also play a crucial role in species identification, population structure, and phylogenetic relationships. Summary of the Invention

[0003] The present invention provides the use of a primer pair and a probe in preparing a kit for detecting canine insect-borne pathogenic microorganisms based on a target sequence of canine ear itch mites.

[0004] One of the purposes of the present invention is to provide a primer pair and a probe for use in preparing a kit for detecting canine insect-borne pathogenic microorganisms, wherein the detection comprises:

[0005] Extract DNA samples of canine insect-borne pathogenic microorganisms;

[0006] Performing fluorescent quantitative PCR amplification on the substrate using the primer pair and the probe;

[0007] Determining the cycle threshold of the DNA sample according to the fluorescence curve;

[0008] The DNA content of canine insect-borne pathogenic microorganisms is determined according to the cycle threshold and the standard equation, wherein the standard equation is y=-1.2x+39.078;

[0009] The reaction system of fluorescent PCR includes:

[0010] 10.0 μL 2× Probe qPCR Premix Ex Taq™,

[0011] 0.2 μL ROX Reference Dye II,

[0012] 1.2 μL, 10 μM F1 as shown in SEQ ID NO: 3,

[0013] 1.2 μL, 10 μM of R1 as shown in SEQ ID NO: 4,

[0014] 1.2 μL, 10 μM of P1 as shown in SEQ ID NO: 5,

[0015] 1.2 μL, 10 μM of P2 as shown in SEQ ID NO: 6,

[0016] 2.0 μL of a standard plasmid carrying the sequence shown in SEQ ID NO: 1, and

[0017] 3 μL of sterile water;

[0018] The reaction procedure of fluorescent PCR is as follows:

[0019] Pre-denaturation at 94°C for 5 min;

[0020] Denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 1 minute constituted one cycle, and 40 cycles were performed in total;

[0021] Extension at 72°C for 5 min.

[0022] The steps of extracting DNA samples of canine insect-borne pathogenic microorganisms include:

[0023] (1) Add 600 μL of lysis buffer to each 2 mL EP tube containing the sample, mix by pipetting or oscillating, incubate the EP tube at 65°C for 25-30 min, remove it, cool it to room temperature, add 400 μL of chloroform, shake vigorously for 15 s, let it stand for 3 min, and centrifuge it at 12,000 rpm and 4°C for 5 min;

[0024] (2) Combine and take as much clean supernatant as possible into a new 2 mL EP tube, add an equal volume of binding solution and 40 μL 30 mg / mL silanol magnetic beads, mix by inversion for 1 min, let it stand for 3 min, place the EP tube on a magnetic separation rack for magnetic separation, and discard the waste liquid;

[0025] (3) Wash and add 600 μL of rinse solution, mix gently for 1-2 minutes, place the EP tube on a magnetic separation rack for magnetic separation, discard the waste liquid, and repeat this step once;

[0026] (4) Elution: Dry at room temperature for 5-10 minutes until the ethanol is completely evaporated, add 50-100 μL of elution buffer, slowly pipette to mix, place in a 65°C water bath for 10 minutes, place the EP tube on a magnetic separation rack for magnetic separation, carefully pipette the supernatant into a new EP tube, and store in a –20°C environment for later use;

[0027] (5) Take 2 μL of the extracted DNA sample and inject it into the sample well of the ultra-micro spectrophotometer. Read the reading. Measure each sample 3 times and take the average value. Record the concentration and purity information of the genomic DNA. Then, add 0.45 g of agarose to the conical flask, measure 30 mL of 0.5×TBE buffer and pour it into the flask. Heat it on high heat in a microwave oven for 2 minutes and then take it out. After cooling to room temperature, add 2 μL of NaRed and shake it gently to mix. Pour it into the gelatin plate and put the comb in place. After cooling and solidifying, take 5 μL of DNA sample and mix it with 1 μL of loading buffer and add it to the sample well. Take another 5 μL of DL15000 DNA Marker and add it to the sample well. Turn on the power and adjust the voltage to 90 V. Perform electrophoresis for 50 minutes. After the end, observe the results under ultraviolet light.

[0028] One of the objects of the present invention is to provide a primer pair for use in preparing a kit for detecting canine insect-borne pathogenic microorganisms, wherein the detection comprises:

[0029] Extract DNA samples of canine insect-borne pathogenic microorganisms;

[0030] PCR amplification was performed using the primer pair, wherein the primer pair was F5: 5'-Dig-ggtatttgaagaggaatgttggggactag, digoxigenin-labeled, as shown in SEQ ID NO: 14, and R5: 5'-Bio-gatgaccaaaaaaccaaaataaatgttgataaagaatagg, biotin-labeled, as shown in SEQ ID NO: 15;

[0031] Take 4 μL of PCR product, 4 μL of 10g / 100mL BSA and 80 μL of PBS buffer solution, pH 7.4, 0.01 μM, and place it on the sample pad of the immunochromatographic test strip. After 10 minutes, the fluorescence signal value passing through the test line and the quality control line is

[0032] The DNA content of canine insect-borne pathogenic microorganisms was determined based on the fluorescence signal value;

[0033] The PCR reaction system includes:

[0034] 1 μL, 0.1 μM F5,

[0035] 1 μL, 0.1 μM R5,

[0036] 10μL of 2×SYBR®PremixExTaq™,

[0037] 7 μL ddH2O

[0038] 1 μL of DNA sample;

[0039] The amplification reaction procedure is:

[0040] Pre-denaturation at 95°C for 10 min;

[0041] One cycle consisted of denaturation at 95°C for 5 s, annealing at 62°C for 60 s, and extension at 72°C for 20 s, and a total of 42 cycles were performed;

[0042] Extension at 72°C for 10 min.

[0043] The steps of preparing the immunochromatographic test strip include:

[0044] 1) Use hydrophilic glass fiber to make the sample pad and conjugate pad. Soak the sample pad and conjugate pad in a mixed solution of buffer and effector. After soaking, take them out and dry them in a drying room for 4-5 hours. The temperature of the drying room is 18°C-26°C and the humidity of the drying room is 20%-28%.

[0045] 2) Spraying 5 mg / mL of fluorescent microspheres labeled with streptavidin onto the conjugate pad at a rate of 5 µL / cm and drying in a drying room overnight to prepare a conjugate pad immobilized with the fluorescent microspheres labeled with streptavidin and a treated sample pad; wherein the buffer base component is a pH 9.0, 0.02 M phosphate buffer, and the effector main components are 1 wt % of PEG with a molecular weight of 4000-20000, 0.05 wt % of casein, 0.05 wt % of fetal bovine serum albumin, and 0.01 wt % of trehalose;

[0046] 3) Use a nitrocellulose membrane with a water flow rate of 90 seconds. Equilibrate the membrane in a sealed chamber at a humidity of 25%-65% for 1 hour. Dilute 48 μg of anti-digoxigenin antibody to 2 mg / mL in Tris-HCl buffer (pH 6.5). Dilute 0.48 μg of biotin-modified BSA to 20 μg / mL in Tris-HCl buffer. Streak the membrane with both antibodies at a streak volume of 0.8 μL / cm. After streaking, dry the membrane and set aside. The buffer used to dilute the antibody and BSA is phosphate buffer (pH 7.4, 0.05 M).

[0047] 4) Attach the nitrocellulose membrane prepared in step 3) to the center of the adhesive PVC baseplate. Attach absorbent paper to one end of the membrane, with a 2mm overlap. Attach the conjugate pad to the other end of the membrane, with a 4mm overlap. Place the sample pad over the conjugate pad, secure the baseplate, and cut into immunochromatographic test strips.

[0048] 5) Add the cut immunochromatographic test strips to the card holder, and set the sample loading window and signal reading window on the upper cover. The sample loading window corresponds to the sample pad, and the signal reading window corresponds to the detection line and quality control line.

[0049] The primer pairs and probes provided by the present invention amplify specific sequences and can perform fluorescence quantitative detection, thereby improving detection sensitivity and specificity.

[0050] The primer pairs provided by the present invention can also be combined with immunochromatographic test strips to provide a fluorescent immunochromatographic test strip detection method. This method can not only effectively distinguish between canine ear scabies and rabbit itch mites, but can also detect the target sequence at 1 copy / μL, with very high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The electrophoresis diagram of the standard plasmid carrying the target sequence is shown in Figure 1. Lane M is the standard, lane 1 is pMD18-T, lane 2 is the enzyme digestion product of the standard plasmid carrying the target sequence shown in Figure 1, and lane 3 is the enzyme digestion product of the standard plasmid carrying the target sequence shown in Figure 2.

[0052] Figure 2 The standard curve of fluorescence quantitative PCR detection of 10 standard plasmids with different concentration gradients in the experimental group was used.

[0053] Figure 3 The standard curve of fluorescence quantitative PCR detection was used for the control group using 10 concentration gradients of standard plasmids.

[0054] Figure 4 The amplification curves are obtained by using different primer pairs and probes to detect different samples using fluorescence quantitative PCR.

[0055] Figure 5 This is an electrophoresis diagram of PCR amplification products using the F2\R2 primer pair. Lane 1 is a DNA sample from a dog earwax sample, lane 2 is a DNA sample from a rabbit itch mite sample, lane 3 is a DNA sample from a buffalo itch mite sample, lane 4 is a DNA sample from a scabies mite sample, lane 5 is a DNA sample from a folliculorum mite sample, and lane 6 is a DNA sample from a sebaceous mite sample.

[0056] Figure 6 The figure shows the fluorescence signal bar graph of six mite samples detected by using F5\R5 or F6\R6 combined with fluorescent immunochromatographic test paper.

[0057] Figure 7 The figure shows the fluorescence signal bar graph of five concentration gradients of standard plasmids detected by using F5\R5 or F6\R6 combined with fluorescent immunochromatographic test paper. DETAILED DESCRIPTION

[0058] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are merely illustrative of the present invention and are not intended to limit the present invention. Reagents not described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and are known in the art.

[0059] 1. Main reagents

[0060] Standard agarose gel DNA purification kit (D1200, Suobaolai, Beijing). High-purity plasmid miniprep kit (DP104, Tiangen, Beijing). EZNA® Mollus cDNA Kit (D3373-01), OMEGA Bio-Tek. dDTP Mixture (2.5 mM), TakaRa. Ethidium bromide (EB) and pMD19 vector, Bio-Rad Biotechnology (Beijing) Co., Ltd. Taq DNA polymerase (CW0680), Kangwei Century Biotechnology Co., Ltd. Agar standards (DL2000, etc.), Beijing Qingke Xinye Biotechnology Co., Ltd. Escherichia coli JM109 competent cells (G6014-20), Shanghai Angyu Biotechnology Co., Ltd. Ampicillin stock solution (A1170), LB solid medium powder (L1015), and LB liquid medium powder (L1010) were products of Beijing Soleb Biotechnology Co., Ltd.; other reagents such as KOH were of domestic analytical grade.

[0061] 2. Main instruments

[0062] 50i microscope (ECLIPSE), a product of Ni-KON Co., Ltd., Japan; vortex mixer (VORTEX-5), a product of Haimen Qilin Bell Instrument Manufacturing Co., Ltd.; high-speed centrifuge (HC-2514), a product of Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.; digital constant temperature water bath (HH-3A), a product of Changzhou Guohua Electric Co., Ltd.; small centrifuge (D1008E), a product of Shanghai Tongshan Biotechnology Co., Ltd.; PCR instrument (A24812), a product of Thermo Fisher Scientific (Shanghai) Instrument Co., Ltd.; vertical pressure steam sterilizer Bacteria collection device (BXM-30R), a product of Shanghai Boxun Medical Biological Instrument Co., Ltd.; microanalytical balance (AUY200), a product of Shimadzu Enterprise Management (China) Co., Ltd.; electrophoresis apparatus (DYY-7C), a product of Beijing Liuyi Biotechnology Co., Ltd.; fully automatic UV gel imaging analysis system (CHAMPGEL5000), a product of Beijing Saizhi Venture Technology Co., Ltd.; ultra-clean workbench (SW-CJ-1F), a product of Sujing Antai Company; micro spectrophotometer (NANO-100), a product of Hangzhou Aosheng Instrument Co., Ltd.

[0063] 3. Microscopic examination

[0064] Place an appropriate amount of sample on a glass slide, add one drop of 10% KOH solution, and cover with a coverslip. Place the slide over an alcohol lamp to gently heat it, then gently press the coverslip to expel any bubbles. Examine the slide under an optical microscope. A positive result is determined if mites or their eggs are found.

[0065] 4. DNA Extraction

[0066] (1) Add 600 μL of lysis buffer (1.4 M NaCl, 2% (w / v) CTAB, 2% (w / v) PVP, 100 mM Tris-HCl (pH 8.0), 20 mM EDTA, 0.2% (v / v) β-mercaptoethanol, 20 mg / mL proteinase K) to each 2 mL EP tube containing the sample and mix by pipetting or vortexing. Incubate the EP tube at 65°C for 25-30 minutes. Remove the tube, cool it to room temperature, add 400 μL of chloroform, shake vigorously for 15 seconds, let it stand for 3 minutes, and centrifuge it at 12,000 rpm at 4°C for 5 minutes.

[0067] (2) Combine and remove as much of the supernatant as possible into a new 2 mL EP tube. Add an equal volume of binding solution (25 mM MPEG-4000, 3 M NaCl) and 40 μL of silanol magnetic beads (30 mg / mL). Mix by inversion for 1 min and let stand for 3 min. Place the EP tube on a magnetic separation rack for magnetic separation and discard the waste liquid (pay attention to the tube cap and residual liquid at the bottom of the tube).

[0068] (3) Washing: Add 600 μL of rinsing solution (70% ethanol solution), mix gently for 1-2 minutes, place the EP tube on a magnetic separation rack for magnetic separation, and discard the waste liquid (pay attention to the tube cap and the residual liquid at the bottom of the tube). Repeat this step once.

[0069] (4) Elution: Air-dry at room temperature for 5-10 minutes until the ethanol is completely evaporated. Add 50-100 μL of elution buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), mix gently by pipetting, and place in a 65°C water bath for 10 minutes (gently shake the EP tube every 1-2 minutes). Place the EP tube on a magnetic separation rack for magnetic separation. Carefully pipette the supernatant into a new EP tube, aliquot, and store at –20°C until ready for use.

[0070] (5) Take 2 μL of the extracted DNA sample and inject it into the sample well of the ultra-micro spectrophotometer. Read the result. Measure each sample 3 times, take the average value, and record the concentration and purity information of the genomic DNA. Next, add 0.45 g of agarose to the conical flask, measure 30 mL of 0.5×TBE buffer (44.5 mM Tris-Borate, 1 mM EDTA, pH 8.0) and pour it into the flask. Heat it in a microwave oven on high for 2 minutes, then remove it. After cooling to room temperature, add 2 μL of NaRed and shake it gently to mix. Pour it into the gelatin plate and place the comb. After cooling and solidification, take 5 μL of DNA sample and mix it with 1 μL of loading buffer (6× Loading Buffer) and add it to the sample well. Take 5 μL of DL15000 DNA Marker and add it to the sample well. Turn on the power, adjust the voltage to 90 V, and run the electrophoresis for 50 minutes. After the end, observe the results under UV light.

[0071] 5. Primers and probes

[0072] The primers and probes shown in Table 1 were designed and synthesized.

[0073] Table 1

[0074]

[0075] 6. Preparation of standard plasmid

[0076] Prepare standard plasmids carrying the target sequences shown in SEQ ID NO: 1 and 2 respectively. Use F3 and R3 as primers to perform PCR amplification to obtain the target sequence shown in SEQ ID NO: 1. Use F4 and R4 as primers to perform PCR amplification to obtain the target sequence shown in SEQ ID NO: 2. Eco RI and HindThe enzyme-digested fragment was digested with the double-digested fragment of the pMD18-T vector (TaKaRa) by T4 ligation. The ligation product was transformed into Escherichia coli DH5α competent cells to achieve positive clones. The plasmid was extracted and sequenced to obtain a standard plasmid carrying the target sequence shown in SEQ ID NO: 1 and 2. The concentration was determined using a nucleic acid protein analyzer and converted to copy number. The plasmid was diluted in 10-fold gradients to 1.0×10 9 ~1 copy / μL was used as a standard and stored at -20℃ until use.

[0077] like Figure 1 As shown, it can be seen that the target sequences shown in SEQ ID NO: 1 and 2 are cloned into the pMD18-T vector.

[0078] 7. Fluorescence PCR

[0079] (1) Fluorescence PCR reaction system

[0080] Experimental group: 10.0 μL 2×Probe qPCR Premix Ex Taq TM , 0.2 μL ROXReferenceDyeⅡ, 1.2 μL (10 μM) F1, 1.2 μL (10 μM) R1, 1.2 μL (10 μM) P1, 1.2 μL (10 μM) P2, 2.0 μL of the standard plasmid carrying the sequence shown in SEQ ID NO: 1, and the volume was made up to 20.0 μL with sterile water. The fluorescence PCR reaction program was: 94°C for 5 min; 60 cycles (94°C for 30 s, 58°C for 30 s, 72°C for 1 min); 72°C for 5 min. The standard plasmid concentration was 10 steps (1.0 × 10 9 ~1.0×10 0 copies / μL).

[0081] Control group: 10.0 μL 2×Probe qPCR Premix Ex Taq TM , 0.2 μL ROXReferenceDyeⅡ, 1.2 μL (10 μM) F2, 1.2 μL (10 μM) R2, 1.2 μL (10 μM) P3, 2.0 μL of the standard plasmid carrying the sequence shown in SEQ ID NO: 2, and sterilized water was added to 20.0 μL. The fluorescence PCR reaction program was: 94°C for 5 minutes; 70 cycles (94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute); 72°C for 5 minutes. The standard plasmid concentration was 10 steps (1.0 × 10 9 ~1.0×10 0 copies / μL).

[0082] (2) Detection

[0083] Optimized fluorescence quantitative PCR was used for detection. Data analysis software was used to establish a standard curve and linear regression equation between the logarithm of the starting template copy number (x) and the Ct value (y). The quantitative range and sensitivity of the method were determined based on the amplification results of each standard DNA.

[0084] like Figure 2 The standard curve of the experimental group can be obtained at 1.0×10 9 ~1.0×10 0 A linear standard curve (y = -1.2x + 39.078, R 2 =0.981), the standard curve has a high linear fit, and for 1.0×10 0 The cycle threshold of the standard plasmid with a copy / μL value is lower, indicating its high sensitivity.

[0085] like Figure 3 The control group was set at 1.0×10 9 ~1.0×10 0 The gradient range of copies / μL could not form an effective linear standard curve (y=-3.6821x+63.65, R 2 =0.7342). Furthermore, the control group was unable to form an effective S-shaped amplification curve for the 1 copy / μL and 10 copies / μL standard plasmids during 70 amplification cycles, and the cycle threshold could not be determined. This indicates that the control group was unable to detect the 1 copy / μL and 10 copies / μL standard plasmids, and its sensitivity was lower than that of the experimental group.

[0086] 8. Specificity Verification

[0087] (1) Test samples

[0088] Rabbit itch mite ( Psoroptescuniculi ), buffalo itch mite ( Psoroptesnatalensis ), Sarcoptescabiei, Sarcoptes canis ( Otodectescynotis ) earwax samples were purchased from the Parasitology Laboratory of Sichuan Agricultural University. Demodexfolliculorum )、Demodex sebaceus( Demodexbrevis Earwax samples were purchased from the Laboratory of Pathogenic Biology at Xi'an Jiaotong University. DNA samples were prepared according to the above method and stored at -20°C until use.

[0089] (2) Detection

[0090] PCR amplification of these DNA samples was performed using F1\R1 as primer pairs and P1 and P2 as probes. 10.0 μL 2× Probe qPCR Premix Ex Taq TM , 0.2 μL ROX Reference Dye II, 1.2 μL (10 μM) F1, 1.2 μL (10 μM) R1, 1.2 μL (10 μM) P1, 1.2 μL (10 μM) P2, 2.0 μL of each DNA sample, and the volume was made up to 20.0 μL with sterile water. The fluorescence PCR reaction program was: 94°C for 5 min; 40 cycles (94°C for 30 s, 58°C for 30 s, 72°C for 1 min); 72°C for 5 min.

[0091] PCR amplification of these DNA samples was performed using F1\R1 as primer pair and P3 as probe. The amplification reaction system was: 10.0 μL 2×ProbeqPCRPremixEx Taq TM , 0.2 μL ROX Reference Dye II, 1.2 μL (10 μM) F1, 1.2 μL (10 μM) R1, 1.2 μL (10 μM) P3, 2.0 μL of each DNA sample, and the volume was made up to 20.0 μL with sterile water. The fluorescence PCR reaction program was: 94°C for 5 min; 40 cycles (94°C for 30 s, 58°C for 30 s, 72°C for 1 min); 72°C for 5 min.

[0092] PCR amplification of these DNA samples was performed using F2\R2 as primer pairs. 16.375 μL sterile double-distilled water, 2.5 μL 10× PCR buffer, 2 μL dNTP Mixture, 0.125 μL Taq DNA polymerase (CW0680, Kangwei Century Biotechnology Co., Ltd.), 2 μL template, 1 μL (10 μM) F4, 1 μL (10 μM) R4. The reaction program was: 94°C for 5 min; 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min; and 72°C for 5 min.

[0093] Figure 4The red line is the fluorescence curve for a DNA sample from a dog earwax sample using the primer pair F1\R1 and the probes P1 and P2. The blue line is the fluorescence curve for a DNA sample from a dog earwax sample using the primer pair F1\R1 and the probe P3. The remaining curves are the fluorescence curves for amplification using the primer pair F1\R1 and the probes P1 and P2 for a DNA sample from a rabbit itch mite sample and five common mites, as well as the fluorescence curves for amplification using the primer pair F1\R1 and the probes P1 and P2 for a DNA sample from a rabbit itch mite sample and five common mites. As can be seen, the primer pair F1\R1 and the probes P1 and P2 are able to effectively detect the dog earwax sample, but not the negative sample or the other five common mites, demonstrating the very high specificity of the primers and probes provided by the present invention. Furthermore, while the primer pair F1\R1 and probe P3 also produced a fluorescence curve, the cycle threshold was too high, preventing a S-shaped curve from forming. This again demonstrates that the sensitivity is inferior to that of the probes P1 and P2. Furthermore, substituting the Ct value (24.6) of the red line into the standard equation (y = -1.2x + 39.078) yields a calculated DNA content of 1.16144861e + 12E copies / μL in the canine earwax sample. The Ct value of the blue line is unreadable, and the standard curve is nonlinear, making it unsuitable for quantitative detection.

[0094] In addition, the F2\R2 primer pair was used for PCR amplification, and the amplified product was detected by electrophoresis, such as Figure 5 As shown in the figure, the DNA sample of the dog earwax sample (lane 1) and the DNA sample of the rabbit itch mite sample (lane 2) were detected (352bp), while the target bands were not found in other samples, indicating that the DNA sample of the dog earwax sample and the rabbit itch mite sample could not be distinguished.

[0095] Fluorescence immunochromatography

[0096] Primers

[0097] Table 2

[0098]

[0099] (2) Preparation of DNA samples for testing

[0100] Swabs were squeezed, and genomic DNA was extracted using a magnetic bead DNA extraction kit (50T, Nanjing Dongna Biotechnology Co., Ltd.) according to the manufacturer's instructions. Viral genomic DNA was extracted using the TRNzol reagent according to the manufacturer's instructions. The concentration and purity of all DNA samples were determined using a Nano-300 micro-volume spectrophotometer. After extraction, DNA and samples were stored at –80°C until used in the next step.

[0101] (3) Preparation of streptavidin fluorescent microspheres

[0102] 15 μL (8 μM) fluorescent microspheres (EmissionPeak 550 nm) were mixed with 540 μL borate buffer (0.05 μM, pH 5.0), followed by the addition of 1.70 mg EDC and 2.3 mg NHSS, and magnetic stirring for 30 minutes. 7.2 μL SA (500 μg / mL streptavidin) was added, and the pH was adjusted to 7.4 with NaOH (0.1 μM). After a 15-minute reaction, the mixture was blocked with 10% glucosamine for 30 minutes. Centrifuge at 20,000 rpm at 4°C for 30 minutes, discard the supernatant, and reconstitute the pellet in phosphate buffer (pH 7.4) containing 0.1% NaNO₃ and store at 4°C until use.

[0103] (4) Biotin-conjugated BSA

[0104] Remove the activated esterified biotin from the refrigerator and return it to room temperature. Prepare a 10 mμM biotin stock solution in phosphate buffer (pH 5.0) and mix it with BSA (5 mg / mL, pH 7.8) at a 20:1 molar ratio. Incubate at 180 rpm for 45 minutes at room temperature (25°C). Place the mixture into a pre-treated dialysis bag and dialyze at 4°C for 2–3 days, changing the dialysate every 6 hours to remove unbound biotin.

[0105] (5) Preparation of immune layer test strips

[0106] 1) Use hydrophilic glass fiber to make the sample pad and conjugate pad. Soak the sample pad and conjugate pad in a mixture of buffer and effector (39 mL per pad). After complete saturation, remove the pad and dry it in a desiccant (18°C-26°C, 20%-28% humidity) for 4-5 hours. Spray streptavidin-labeled fluorescent microspheres (5 mg / mL) onto the conjugate pad at a rate of 5 µL / cm and dry overnight in a desiccant to prepare the probe-immobilized conjugate pad and treated sample pad. The buffer base is pH 9.0, 0.02 M phosphate buffer. The effector main components are 1 wt% PEG (molecular weight 4,000-20,000), 0.05 wt% casein, 0.05 wt% fetal bovine serum albumin, and 0.01 wt% trehalose.

[0107] 2) Use a nitrocellulose membrane with a water flow rate of 90 seconds. Equilibrate the membrane in a sealed chamber at a humidity of 25%-65% for 1 hour. Dilute 48 μg of anti-digoxigenin antibody (Wuxi Zhongdeber Biotechnology Co., Ltd.) to 2 mg / mL in Tris-HCl buffer (pH 6.5). Dilute 0.48 μg of biotin-modified BSA to 20 μg / mL in Tris-HCl buffer (pH 6.5). Streak the nitrocellulose membrane with both antibodies at a streak volume of 0.8 μL / cm. After streaking, dry the membrane and set aside. The antibody and BSA were diluted in 0.05 M phosphate buffer (pH 7.4).

[0108] 3) Attach the nitrocellulose membrane prepared in step 2) to the center of the adhesive PVC baseplate. Attach absorbent paper to one end of the nitrocellulose membrane, with a 2 mm overlap. Attach the conjugate pad to the other end of the nitrocellulose membrane, with the two interlaced and a 4 mm overlap. Place the sample pad over the conjugate pad, secure the baseplate, and cut into immunochromatographic test strips.

[0109] 4) Add the cut immunochromatographic test strips to the card holder, and set the sample loading window and signal reading window on the upper cover. The sample loading window corresponds to the sample pad, and the signal reading window corresponds to the detection line and quality control line.

[0110] (6) Fluorescent immunochromatographic test strip detection

[0111] The upstream primer is labeled with digoxigenin at its 5' end, and the downstream primer is labeled with biotin at its 5' end. PCR amplification results in double-stranded DNA with one strand labeled with digoxigenin and the other with biotin. During the chromatography process on the test strip, the double-labeled DNA double-strands bind to streptavidin-labeled quantum dots (QDs-SA), forming a QDs-SA-biotin-DNA complex. On the test line (T line), the double-labeled DNA double-strands bind to digoxigenin monoclonal antibody (Digoxigenin) to form a QDs-SA-biotin-DNA-Digoxigenin-Digoxigenin monoclonal antibody complex, resulting in color development on the T line. On the control line (C line), the QDs-SA binds to biotin conjugated to BSA to form a QDs-SA-biotin-BSA complex, resulting in color development on the C line. This method ensures highly specific test results.

[0112] PCR reaction system: 1 μL each of F5 / R5 or F6 / R6 (0.1 μM), 10 μL 2× SYBR® Premix ExTaq™, 7 μL ddH2O, and 1 μL template DNA. The amplification program was as follows: 95°C denaturation for 10 min; 42 cycles of denaturation at 95°C for 5 s, annealing at 62°C for 60 s, and extension at 72°C for 20 s; and extension at 72°C for 10 min. Each sample was replicated three times.

[0113] 4 μL of PCR product, 4 μL of 10 g / 100 mL BSA, and 80 μL of PBS (0.01 μM, pH 7.4) buffer solution were applied to the sample pad of the immunolayer test strip. After 10 minutes, images of the test line (T) and control line (C) were recorded using a fluorescence imager, and the relative fluorescence unit (RFU) of the test strip was read using a Nanoeasy 1700 fluorescence quantitative analyzer. 4 μL of 10 g / 100 mL BSA and 80 μL of PBS (0.01 μM, pH 7.4) buffer solution served as a control (NC).

[0114] (7) Results

[0115] Figure 6 The figure shows the fluorescence signal values ​​of T and C lines when the DNA samples of 6 kinds of mites were amplified by primers F5 and R5 for immunochromatographic detection, and the fluorescence signal values ​​of T and C lines when the DNA samples of 6 kinds of mites were amplified by primers F6 and R6 for immunochromatographic detection. Figure 6 As shown in the figure, the C lines of the immunochromatographic detection of the six mite DNA samples amplified by F5 and R5 as primers were basically consistent, indicating that the test strips were not invalid, and the T line fluorescence signal value of the dog ear scabies was significantly higher than the C line and NC group, indicating that it was clearly detected, while no obvious detection was found for the other five samples. However, the T line and C line fluorescence signal values ​​of the rabbit itch mite DNA samples amplified by F6 and R6 as primers were comparable and significantly higher than the NC group, indicating that the detection using F6 and R6 could not distinguish between dog ear scabies and rabbit itch mites, and the specificity was poor, which is consistent with the above Figure 5 The results remain consistent.

[0116] Furthermore, 5 gradients (1.0×10 6 , 1.0×10 4 , 1.0×10 2 , 10, 1 copy / μL) of the target sequence SEQ ID NO: 1 standard plasmid as a template, using F5 and R5 for PCR amplification, and the amplified products were detected by fluorescent immunochromatographic test paper. 6 , 1.0×10 4 , 1.0×10 2 , 10, and 1 copy / μL) of the target sequence was a standard plasmid of SEQ ID NO: 2 as a template, F6 and R6 were used for PCR amplification, and the amplified products were detected by fluorescent immunochromatographic test paper.

[0117] The results are as follows Figure 7As shown, F5 and R5 were used as primers to amplify the standard plasmid with the target sequence of SEQ ID NO: 1 and the fluorescent immunochromatographic test paper was used for detection. The fluorescent signal value of the T line was not much different from the C line in all 5 gradients, indicating that the F5 and R5 provided by the present invention as primers and the fluorescent immunochromatographic test paper can detect 1 copy / μL of the target sequence, with high sensitivity. However, F6 and R6 were used as primers to amplify the standard plasmid with the target sequence of SEQ ID NO: 2 and the fluorescent immunochromatographic test paper was used for detection. Only the samples of the first 3 gradients were detected, indicating that the use of F6 and R6 as primers and the fluorescent immunochromatographic test paper can only detect a minimum of 100 copies / μL of the target sequence, and the sensitivity is lower than that of the experimental group.

[0118] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. The use of primer pairs, probes and standard plasmids in the preparation of a kit for detecting canine ear mites, characterized in that: The primer pair includes F1 as shown in SEQ ID NO:3 and R1 as shown in SEQ ID NO:4, the probes are shown in SEQ ID NO:5 and SEQ ID NO:6, and the standard plasmid is pMD18-T carrying the sequence shown in SEQ ID NO:1; the detection includes: Extract DNA samples of canine ear mites; Fluorescent quantitative PCR amplification was performed using the primer pair and the probe. The reaction system of fluorescent PCR included 10.0 μL 2×ProbeqPCRPremixExTaqTM, 0.2 μL ROXReferenceDyeⅡ, 1.2 μL, 10 μM F1 as shown in SEQ ID NO: 3, 1.2 μL, 10 μM R1 as shown in SEQ ID NO: 4, 1.2 μL, 10 μM P1 as shown in SEQ ID NO: 5, 1.2 μL, 10 μM P2 as shown in SEQ ID NO: 6, and 2.0 μL of a standard plasmid carrying the sequence shown in SEQ ID NO: 1, and the volume was made up to 20.0 μL with sterile water. The reaction program of fluorescent PCR was: 94°C for 5 min; 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min, constituting one cycle, for a total of 40 cycles; 72°C for 5 min; Determining the cycle threshold of the DNA sample according to the fluorescence curve; The DNA content of the canine ear mite was determined according to the cycle threshold value and the standard equation, wherein the standard equation is y=-1.2x+39.

078.

2. The use according to claim 1, characterized in that The step of extracting the DNA sample of canine ear itch mites comprises: (1) Add 600 μL of lysis buffer to each 2 mL EP tube containing the sample, mix by pipetting or oscillating, incubate the EP tube at 65°C for 25-30 min, remove it, cool it to room temperature, add 400 μL of chloroform, shake vigorously for 15 s, let it stand for 3 min, and centrifuge at 12000 rpm and 4°C for 5 min; (2) Combine and take as much clean supernatant as possible into a new 2 mL EP tube, add an equal volume of binding solution and 40 μL 30 mg / mL silanol magnetic beads, mix by inversion for 1 min, let it stand for 3 min, place the EP tube on a magnetic separation rack for magnetic separation, and discard the waste liquid; (3) Wash and add 600 μL of rinse solution, mix gently for 1-2 minutes, place the EP tube on a magnetic separation rack for magnetic separation, discard the waste liquid, and repeat this step once; (4) Elution: Dry at room temperature for 5-10 minutes until the ethanol is completely evaporated, add 50-100 μL of elution buffer, slowly pipette to mix, place in a 65°C water bath for 10 minutes, place the EP tube on a magnetic separation rack for magnetic separation, carefully pipette the supernatant into a new EP tube, and store in a –20°C environment for later use; (5) Take 2 μL of the extracted DNA sample and inject it into the sample well of the ultra-micro spectrophotometer. Read the reading. Measure each sample 3 times and take the average value. Record the concentration and purity information of the genomic DNA. Then, add 0.45 g of agarose to the conical flask, measure 30 mL of 0.5×TBE buffer and pour it into the flask. Heat it on high heat in a microwave oven for 2 minutes and then take it out. After cooling to room temperature, add 2 μL of NaRed and shake it gently to mix. Pour it into the gelatin plate and put the comb in place. After cooling and solidifying, take 5 μL of DNA sample and mix it with 1 μL of loading buffer and add it to the sample well. Take another 5 μL of DL15000 DNA Marker and add it to the sample well. Turn on the power and adjust the voltage to 90 V. Perform electrophoresis for 50 minutes. After the end, observe the results under ultraviolet light.

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