Application of primer pair and probe in preparation of kit for detecting canine entomophilous pathogenic microorganisms

Abstract

The invention relates to the field of detection of canine entomophilous pathogenic microorganisms, in particular to application of a primer pair and a probe in preparation of a kit for detecting the canine entomophilous pathogenic microorganisms. The primer pair is as shown in SEQ ID NO: 3 and 4, and the probe is as shown in SEQ ID NO: 5 and 6. The kit comprises the primer pair and the probe. According to the primer pair and the probe pair provided by the invention, specific sequences are amplified, fluorescent quantitative detection can be carried out, and the detection sensitivity and specificity are improved.

Description

Technical Field

[0001] The 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 pathogens are canine ear mites ( Otodectescynotis ), which is a non-burrowing, monospecific mite that parasitizes the vertical ear canal and horizontal ear canal skin surface of the host. It has a wide host range and is distributed all over the world. Canine and feline animals can be infected. There have also been reports of canine ear mites infecting ruminants, and there is a risk of zoonotic infection. Animals carrying mites are the main source of infection. It can be transmitted through direct contact or indirect contact through utensils, objects, etc., especially during the lactation period, when young dogs and cats frequently come into contact with mother dogs and cats and are prone to infection. After infection, sick animals show symptoms such as itching and external otitis, and there is an increased possibility of animals suffering from cerumen gland tumors, thereby reducing the quality of life of animals and causing economic losses. Microscopic examination is currently the gold standard for clinical detection of ear mites in dogs and cats. The external ear canal secretions of sick animals are collected with cotton swabs, and then the diseased material is placed on a slide and a few drops of 10% KOH solution are added for transparent treatment and then microscopic examination is performed. If one or more worm bodies or eggs are found, the diagnosis can be confirmed, with a sensitivity of about 57%. This method is affected by many subjective factors, and the professional knowledge and technical level of clinical laboratories vary, which may lead to missed diagnosis or misdiagnosis, affecting prevention and treatment. Molecular biological methods have been widely used in the diagnosis of clinical parasitic diseases, with high sensitivity and specificity, and have also played an important role in the identification of species, population structure and phylogenetic relationship. Summary of the invention

[0003] The invention provides application 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: Extract DNA samples of canine vector-borne pathogenic microorganisms; Performing fluorescent quantitative PCR amplification on the substrate using the primer pair and the probe; Determine the cycle threshold of the DNA sample according to the fluorescence curve; The DNA content of canine insect-borne pathogenic microorganisms is determined according to the cycle threshold value and the standard equation, wherein the standard equation is y=-1.2x+39.078; The reaction system of fluorescent PCR includes: 10.0 μL 2× Probe qPCR Premix Ex Taq™, 0.2 μL ROX Reference Dye II, 1.2 μL, 10 μM of F1 as shown in SEQ ID NO: 3, 1.2 μL, 10 μM of R1 as shown in SEQ ID NO: 4, 1.2 μL, 10 μM of P1 as shown in SEQ ID NO: 5, 1.2 μL, 10 μM of P2 as shown in SEQ ID NO: 6, 2.0 μL of a standard plasmid carrying the sequence shown in SEQ ID NO: 1, and 3 μL of sterile water; The reaction procedure of fluorescent PCR is as follows: Pre-denaturation at 94°C for 5 min; Denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min constituted one cycle, and a total of 40 cycles were performed; and Extension at 72°C for 5 min.

[0005] The steps of extracting DNA samples of canine insect-borne pathogenic microorganisms include: (1) Add 600 μL of lysis buffer to each 2 mL EP tube that has been loaded with samples, mix by pipetting or shaking, incubate the EP tube at 65°C for 25-30 min, take it out, 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; (2) Take as much clean supernatant as possible and put it into a new 2 mL EP tube. Add an equal volume of binding solution and 40 μL of 30 mg / mL silanol magnetic beads. Mix by inversion for 1 min. Let stand for 3 min. Place the EP tube on a magnetic separation rack for magnetic separation and discard the waste liquid. (3) Washing: 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 blow and 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 in a microwave oven on high for 2 min and 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 1 μL of loading buffer and mix it. Then 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 min. After the end, observe the results under ultraviolet light.

[0006] One of the purposes 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: Extract DNA samples of canine vector-borne pathogenic microorganisms; 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; Take 4 μL of PCR product, 4 μL of 10 g / 100 mL 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 detection line and the quality control line is measured; and Determine the DNA content of canine insect-borne pathogenic microorganisms based on the fluorescence signal value; The PCR reaction system includes: 1 μL, 0.1 μM F5, 1μL, 0.1μM R5, 10μL of 2×SYBR®PremixExTaq™, 7 μL ddH2O 1 μL of DNA sample; The amplification reaction procedure is: Pre-denaturation at 95°C for 10 min; Denaturation at 95°C for 5 s, annealing at 62°C for 60 s, and extension at 72°C for 20 s constituted 1 cycle, and a total of 42 cycles were performed; Extension at 72°C for 10 min.

[0007] Wherein, the preparation steps of the immunochromatographic test strip include: 1) Use hydrophilic glass fiber to make sample pad and conjugate pad, soak the sample pad and conjugate pad in the mixed solution of buffer and effector, take them out after soaking, and dry them in a drying room for 4-5 hours. The temperature of the drying room is 18℃-26℃, and the humidity of the drying room is 20%-28%; 2) Spraying 5 mg / mL of fluorescent microspheres labeled with streptavidin on the conjugate pad at a volume of 5 µL / cm, and drying in a drying room overnight to obtain a conjugate pad fixed with the fluorescent microspheres labeled with streptavidin and a treated sample pad; wherein the basic component of the buffer is a phosphate buffer of pH 9.0 and 0.02 M, and the main component of the effector is 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; 3) Select a nitrocellulose membrane with a water flow rate of 90s, place the nitrocellulose membrane in a closed box with a humidity of 25%-65% for 1h, dilute 48μg of anti-digoxigenin antibody to 2mg / mL with Tris-HCl buffer at pH 6.5, and dilute 0.48μg of biotin-modified BSA to 20μg / mL with buffer, and streak the above two antibodies on the nitrocellulose membrane with a streak volume of 0.8μL / cm; after streaking, dry it for later use; the buffer for diluting the antibody and BSA is a phosphate buffer at pH 7.4 and 0.05M; 4) Paste the nitrocellulose membrane prepared in step 3) in the middle of the sticky PVC bottom plate, paste absorbent paper on one end of the nitrocellulose membrane, and the overlap width of the two is 2mm; the other end of the nitrocellulose membrane is connected to the binding pad, and the two are staggered with an overlap width of 4mm. Cover the sample pad on the binding pad, fix the bottom plate, and finally cut it into immunochromatographic test strips; 5) Add the cut immunochromatographic test strips to the card shell, and set the sample loading window and signal reading window on the upper cover, where the sample loading window corresponds to the sample pad, and the signal reading window corresponds to the detection line and quality control line.

[0008] 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.

[0009] The primer pair provided by the present invention can also be combined with an immunochromatographic test paper to provide a fluorescent immunochromatographic test paper detection method. The method can not only effectively distinguish between canine ear scabies and rabbit itch mites, but also detect 1 copy / μL of the target sequence with very high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1The electrophoresis diagram of the standard plasmid carrying the target sequence. 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 NO: 1, and lane 3 is the enzyme digestion product of the standard plasmid carrying the target sequence shown in NO: 2.

[0011] Figure 2 The standard curve of fluorescence quantitative PCR detection of 10 standard plasmids with different concentration gradients was used for the experimental group.

[0012] Figure 3 The standard curve of fluorescence quantitative PCR detection of 10 standard plasmids with different concentration gradients was used as the control group.

[0013] Figure 4 The amplification curves are obtained by using different primer pairs and probes to detect different samples using fluorescence quantitative PCR.

[0014] Figure 5 The electrophoresis detection diagram of PCR amplification products using the F2\R2 primer pair. Lane 1 is the DNA sample of the dog earwax sample, lane 2 is the DNA sample of the rabbit itch mite sample, lane 3 is the DNA sample of the buffalo itch mite, lane 4 is the DNA sample of the scabies mite, lane 5 is the DNA sample of the hair folliculorum Demodex, and lane 6 is the DNA sample of the sebaceous Demodex.

[0015] Figure 6 This is a bar graph of the fluorescence signals of six mite samples detected using F5\R5 or F6\R6 combined with fluorescent immunochromatographic test paper.

[0016] Figure 7 This is a histogram of the fluorescence signals of the standard plasmids with five concentration gradients detected by using F5\R5 or F6\R6 combined with fluorescent immunochromatographic test paper. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The reagents not described separately in detail in the present invention are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0018] 1. Main reagents Agarose gel DNA recovery kit (D1200, Beijing Suobaolai). High-purity plasmid mini-preparation kit (DP104, Beijing Tiangen). EZNA® Mollus cDNA Kit (D3373-01), OMEGA Bio-Tek. dDTP Mixture (2.5mM), TakaRa. Ethidium bromide (EB), pMD19 vector, Bio-Rid Biotechnology (Beijing) Co., Ltd. Taq DNA polymerase (CW0680), Kangwei Century Biotechnology Co., Ltd. Agar standard 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 dry powder (L1015), and LB liquid medium dry powder (L1010) were products of Beijing Solebow Biotechnology Co., Ltd.; other reagents such as KOH were of domestic analytical grade.

[0019] 2. Main instruments 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 (Shanghai) Instrument Co., Ltd.; vertical pressure steam sterilizer Bacteria collector (BXM-30R), a product of Shanghai Boxun Medical Biological Instrument Co., Ltd.; micro analytical balance (AUY200), a product of Shimadzu Enterprise Management (China) Co., Ltd.; electrophoresis instrument (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.; clean bench (SW-CJ-1F), a product of Sujing Antai Company; micro spectrophotometer (NANO-100), a product of Hangzhou Aosheng Instrument Co., Ltd.

[0020] 3. Microscope examination Take an appropriate amount of sample on a glass slide, add 1 drop of 10% KOH solution, cover with a cover glass, place the glass slide on an alcohol lamp to slightly heat it, then gently press the cover glass to expel bubbles, and examine it under an optical microscope. If mites or their eggs are found, it can be judged as positive.

[0021] 4. DNA Extraction (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 filled with samples, and mix by pipetting or shaking. Incubate the EP tube at 65°C for 25-30 min. Remove the tube, 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 at 4°C for 5 min.

[0022] (2) Combine and take as much clean supernatant as possible into a new 2mL EP tube, add an equal volume of binding solution (25mMPEG-4000, 3MNaCl) and 40μL of silanol magnetic beads (30mg / mL), mix by inversion for 1min, and let stand for 3min. Place the EP tube on a magnetic separation rack for magnetic separation, and discard the waste liquid (pay attention to the residual liquid on the tube cap and the bottom of the tube).

[0023] (3) Washing: Add 600 μL of rinse 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 residual liquid at the bottom of the tube). Repeat this step once.

[0024] (4) Elution: Dry at room temperature for 5-10 minutes until the ethanol is completely evaporated, add 50-100μL of elution buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0), mix slowly by pipetting, and place in a 65℃ 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, and store in a –20℃ environment for later use.

[0025] (5) Take 2μL of the extracted DNA sample and inject it into the sample well of the ultra-micro spectrophotometer. Read the results. Measure each sample 3 times, take the average value, and record the concentration and purity information of the genomic DNA. Next, add 0.45g of agarose to the conical flask, measure 30mL of 0.5×TBE buffer (44.5mMTris-Borate, 1mMEDTA, pH8.0) and pour it into the flask. Heat it in a microwave oven at high temperature for 2min, then take it out. After cooling to room temperature, add 2μL of NaRed and shake it gently to mix it. Pour it into the gelatin plate and put the comb in place. After cooling and solidifying, take 5μL of DNA sample and 1μL of loading buffer (6×LoadingBuffer), mix it and add it to the sample well. Take another 5μL DL15000 DNA Marker and add it to the sample well. Turn on the power, adjust the voltage to 90V, and perform electrophoresis for 50min. After the end, observe the results under ultraviolet light.

[0026] 5. Primers and probes The primers and probes shown in Table 1 were designed and synthesized.

[0027] Table 1

[0028] 6. Preparation of standard plasmid 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 Hind The digested fragments were digested with enzymes of pMD18-T vector (TaKaRa) by T4 ligation, and the ligation products were transformed into E. coli DH5α competent cells to achieve positive cloning. The plasmids were extracted for sequence identification to obtain standard plasmids carrying the target sequences shown in SEQ ID NO: 1 and 2. The concentration was determined by a nucleic acid protein analyzer and converted into copy number, and the plasmids were diluted 10 times to 1.0×10 9 ~1 copy / μL was used as a standard and stored at -20℃ for future use.

[0029] 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.

[0030] 7. Fluorescence PCR (1) Fluorescence PCR reaction system Experimental group: 10.0 μL 2×ProbeqPCRPremixEx Taq TM , 0.2μL ROXReferenceDyeⅡ, 1.2μL (10μM) F1, 1.2μL (10μM) R1, 1.2μL (10μM) P1, 1.2μL (10μM) P2, 2.0μL of the standard plasmid carrying the sequence shown in SEQ ID NO: 1, and add sterile water to 20.0μL. The reaction program of fluorescent PCR is: 94℃5min; 60 cycles (94℃30s, 58℃30s, 72℃1min); 72℃5min. Among them, the concentration of the standard plasmid is 10 gradients (1.0×10 9 ~1.0×10 0 copies / μL).

[0031] Control group: 10.0 μL 2×ProbeqPCRPremixEx Taq TM, 0.2μL ROXReferenceDyeⅡ, 1.2μL (10μM) F2, 1.2μL (10μM) R2, 1.2μL (10μM) P3, 2.0μL of the standard plasmid carrying the sequence shown in SEQ ID NO: 2, and add sterile water to 20.0μL. The reaction program of fluorescent PCR is: 94℃5min; 70 cycles (94℃30s, 58℃30s, 72℃1min); 72℃5min. Among them, the concentration of the standard plasmid is 10 gradients (1.0×10 9 ~1.0×10 0 copies / μL).

[0032] (2) Detection The optimized fluorescence quantitative PCR was used for detection. The data analysis software was used for analysis to establish a standard curve and linear regression equation between the logarithm of the starting template copy number (x) and the Ct value (y), and the quantitative range and sensitivity of the method were determined based on the amplification results of each standard DNA.

[0033] like Figure 2 The standard curve of the experimental group can be 1.0×10 9 ~1.0×10 0 A linear standard curve was formed in the gradient range of copies / μL (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 copies / μL is lower, indicating its high sensitivity.

[0034] 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). In addition, the control group could not form an effective S-type amplification curve for the standard plasmids of 1 copy / μL and 10 copies / μL during 70 amplification cycles, and the cycle threshold could not be determined, indicating that the control group could not detect the standard plasmids of 1 copy / μL and 10 copies / μL, and the sensitivity was lower than that of the experimental group.

[0035] 8. Specificity Verification (1) Test samples Rabbit itch mite ( Psoroptescuniculi )、Buffalo itch mite ( Psoroptes natalensis ), Sarcoptescabiei, Sarcoptes canis ( Otodectescynotis) were purchased from the Parasitology Laboratory of Sichuan Agricultural University. Demodexfolliculorum )、Demodex sebaceus( Demodexbrevis The earwax samples were purchased from the Pathogenic Biology Laboratory of Xi'an Jiaotong University. DNA samples were prepared according to the above method and stored at -20℃ for future use.

[0036] (2) Detection PCR amplification was performed on these DNA samples using F1\R1 as primer pairs and P1 and P2 as probes. 10.0μL 2×ProbeqPCRPremixEx Taq TM , 0.2μL ROX Reference DyeⅡ, 1.2μL (10μM) F1, 1.2μL (10μM) R1, 1.2μL (10μM) P1, 1.2μL (10μM) P2, 2.0μL of various DNA samples, add sterile water to 20.0μL. The reaction program of fluorescent PCR is: 94℃5min; 40 cycles (94℃30s, 58℃30s, 72℃1min); 72℃5min.

[0037] PCR amplification was performed on these DNA samples using F1\R1 as primer pair and P3 as probe. The amplification reaction system was: 10.0 μL 2×ProbeqPCRPremixEx Taq TM , 0.2μL ROX Reference DyeⅡ, 1.2μL (10μM) F1, 1.2μL (10μM) R1, 1.2μL (10μM) P3, 2.0μL of various DNA samples, add sterile water to 20.0μL. The reaction program of fluorescent PCR is: 94℃5min; 40 cycles (94℃30s, 58℃30s, 72℃1min); 72℃5min.

[0038] F2\R2 were used as primer pairs to perform PCR amplification on these DNA samples. 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.), 2μL template, 1μL (10μM) F4, 1μL (10μM) R4. The reaction program was: 94℃5min; 94℃30s, 58℃30s, 72℃1min, 35 cycles; 72℃5min.

[0039] Figure 4The red line in the middle is the fluorescence curve of the DNA sample of the dog earwax sample using F1\R1 as a primer pair and P1 and P2 as probes, the blue line is the fluorescence curve of the DNA sample of the dog earwax sample using F1\R1 as a primer pair and P3 as a probe, and the rest are the amplification fluorescence curves of the DNA sample of the rabbit itch mite sample and five common mites using F1\R1 as a primer pair and P1 and P2 as probes, and the amplification fluorescence curves of the DNA sample of the rabbit itch mite sample and five common mites using F1\R1 as a primer pair and P3 as a probe. It can be seen that the use of F1\R1 as a primer pair and P1 and P2 as probes can effectively detect the dog earwax sample, while negative samples and the other five common mites are not detected, indicating that the primers and probes provided by the present invention are very specific. In addition, although the fluorescence curve can be amplified by using F1\R1 as the primer pair and P3 as the probe, the cycle number threshold is high and the S-shaped curve is not amplified, which again shows that its sensitivity is not as good as the group using P1 and P2 as probes. In addition, substituting the Ct value of the red line (24.6) into the standard equation (y=-1.2x+39.078), it can be calculated that the DNA content in the DNA sample of the canine earwax sample is 1.16144861e+12E copies / μL. The Ct value of the blue line cannot be read and its standard curve is not linear, which cannot meet the quantitative detection requirements.

[0040] 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. 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 other samples had no target bands, indicating that it could not distinguish between the dog earwax sample and the rabbit itch mite sample.

[0041] Fluorescence immunochromatography Primers Table 2

[0042] (2) Preparation of DNA samples for testing The swab was squeezed and the sample genomic DNA was extracted using a magnetic bead DNA extraction kit (50T Nanjing Dongna Biotechnology Co., Ltd.) according to the instructions. Viral genomic DNA was extracted using TRNzol reagent according to the instructions. The concentration and purity of all DNA samples were measured by Nano-300 ultra-micro spectrophotometer. After extraction, DNA / samples were stored at –80°C until the next step.

[0043] (3) Preparation of streptavidin fluorescent microspheres Take 15μL (8μM) of fluorescent microspheres (EmissionPeak550nm) and 540μL borate buffer (0.05μM, pH5.0), mix well, add 1.70mg EDC and 2.3mg NHSS, stir magnetically for 30min. Add 7.2μLSA (streptavidin, 500μg / mL), adjust pH to 7.4 with NaOH (0.1μM), react for 15min, and block with 10% glucosamine for 30min. Centrifuge at 20000r / min, 4℃ for 30min, discard the supernatant, and re-dissolve the precipitate with phosphate buffer (pH7.4) containing 0.1% NaNO3, and store at 4℃ for use.

[0044] (4) Biotin-conjugated BSA Take out the active esterified biotin from the refrigerator, return to room temperature, prepare 10mμM biotin stock solution with phosphate buffer (pH5.0), mix with BSA (5mg / mL, pH7.8) at a feed ratio (molar ratio) of 20:1, and incubate at room temperature (25℃) at 180r / min for 45min. Put the mixed solution into the treated dialysis bag and dialyze at 4℃ for 2~3d, changing the dialysis solution every 6h to remove unbound biotin.

[0045] (5) Preparation of immunohistochemical test strips 1) Use hydrophilic glass fiber to make sample pads and conjugate pads, soak the sample pads and conjugate pads in a mixed solution of buffer and effector (each pad corresponds to 39 mL of mixed solution), take them out after they are completely soaked, and dry them in a drying room (temperature 18℃-26℃, humidity 20%-28%) for 4-5 hours. Spray fluorescent microspheres (5 mg / mL) labeled with streptavidin on the conjugate pad at a rate of 5 µL / cm, dry them in a drying room overnight, and obtain a conjugate pad fixed with a probe and a treated sample pad. Among them, the basic component of the buffer is pH 9.0, 0.02M phosphate buffer, and the main components of the effector are 1wt% PEG with a molecular weight of 4000-20000, 0.05wt% casein, 0.05wt% fetal bovine serum albumin, and 0.01wt% trehalose.

[0046] 2) Select a nitrocellulose membrane with a water flow rate of 90s, place the nitrocellulose membrane in a closed box with a humidity of 25%-65% for 1h, dilute 48μg of anti-digoxigenin antibody (Wuxi Zhongdeber Biotechnology Co., Ltd.) to 2mg / mL with Tris-HCl buffer at pH 6.5, and dilute 0.48μg of biotin-modified BSA to 20μg / mL with buffer. Streak the above two antibodies on the nitrocellulose membrane with a streaking volume of 0.8µL / cm. After streaking, dry it for use. The buffer for diluting the antibody and BSA is a phosphate buffer at pH 7.4 and 0.05M.

[0047] 3) Paste the nitrocellulose membrane prepared in step 2) in the middle of the sticky PVC bottom plate, paste absorbent paper on one end of the nitrocellulose membrane, and the overlap width of the two is 2 mm. The other end of the nitrocellulose membrane is connected to the binding pad, and the two are staggered with an overlap width of 4 mm. Cover the sample pad on the binding pad, fix the bottom plate, and finally cut it into immunochromatographic test strips.

[0048] 4) Add the cut immunochromatographic test strips to the card shell, and set the sample loading window and signal reading window on the upper cover, where the sample loading window corresponds to the sample pad, and the signal reading window corresponds to the detection line and quality control line.

[0049] (6) Detection of fluorescent immunochromatographic test strips The 5' end of the upstream primer is labeled with digoxigenin, and the 5' end of the downstream primer is labeled with biotin. In this way, one strand of the double-stranded DNA obtained by PCR amplification is labeled with digoxigenin and the other strand is labeled with biotin. During the chromatography process of the test strip, on the one hand, the double-labeled DNA double strands bind to the quantum dots (QDs-SA) labeled with streptavidin biotin to form a QDs-SA-biotin-DNA complex. On the other hand, on the test line (T line), the double-labeled DNA double strands bind to digoxigenin monoclonal antibody to form a QDs-SA-biotin-DNA-digoxigenin-digoxigenin monoclonal antibody complex, thereby coloring the T line. The quantum dot QDs-SA binds to the biotin coupled to BSA on the quality control line (C line) to form a QDs-SA-biotin-BSA complex, thereby coloring the C line. This method ensures the high specificity of the test results.

[0050] PCR reaction system: 1 μL each of F5\R5 or F6\R6 (0.1 μM), 10 μL 2×SYBR® Premix ExTaq™, 7 μL ddH2O, 1 μL template DNA. Amplification reaction program: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 5 s, 62℃ annealing for 60 s, 72℃ extension for 20 s, for a total of 42 cycles; 72℃ extension for 10 min. Each sample was repeated 3 times.

[0051] Take 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 to the sample pad of the immune layer test strip. After 10 minutes, the images of the test line (test line, T) and the control line (control line, C) were recorded by a fluorescent imager, and the fluorescence signal value (relative fluorescence unit, RFU) of the test strip was read by a fluorescent quantitative analyzer Nanoeasy 1700. 4 μL of 10 g / 100 mL BSA and 80 μL of PBS (0.01 μM, pH 7.4) buffer solution was used as the control (NC).

[0052] (7) Results Figure 6 The figure shows the T-line and C-line fluorescence signal values ​​of the immunochromatographic detection of 6 kinds of mite DNA samples amplified by using F5 and R5 as primers, and the T-line and C-line fluorescence signal values ​​of the immunochromatographic detection of 6 kinds of mite DNA samples amplified by using F6 and R6 as primers. Figure 6 As shown in the figure, the C line of the immunochromatographic test of the six mite DNA samples amplified by using F5 and R5 as primers was 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 there was no obvious detection for the other five samples. However, the T line and C line fluorescence signal values ​​of the immunochromatographic test of the rabbit itch mite DNA samples amplified by using F6 and R6 as primers were comparable and significantly higher than the NC group, indicating that the use of F6 and R6 for detection 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.

[0053] Furthermore, five gradients (1.0×10 6 , 1.0×10 4 , 1.0×10 2 , 10, 1 copy / μL) standard plasmid with the target sequence of SEQ ID NO: 1 was used as a template, F5 and R5 were used for PCR amplification, and the amplified products were detected by fluorescent immunochromatographic test paper. 6 , 1.0×10 4 , 1.0×10 2 , 10, 1 copy / μL) standard plasmid with the target sequence of SEQ ID NO: 2 was used as a template, F6 and R6 were used for PCR amplification, and the amplified products were detected by fluorescent immunochromatographic test paper.

[0054] The results are as follows Figure 7 As shown, F5 and R5 were used as primer pairs 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 that was not much different from the C line was detected in 5 gradients, indicating that the F5 and R5 provided by the present invention as primer pairs and the fluorescent immunochromatographic test paper can detect 1 copy / μL of the target sequence, and the sensitivity is high. However, F6 and R6 were used as primer pairs to amplify the standard plasmid with the target sequence of SEQ ID NO: 2 and the fluorescent immunochromatographic test paper was used for detection, and only the samples of the first 3 gradients could be detected, indicating that the use of F6 and R6 as primer pairs and the fluorescent immunochromatographic test paper can only detect the target sequence of 100 copies / μL at the lowest, and the sensitivity is not as good as that of the experimental group.

[0055] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. Use of primer pairs and probes in the preparation of a kit for detecting canine insect-borne pathogenic microorganisms, the detection comprising: Extract DNA samples of canine vector-borne pathogenic microorganisms; Performing fluorescent quantitative PCR amplification on the substrate using the primer pair and the probe; Determine the cycle threshold of the DNA sample according to the fluorescence curve; The DNA content of canine insect-borne pathogenic microorganisms is determined according to the cycle threshold value and the standard equation, wherein the standard equation is y=-1.2x+39.078; The reaction system of fluorescent PCR includes: 10.0 μL 2× Probe qPCR Premix Ex Taq ™, 0.2 μL ROX Reference Dye II, 1.2 μL, 10 μM of F1 as shown in SEQ ID NO: 3, 1.2 μL, 10 μM of R1 as shown in SEQ ID NO: 4, 1.2 μL, 10 μM of P1 as shown in SEQ ID NO: 5, 1.2 μL, 10 μM of P2 as shown in SEQ ID NO: 6, 2.0 μL of a standard plasmid carrying the sequence shown in SEQ ID NO: 1, and 3 μL of sterile water; The reaction procedure of fluorescent PCR is as follows: Pre-denaturation at 94°C for 5 min; Denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min constituted one cycle, and a total of 40 cycles were performed; and Extension at 72°C for 5 min.

2. The method according to claim 1, wherein the step of extracting DNA samples of canine insect-borne pathogenic microorganisms comprises: (1) Add 600 μL of lysis buffer to each 2 mL EP tube that has been loaded with samples, mix by pipetting or shaking, incubate the EP tube at 65°C for 25-30 min, take it out, wait for it to cool 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) Take as much clean supernatant as possible and put it into a new 2 mL EP tube. Add an equal volume of binding solution and 40 μL of 30 mg / mL silanol magnetic beads. Mix by inversion for 1 min. Let stand for 3 min. Place the EP tube on a magnetic separation rack for magnetic separation and discard the waste liquid. (3) Washing: 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 blow and 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 in a microwave oven on high for 2 min and 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 1 μL of loading buffer and mix it. Then 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 min. After the end, observe the results under ultraviolet light.

3. Use of a primer pair in the preparation of a kit for detecting canine insect-borne pathogenic microorganisms, the detection comprising: Extract DNA samples of canine vector-borne pathogenic microorganisms; PCR amplification was performed using the primer pair, wherein the primer pair was F5: 5'-Dig-ggtatttgaagaggaatgttggggactag, digoxigenin-labeled, and R5: 5'-Bio-gatgaccaaaaaaccaaaataaatgttgataaagaatagg, biotin-labeled; Take 4 μL of PCR product, 4 μL of 10 g / 100 mL 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 detection line and the quality control line is measured; and Determine the DNA content of canine insect-borne pathogenic microorganisms based on the fluorescence signal value; The PCR reaction system includes: 1 μL, 0.1 μM F5, 1μL, 0.1μM R5, 10μL of 2×SYBR®PremixExTaq™, 7 μL ddH2O 1 μL of DNA sample; The amplification reaction procedure is: Pre-denaturation at 95°C for 10 min; Denaturation at 95°C for 5 s, annealing at 62°C for 60 s, and extension at 72°C for 20 s constituted one cycle, and a total of 42 cycles were performed; Extension at 72°C for 10 min.

4. The use according to claim 3, wherein the preparation steps of the immunochromatographic test strip include: 1) Use hydrophilic glass fiber to make sample pad and conjugate pad, soak the sample pad and conjugate pad in the mixed solution of buffer and effector, take them out after soaking, and dry them in a drying room for 4-5 hours. The temperature of the drying room is 18℃-26℃, and the humidity of the drying room is 20%-28%; 2) Spraying 5 mg / mL of fluorescent microspheres labeled with streptavidin on the conjugate pad at a volume of 5 µL / cm, and drying in a drying room overnight to obtain a conjugate pad fixed with the fluorescent microspheres labeled with streptavidin and a treated sample pad; wherein the basic component of the buffer is a phosphate buffer of pH 9.0 and 0.02 M, and the main component of the effector is 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; 3) Select a nitrocellulose membrane with a water flow rate of 90s, place the nitrocellulose membrane in a closed box with a humidity of 25%-65% for 1h, dilute 48μg of anti-digoxigenin antibody to 2mg / mL with Tris-HCl buffer at pH 6.5, and dilute 0.48μg of biotin-modified BSA to 20μg / mL with buffer, and streak the above two antibodies on the nitrocellulose membrane with a streak volume of 0.8μL / cm; after streaking, dry it for later use; the buffer for diluting the antibody and BSA is a phosphate buffer at pH 7.4 and 0.05M; 4) Pasting the nitrocellulose membrane prepared in step 3) on the middle of the sticky PVC bottom plate, pasting absorbent paper on one end of the nitrocellulose membrane, with the overlap width of the two being 2 mm; connecting the binding pad on the other end of the nitrocellulose membrane, with the two interlaced with each other, with an overlap width of 4 mm; covering the binding pad with the sample pad, fixing the bottom plate, and finally cutting into immunochromatographic test strips; 5) Add the cut immunochromatographic test strips to the card shell, and set the sample loading window and signal reading window on the upper cover, where the sample loading window corresponds to the sample pad, and the signal reading window corresponds to the detection line and quality control line.

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