Primer probe composition for detecting chicken coccidiosis oocysts based on RAA-LFD and application of primer probe composition

Abstract

The invention relates to a primer probe composition for detecting chicken coccidiosis oocysts based on RAA-LFD and application of the primer probe composition. The specific primer and the probe are designed according to the specific sequence of the chicken coccidiosis 18S rRNA gene, and the chicken coccidiosis 18S rRNA gene in a chicken manure sample can be qualitatively detected. Firstly, a target gene is subjected to isothermal amplification through an RAA method, then a lateral flow chromatography test strip is combined, finally, an amplification product is subjected to visual analysis, and the purpose of rapid detection can be achieved. The method has the advantages of being simple and convenient to operate, rapid in reaction, high in sensitivity, good in specificity, visual in reaction result and the like, is suitable for rapid diagnosis of clinical samples and laboratories, and has a good application prospect in prevention and control of chicken coccidiosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular diagnosis of animal diseases, and in particular relates to a chicken coccidia fecal oocyst visualization detection test strip based on recombinase-mediated isothermal amplification (RAA) combined with a lateral flow chromatography test strip (LFD) and its application. Background Art

[0002] Coccidiosis is a parasitic disease caused by one or more coccidia species of the genus Eimeria that infect intestinal epithelial cells, severely threatening the intestinal health of chickens. It is a major disease that severely impacts the development of the poultry industry. Seven recognized pathogens are E. tenella, E. necatrix, E. maxima, E. acervulina, E. mitis, E. praecox, and E. brunetti. Coccidiosis is a global disease, causing economic losses exceeding £10 billion annually. In the United States, coccidiosis ranked first among the major diseases affecting the broiler industry for seven consecutive years (2016-2022). In the United Kingdom, coccidiosis ranks among the top three most economically devastating poultry diseases.

[0003] Rapid detection of chicken coccidiosis is a crucial tool for timely disease prevention and control, minimizing economic losses. Currently, the clinical diagnosis of chicken coccidiosis relies primarily on traditional fecal examinations. While feasible, these tests require specialized personnel, are complex, and time-consuming. Molecular diagnostic techniques include PCR and fluorescent quantitative PCR. These techniques place relatively high demands on the associated instrumentation and equipment. Therefore, new, simple, and rapid detection methods are urgently needed.

[0004] The 18S genomic sequence is located in the coding region and performs coding functions. It is subject to strong selective pressure and has a slow evolutionary rate. Its gene sequence is highly conserved in most organisms. Studies have shown that the 18S rRNA gene has an interspecies homology of 92.9% to 99.4% in chicken coccidia. The 18S rRNA gene is a good target for molecular detection.

[0005] Recombinase-mediated isothermal amplification (RAA) technology utilizes recombinase, single-stranded binding proteins, and polymerase to carry out the reaction. It does not require thermostable enzymes or complex thermal cyclers, and boasts rapid reaction speed, strong specificity, and high sensitivity. Combining RAA with lateral flow dipsticks (LFD) allows for visualization of test results.

[0006] In existing technologies, traditional fecal examinations rely on professionals to identify the morphological characteristics of chicken coccidia (such as oocyst size and degree of sporulation), which is time-consuming and prone to misjudgment. In terms of molecular detection, conventional PCR takes more than 3 hours and relies on a thermal cycler (equipment cost > 50,000 yuan / unit), and the fluorescent quantitative PCR method also requires expensive instruments and equipment. Summary of the Invention

[0007] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and to provide a method and test strip for visual detection of chicken coccidia fecal oocysts based on the combination of RAA and LFD.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] A primer-probe combination for detecting chicken coccidia or their oocysts by RAA-LFD, wherein the upstream primer is shown as SEQ ID NO.1, the downstream primer is shown as SEQ ID NO.2, and the probe sequence is FAM-TAGGGTATTGGCCTACCGTGGCAGTGACGG[THF]TAACGGGGAATTAGG-C3, wherein the downstream primer is labeled with biotin at its 5' end; the 5' end of the probe is labeled with FAM fluorescein, the +31 position is labeled with a THF residue, and a blocking group C3 is added to the 3' end.

[0010] The primer-probe combination is used in preparing a reagent for detecting chicken coccidia using RAA-LFD.

[0011] As a preferred embodiment of the present invention, the primer-probe combination is used in the preparation of a reagent for detecting chicken coccidia oocysts using RAA-LFD.

[0012] As a further preferred embodiment of the present invention, the primer-probe combination is used in the preparation of a reagent for detecting chicken coccidia oocysts in chicken feces using RAA-LFD.

[0013] A method for detecting chicken coccidia oocysts in chicken feces by using RAA-LFD, wherein the primer-probe combination is used to detect DNA samples extracted from the chicken feces by using RAA-LFD.

[0014] As a preferred embodiment of the present invention, the method comprises a method for extracting DNA samples from chicken feces:

[0015] (1) 1 g of chicken coccidia fecal oocyst sample was mixed with 15 mL of deionized water, stirred thoroughly, and filtered using a funnel. The filtrate was collected in a 15 mL centrifuge tube and centrifuged at 2000 g for 5 min.

[0016] (2) Discard the supernatant, resuspend the pellet in 3 mL of 20% NaClO solution, and incubate at room temperature for 10 min.

[0017] (3) After incubation, add 10 mL of deionized water to the centrifuge tube and centrifuge at 2000 g for 5 min. Discard the supernatant. Repeat twice. Resuspend the precipitate in 1 mL of deionized water and transfer it to a 1.5 mL centrifuge tube and centrifuge at 2000 g for 5 min.

[0018] (4) Discard the supernatant, add an equal volume of glass beads and 100 μL of oocyst lysis buffer to the precipitate, and vortex for 5 min until the oocysts are completely ruptured. The formula of the oocyst lysis buffer is 10 mM Tris-base, 10 mM Na2EDTA·2H2O, 1.5 mg / mL SDS, 100 mM NaCl, pH = 8.0;

[0019] (5) Centrifuge at 10700 g for 10 min at room temperature. The supernatant is the DNA sample, which is used as a template for subsequent PCR and RAA amplification.

[0020] As a further preferred embodiment of the present invention, the method comprises the following steps:

[0021] (1) extracting DNA samples from chicken feces according to the method described in claim 6;

[0022] (2) Using 1 μL of DNA sample as a template, the primer-probe combination of claim 1 was used for RAA reaction at 37°C for 25 min. The reaction system was as follows: total volume 25 μL, buffer 12 μL, upstream primer F (1 μM) 1 μL, downstream primer R (1 μM) 1 μL, probe 1 μL, DNA template 1 μL, purified water 7 μL, and magnesium acetate 2 μL;

[0023] (6) Take 10 μL of the reaction product and add it to 90 μL of 1× PBS, mix well, insert the test strip, and read the result after 2 minutes. A negative result is a red strip only on the quality control line, and a positive result is a red strip on both the quality control line and the test line.

[0024] A RAA-LFD kit for detecting chicken coccidia oocysts in chicken feces, comprising:

[0025] (1) DNA extraction reagents: deionized water, 20% NaClO aqueous solution, 0.4-0.6 mm glass beads, oocyst lysis buffer, the formula of the oocyst lysis buffer is 10 mM Tris-base, 10

[0026] mMNa2EDTA·2H2O, 1.5mg / mLSDS, 100mMNaCl, pH=8.0;

[0027] (2) The primer-probe combination of claim 1;

[0028] (3) Other reagents for the RAA reaction;

[0029] (4)LFD test strips.

[0030] The present invention is based on the following findings:

[0031] RAA-LFD is a convenient and rapid detection technology with promising application prospects in animal disease diagnosis. However, the use of the 18S rRNA gene, a component of the chicken coccidia genome, in the preparation of RAA-LFD rapid detection kits has not been reported.

[0032] The present invention has the following advantages and effects:

[0033] 1. The present invention designs specific primers and probes based on the specific conserved target sequence of the 18S rRNA gene of chicken coccidia, which can be used to qualitatively detect the 18S rRNA gene of chicken coccidia oocysts in chicken feces.

[0034] 2. By adopting RAA-LFD, rapid visual inspection is possible.

[0035] 3. This invention combines recombinase-mediated isothermal amplification (RAA) with lateral flow chromatography (LFD) for the first time.

[0036] Combined with chicken coccidiosis detection, this technology achieves the following breakthroughs: ① On-site visual detection, with the entire test process taking less than half the time required for conventional PCR; ② Low cost, requiring no specialized equipment, with a single test costing less than 10 yuan. This patent enables immediate, on-site detection of chicken coccidiosis at the grassroots level. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 PCR amplification products of four pairs of RAA primers

[0038] M: DL-2000 Marker; N1 to N4: negative control

[0039] Figure 2 RAA amplification products of four pairs of RAA primers

[0040] M: DL-2000 Marker; N1 to N4: negative control

[0041] Figure 3 The results of five species of chicken coccidia F1 / R1 primers RAA amplification

[0042] M: DL-2000 Marker; 1-5: E. tenella, E. necatrix, E. acervulina, E. maxima, E. mitis; N: negative control

[0043] Figure 4 The results of RAA amplification using primers F1 / R1 at different times

[0044] M: DL-2000 Marker, N: negative control

[0045] Figure 5 Comparison of PCR amplification results for fecal oocyst DNA extracted using methods 1 and 2. M: DL-2000 Marker; 1: DNA extracted using method 1; 2: DNA extracted using method 2; N: negative control.

[0046] Figure 6 Comparison of RAA amplification results for fecal oocyst DNA extracted using methods 1 and 2. M: DL-2000 Marker; 1: DNA extracted using method 1; 2: DNA extracted using method 2; N: negative control.

[0047] Figure 7 Sensitive PCR results of chicken coccidia fecal oocyst DNA extracted for method 2

[0048] M: DL-2000 Marker; P: positive control; 1-5 are DNA extracted from 50, 100, 300, 700, and 1100 oocysts added to 1 g of feces, respectively; N: negative control

[0049] Figure 8 Schematic diagram of RAA-LFD test strip

[0050] Figure 9 Schematic diagram for interpreting RAA-LFD test results

[0051] Figure 10 Comparison of RAA-LFD detection with different probe concentrations

[0052] P: positive group; T: experimental group; P1: DNA template added, probe concentration 1μM; TI: no DNA template added, probe concentration 1μM; P2: DNA template added, probe concentration 100nM; T2: no DNA template added, probe concentration 100nM; P3: DNA template added, probe concentration 10nM; T3: no DNA template added, probe concentration 10nM

[0053] Figure 11 Optimal reaction time selection for RAA-LFD

[0054] N: Negative control

[0055] Figure 12 Evaluation of the specificity of the RAA-LFD method

[0056] 1-9: E. tenella, E. necatrix, E. acervulina, E. maxima, E. mitis, chicken intestinal tissue, E. coli, Clostridium perfringens, negative fecal genomic DNA; N: negative control

[0057] Figure 13 Sensitive detection of genomic DNA for RAA-LFD

[0058] 1-5: 86 ng / μL-8.6 pg / μL chicken coccidia genomic DNA; N: negative control

[0059] Figure 14 Results 1-5 are DNA extracted from 50, 100, 300, 700, and 1100 oocysts added to 1 g of feces using the RAA-LFD method; N: negative control

[0060] Figure 15 Repeatability test for RAA-LFD

[0061] P1: positive control for the first test; N1: negative control for the first test; P2: positive control for the second test; N2: negative control for the second test; P3: positive control for the third test; N3: negative control for the third test DETAILED DESCRIPTION

[0062] Basic Materials:

[0063] 1. Chicken coccidia, chicken intestinal tissue, Escherichia coli, and Clostridium perfringens genomic DNA were provided by the Veterinary Parasitology Laboratory of Nanjing Agricultural University.

[0064] 2. Reagents: 2× Magic Green Taq SuperMix was purchased from Tolu Biotechnology Co., Ltd.; DL2000 Plus DNA Maker was purchased from Nanjing Novozymes Biotechnology Co., Ltd.; agarose was purchased from White Shark Biotechnology; RAA Nucleic Acid Amplification Kit, RAA Nucleic Acid Amplification Kit (Test Strip Method), and Nucleic Acid Detection Strips (Biotin / FAM) were all purchased from Jiangsu Qitian Biotechnology Co., Ltd.; Tris-phenol chloroform isoamyl alcohol (25:24:1) was purchased from Shanghai Meigen Biotechnology Co., Ltd.; 0.4-0.6 mm glass beads and OMEGA Stool DNA Kit were both purchased from Omega Biotechnology; sodium hypochlorite and Tris-base were purchased from White Shark Biotechnology; sodium chloride and Na2EDTA·2H2O were both purchased from Sinopharm Chemical Reagent Co., Ltd.; and SDS was purchased from Sangon Biotechnology.

[0065] 3. Clinical test samples: A total of 14 chicken feces samples were collected from Zhangli Breeder Farm in Tai'an, Shandong, Hutun Breeder Farm in Tai'an, Shandong, and Xinbei Breeder Farm in Changzhou, Jiangsu.

[0066] 4. Main instruments and equipment: digital constant temperature water bath (Changzhou Noki Instrument Co., Ltd.); PCR instrument (Hangzhou Bioer Technology Co., Ltd.); micropipette (Thermo Fisher Scientific, USA); vortex mixer (Dalong Instrument); handheld centrifuge (Scilogex); desktop low-speed centrifuge (Kecheng Instrument); high-speed centrifuge (Eppendorf), and funnel.

[0067] Example 1 Primer design and screening

[0068] 1. Primer design

[0069] The present invention used DNAMAN to compare and analyze the homology of the 18S rRNA gene sequences of seven species of chicken coccidia and determined the conserved region (SEQ ID NO.7). Based on the conserved region, four pairs of RAA primers were designed using Primer Premier 6. The designed primer pairs and product sizes are shown in Table 1.

[0070] Table 1 RAA primers

[0071]

[0072] 2. Primer screening

[0073] The designed RAA primers based on 18S rRNA gene of chicken coccidia were screened by PCR and RAA-AGE to determine the best reaction primers.

[0074] (1) Add 12.5 μL of 2× Taq enzyme, 1 μL of upstream and downstream primers, 1 μL of E. tenella gDNA, and 9.5 μL of water to a PCR tube. Place the tube in a PCR instrument and set the program to pre-denaturation at 95°C for 3 min, denaturation at 95°C for 10 s, annealing at 58°C for 10 s, extension at 72°C for 15 s, and complete extension at 72°C for 5 min for 30 cycles. After the PCR reaction is completed, perform electrophoresis on a 2% agarose gel.

[0075] The results are as follows Figure 1 As shown, all four primer pairs amplified the target bands, and all four primer pairs showed good amplification efficiency.

[0076] (2) Use RAA combined with AGE to screen amplification primers. The RAA reaction was performed according to the instructions with slight improvements. 25 μL of buffer, 2 μL of upstream and downstream primers, and 14 μL of purified water. After thorough mixing, pipette all of the mixture into the reaction tube, flick the tube wall lightly with your hand, and centrifuge for 5 seconds to mix the system. Then divide it evenly into two enzyme-free PCR tubes, and add 1 μL of purified water to the negative control group. Then, add 2.5 μL of magnesium acetate solution to the cap of each centrifuge tube, cover the negative control group, and add 1 μL of E. tenella gDNA to the test group and cover it. Flick the tube wall lightly and centrifuge briefly, then place the reaction tube in a water bath at 37°C for 30 minutes. After the reaction is complete, add 25 μL of phenol / chloroform / isoamyl alcohol (V:V:V=25:24:1) to the centrifuge tube, shake it thoroughly, centrifuge it for 2 minutes, and take the supernatant for 2% agarose gel electrophoresis detection. The performance of the primers was evaluated based on whether the amplified bands of the four primer pairs were single and bright, so as to screen out the best primers.

[0077] The results are as follows Figure 2 As shown, all four pairs of primers amplified the target bands. The amplified band of the first pair of primers was single and bright, while the bands amplified by the second, third, and fourth pairs of primers were weaker. The amplification efficiency of the first pair of RAA primers was good, so the first pair of primers (SEQ ID NO.1, SEQ ID NO.2) was selected for subsequent experiments.

[0078] (3) Verify the ability of the selected primers to amplify five species of chicken coccidia and the reaction time. To verify the ability of the selected primers to amplify five species of chicken coccidia, genomic DNA of tender, toxic, pile-shaped, giant, and mild Eimeria oocysts were added to the RAA systems of the five test groups respectively; for the reaction time, the prepared RAA reaction system was reacted in a water bath at 37°C for 10 min, 15 min, 20 min, 25 min, and 30 min. After the reaction, 25 μL of phenol / chloroform / isoamyl alcohol (25:24:1) was added to the centrifuge tube and fully shaken. The tube was centrifuged for 2 min in a handheld centrifuge, and the supernatant was used for 2% agarose gel electrophoresis.

[0079] The results are as follows Figure 3 、 Figure 4 As shown, both primer pairs SEQ ID NO. 1 and SEQ ID NO. 2 were able to amplify the 18S rRNA target gene of five species of Gallus gallus. Both primer pairs SEQ ID NO. 1 and SEQ ID NO. 2 amplified bands at 10, 15, 20, 25, and 30 minutes, and the bands gradually brightened. The bands reached their brightest after 20 minutes, and there was no significant difference in the brightness of the bands at 20, 25, and 30 minutes.

[0080] Example 2. Extraction of Oocyst DNA from Chicken Feces Samples

[0081] 1. Extraction of oocyst DNA from chicken coccidia feces (Method 1):

[0082] (1) Use OMEGA Stool DNA Kit to extract chicken coccidia fecal oocyst DNA. Weigh 200 mg of chicken coccidia-positive feces into a 2 mL centrifuge tube, add 200 mg of Glass beads X and 540 μL of SLX-Mlus Buffer, and vortex at maximum speed for 10 min.

[0083] (2) Add 60 μL DS Buffer and 20 μL Proteinase K, vortex mix for 2 min, and incubate in a 70°C water bath for 10 min, vortexing every 5 min.

[0084] (3) Add 200 μL SP2 Buffer and vortex mix at high speed for 30 seconds, place on ice for 5 minutes to facilitate precipitation, and then centrifuge at 13,000 g for 5 minutes at room temperature;

[0085] (4) Transfer 400 μL of supernatant to a new 1.5 mL centrifuge tube. Do not transfer to the precipitate. Add 200 μL of cHTR Reagent to the centrifuge tube and vortex for 10 seconds to mix.

[0086] (5) Place at room temperature for 2 min, then centrifuge at 13000g for 2 min at room temperature. Transfer 250 μL of supernatant to a new 1.5 mL centrifuge tube, add 250 μL of BL Buffer and 250 μL of anhydrous ethanol, and vortex for 10 s to mix.

[0087] (6) Place the HiBind DNA Mini Column in a 2 mL collection tube, transfer all the mixed solution to the HiBind DNA Mini Column, centrifuge at 13,000 g for 1 min, and discard the filtrate;

[0088] (7) Add 500 μL of VHB Buffer to the HiBind DNA Mini Column, centrifuge at 13,000 g for 1 min, and discard the filtrate;

[0089] (8) Add 700 μL DNA Wash Buffer, centrifuge at 13,000 g for 1 min, and discard the filtrate;

[0090] (9) Repeat step 8;

[0091] (10) Place the HiBind DNA Mini Column back into a 2 mL collection tube, centrifuge the empty column at 13,000 g for 2 min, and dry the column.

[0092] (11) Finally, place the HiBind DNA Mini Column in a new 1.5 mL centrifuge tube, add 50 μL of Elution Buffer preheated to 65 °C, let stand at room temperature for 2 min, and centrifuge at 13,000 g for 1 min to elute the DNA.

[0093] (12) The products were subjected to PCR and RAA detection, respectively. The reaction systems are shown in Tables 2 and 3.

[0094] Extraction of oocyst DNA from chicken feces (Method 2):

[0095] (1) 1 g of chicken coccidia fecal oocyst sample was mixed with 15 mL of deionized water, stirred thoroughly, and filtered using a funnel. The filtrate was collected in a 15 mL centrifuge tube and centrifuged at 2000 g for 5 min.

[0096] (2) Discard the supernatant, resuspend the pellet in 3 mL of 20% NaClO solution, and incubate at room temperature for 10 min.

[0097] (3) After incubation, add 10 mL of deionized water to the centrifuge tube and centrifuge at 2000 g for 5 min. Discard the supernatant. Repeat twice. Resuspend the precipitate in 1 mL of deionized water and transfer it to a 1.5 mL centrifuge tube and centrifuge at 2000 g for 5 min.

[0098] (4) Discard the supernatant, add an equal volume of glass beads and 100 μL of oocyst lysis buffer to the precipitate, and vortex for 5 min until the oocysts are completely ruptured. The formula of the oocyst lysis buffer is 10 mM Tris-base, 10 mM Na2EDTA·2H2O, 1.5 mg / mL SDS, 100 mM NaCl, pH = 8.0;

[0099] (5) Centrifuge at 10700 g for 10 min at room temperature. The supernatant is used as a template for subsequent PCR and RAA amplification. The reaction systems are shown in Tables 2 and 3.

[0100] Table 2 DNA extraction method comparison PCR reaction system (25μL)

[0101]

[0102] Table 3 Comparison of DNA extraction methods RAA reaction system (25 μL)

[0103]

[0104] The results are as follows Figure 5 、 Figure 6As shown, the results show that both method 1 and method 2 can successfully extract fecal egg DNA, and the bands are bright. Compared with method 1, method 2 does not require too many steps and instruments, is simple to operate and takes less time. Method 2 was used to extract DNA in subsequent experiments.

[0105] 2. Sensitivity test of the method for extracting oocyst DNA from chicken feces

[0106] After washing, laboratory-stored oocysts were added to 1 g of negative chicken feces at the number of 50, 100, 300, 700, and 1100 to make positive chicken feces samples. DNA was extracted from 1 g of negative chicken feces samples using the same method as a negative control. DNA was extracted from positive chicken feces samples using the OMEGA Stool DNA Kit as a positive control to test the sensitivity of DNA extraction using method 2. PCR was used to detect the extracted DNA.

[0107] Add 12.5 μL of 2× Taq enzyme, 1 μL of upstream and downstream primers, 1 μL of template, and 9.5 μL of water into the PCR tube, place it in the PCR instrument, set the program to pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 10 seconds, annealing at 58°C for 10 seconds, extension at 72°C for 15 seconds, and complete extension at 72°C for 5 minutes, 30 cycles, and perform electrophoresis on 2% agarose gel after the PCR reaction is completed.

[0108] The results are as follows Figure 7 As shown, after 100 oocysts were counted, a band could be observed, and as the number of oocysts increased, the band gradually became brighter.

[0109] Example 3 Optimization of RAA-LFD reaction conditions

[0110] 1. Test strip composition and result interpretation

[0111] Lateral flow cytometry test strips (LFD) Figure 8 ) consists of a sample pad, a conjugate pad, an absorbent pad, a backing, an NC membrane test line (T line), and a quality control line (C line). When the target molecule is amplified by specifically labeled primers, a large number of amplicons labeled with FAM and biotin are produced. After the dual-labeled amplicons are dripped onto the sample pad of the test strip, they migrate from the sample pad to the absorbent pad under capillary action. Once they move to the conjugate pad of the test strip, the biotin on the amplicons binds to gold colloidal particles (AuNPs) conjugated with streptavidin, forming a FAM-dsDNA-biotin-streptavidin-AuNP complex. When this complex moves to the test line (T line), it binds to the FAM antibody applied to the test line, thereby intercepting the gold colloidal particles and displaying a red band. Excess streptavidin-conjugated AuNPs will continue to migrate to the test line (C line) and bind to the secondary antibody (biotinylated bovine serum albumin), displaying a red band.

[0112] Add the sample to be tested to the sample pad and observe the color of the T line and C line of the test strip ( Figure 9 Positive (+): The control line (C line) of the test strip is colored, and the test line (T line) is colored visible to the naked eye; Negative (-): The control line (C line) of the test strip is colored, and the test line (T line) is not colored; Invalid: The control line (C line) of the test strip is not colored, regardless of whether the test line (T line) is colored.

[0113] 2. Probe design

[0114] To visualize RAA amplification products on lateral flow test strips, a DNA probe was designed based on the appropriate target sequence between the optimal RAA upstream and downstream primers. The probe was labeled with a FAM antigen group at its 5' end, had a tetrahydrofuran (THF) abasic site incorporated internally, and had a blocker at its 3' end. The downstream primer was labeled with biotin at its 5' end, as shown in Table 4. Both primer and probe sequences were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0115] Table 4 RAA-LFD primers and probes

[0116]

[0117] 3. RAA-LFD probe concentration screening

[0118] The probe was diluted from 1 μM to 100 nM and 10 nM for reaction. The positive control group (P group) used E. tenella gDNA as template. The experimental group (T group) did not add template but only added the probe and primers. The T group concentration range in which no T line appeared was selected for subsequent studies. The T group reaction system included a total volume of 25 μL, 12 μL buffer, 1 μL upstream primer F (1 μM), 1 μL downstream primer R (1 μM), 1 μL probe (1 μM, 100 nM, 10 nM), 8 μL purified water, and 2 μL of magnesium acetate added to the tube cap. The P group reaction system included a total volume of 25 μL, 12 μL buffer, 1 μL upstream primer F (1 μM), 1 μL downstream primer R (1 μM), 1 μL probe (1 μM, 100 nM, 10 nM), 1 μL DNA template, 7 μL purified water, and 2 μL of magnesium acetate added to the tube cap. Preheat a water bath to 37°C, add the above reagent components, label each reaction tube with the probe concentration, and centrifuge for 10 seconds to thoroughly mix the components. The reaction time is 30 minutes. After the reaction, add 10μL of the sample to 90μL of 1× PBS, shake to mix, insert the test strip, and wait for 2 minutes for the liquid to be drawn upwards before reading the result.

[0119] The results are as follows Figure 10As shown, a probe concentration of 100 nM was selected as suitable for use, and subsequent studies were carried out using the probe at this concentration.

[0120] 4. RAA-LFD reaction time optimization

[0121] Experiments were designed to verify RAA amplification at reaction times of 10, 15, 20, 25, and 30 minutes. To each component, 12 μL of buffer, 1 μL each of upstream and downstream primers and probes, 1 μL of E. tenella gDNA, 7 μL of purified water, and 2 μL of magnesium acetate were added to the cap of the tube. Preheat a water bath to 37°C and add the above reagent components. Mark the time for each reaction tube and centrifuge briefly for 10 seconds to thoroughly mix the components. The reaction time was 10-30 minutes. After the reaction, 10 μL of the solution was added to 90 μL of 1× PBS. Shake to mix thoroughly, insert the test strip, and wait for 2 minutes for the liquid to aspirate upwards before reading the result.

[0122] The results are as follows Figure 11 As shown in the figure, the band on the T line gradually brightened. Although the band appeared after 10 minutes of reaction, the band was dark and the detection stability was poor. The band on the T line reached the brightest after 25 minutes of reaction. Since there was no significant difference in the brightness of the band on the T line between the reaction times of 25 minutes and 30 minutes, in order to shorten the detection time, 25 minutes was used as the optimal reaction time in the subsequent reactions.

[0123] Example 4 Performance Analysis of RAA-LFD Method for Detecting Chicken Coccidia Fecal Oocyst DNA

[0124] 1. Evaluation of the specificity of the RAA-LFD detection method

[0125] The RAA-LFD assay was used to detect DNA from five chicken coccidia species and genomic DNA from other microorganisms present in the chicken intestine (chicken intestinal tissue, Escherichia coli, and Clostridium perfringens) to evaluate the specificity of the assay. To each component, 12 μL of buffer, 1 μL each of upstream and downstream primers and probes, 1 μL of DNA template, 7 μL of purified water, and 2 μL of magnesium acetate were added to the cap of the tube. The experimental steps are as follows:

[0126] (1) Preheat the water bath to 37°C and add the above reagent components.

[0127] (2) After marking the number of each reaction tube, use a handheld centrifuge to centrifuge for 10 seconds to fully mix the components. The reaction time is 25 minutes.

[0128] (3) After the reaction is complete, take 10 μL and add it to 90 μL of 1× PBS. Shake to mix well and insert the test strip. Wait for the liquid to be drawn upward for 2 minutes and then read the result.

[0129] The results are as follows Figure 12 As shown in the figure, the RAA-LFD method was unable to detect the DNA of several species except the DNA of chicken coccidia, which indicated that the RAA-LFD detection method had good specificity.

[0130] 2. Sensitivity evaluation of RAA-LFD detection method

[0131] (1) Using chicken coccidia genomic DNA as a template

[0132] The concentration of laboratory-stored E. tenella gDNA was determined to be 86 ng / μL using an ultramicrospectrophotometer. This DNA was diluted 10-fold to 8.6 pg / μL as a DNA template to test the sensitivity of the RAA-LFD assay. A negative control group was set up. To each component, 12 μL of buffer, 1 μL each of upstream and downstream primers and probes, 1 μL of DNA template, 7 μL of purified water, and 2 μL of magnesium acetate were added to the tube cap. The experimental steps are as follows:

[0133] (1.1) Preheat a water bath to 37°C and add the above reagent components.

[0134] (1.2) After marking each reaction tube, centrifuge for 10 seconds to thoroughly mix the components. The reaction time is 25 minutes.

[0135] (1.3) After the reaction is complete, take 10 μL and add it to 90 μL of 1× PBS. After shaking to mix, use a pipette to draw up 50 μL of liquid and drop it onto the sample pad of the test strip. Wait for 2 minutes before the liquid is drawn up and the result is read.

[0136] The results are as follows Figure 13 As shown in the figure, the method can detect a minimum of 8.6 pg / μL of chicken coccidia genomic DNA.

[0137] (2) DNA extracted from different oocyst numbers as templates

[0138] Use method 2 to extract DNA from chicken coccidia fecal oocysts. Add 50-1100 oocysts to 1g of negative chicken feces as a simulated sample for DNA extraction. The sensitivity of the RAA-LFD assay is then evaluated, and a negative control is set up. The negative control uses DNA extracted from negative feces as a template. To each component, add 12μL of buffer, 1μL each of upstream and downstream primers and probes, 1μL of DNA template, 7μL of purified water, and 2μL of magnesium acetate to the tube cap. The steps are as follows:

[0139] (1.1) Preheat a water bath to 37°C and add the above reagent components.

[0140] (1.2) After marking each reaction tube, centrifuge for 10 seconds to thoroughly mix the components. The reaction time is 25 minutes.

[0141] (1.3) After the reaction is complete, take 10 μL and add it to 90 μL of 1× PBS. After shaking to mix, use a pipette to draw up 50 μL of liquid and drop it onto the sample pad of the test strip. Wait for 2 minutes before the liquid is drawn up and the result is read.

[0142] The results are as follows Figure 14 As shown in the figure, the method can detect a stool sample containing at least 300 oocysts in 1 g of stool, indicating that it has good sensitivity.

[0143] 3. Repeatability evaluation of RAA-LFD detection method

[0144] Oocyst DNA extracted from positive chicken feces samples was subjected to RAA-LFD three times, with a negative control group set up each time. The results were observed to determine the repeatability of the RAA-LFD method. The reaction system and experimental steps are as follows:

[0145] Table 5 Repeatability evaluation reaction system of RAA-LFD detection method (25 μL)

[0146]

[0147] The experimental steps are as follows:

[0148] (1) Preheat the water bath to 37°C and add the above reagent components.

[0149] (2) After marking the number of each reaction tube, use a handheld centrifuge to centrifuge for 10 seconds to fully mix the components. The reaction time is 25 minutes.

[0150] (3) After the reaction is complete, take 10 μL and add it to 90 μL of 1× PBS. Shake to mix, then insert the test strip and wait for the liquid to be drawn upward for 2 minutes before reading the result.

[0151] The results are as follows Figure 15 As shown, the results of the positive and negative groups in three tests were stable, and the bands in the positive group were bright.

[0152] Example 5. Preliminary application of the RAA-LFD detection method for chicken coccidia fecal oocysts

[0153] The RAA-LFD test strips prepared by the present invention were used to test 14 clinical samples and compared with the corresponding stool test results and PCR test results (Table 6, Table 7). Among the 14 clinical samples, the stool test detected 1 positive sample and 13 negative samples, the PCR method detected 2 positive samples and 12 negative samples, and the RAA-LFD method of the present invention detected 1 positive sample and 13 negative samples. The total concordance rate between the RAA-LFD test results and the stool test results was 100%; the total concordance rate between the RAA-LFD test results and the PCR test results was 92.86%. The specific test results are shown in Table 8.

[0154] Table 6 Comparison of RAA-LFD and microscope detection results

[0155]

[0156] Table 7 Comparison of RAA-LFD and PCR detection results

[0157]

[0158]

[0159] Table 8 Detection of the sample by the test strips of the present invention, stool examination, and PCR

[0160]

[0161] Note: 1: “P” stands for Positive; “N” stands for Negative.

Claims

1. A primer-probe combination for detecting chicken coccidia or their oocysts by RAA-LFD, characterized in that: The upstream primer is shown as SEQ ID NO.1, the downstream primer is shown as SEQ ID NO.2, and the probe sequence is FAM-TAGGGTATTGGCCTACCGTGGCAGTGACGG[THF]TAACGGGGAATTAGG-C3, wherein the downstream primer is labeled with biotin at its 5' end; the 5' end of the probe is labeled with FAM fluorescein, the +31 position is labeled with THF residue, and a blocking group C3 is added to the 3' end.

2. Use of the primer-probe combination according to claim 1 in preparing a reagent for detecting chicken coccidia by RAA-LFD.

3. The use according to claim 2, characterized in that Use of the primer-probe combination according to claim 1 in preparing a reagent for detecting chicken coccidia oocysts by RAA-LFD.

4. The use according to claim 3, characterized in that Use of the primer-probe combination according to claim 1 in preparing a reagent for detecting chicken coccidia oocysts in chicken feces using RAA-LFD.

5. A method for detecting chicken coccidia oocysts in chicken feces using RAA-LFD, characterized in that: The primer-probe combination according to claim 1 is used to detect DNA samples extracted from chicken feces by RAA-LFD.

6. The method according to claim 5, characterized in that Contains a method for extracting DNA samples from chicken feces: (1) 1 g of chicken coccidia fecal oocyst sample was mixed with 15 mL of deionized water, stirred thoroughly, and filtered using a funnel. The filtrate was collected in a 15 mL centrifuge tube and centrifuged at 2000 g for 5 min. (2) Discard the supernatant, resuspend the pellet in 3 mL of 20% NaClO solution, and incubate at room temperature for 10 min. (3) After incubation, add 10 mL of deionized water to the centrifuge tube and centrifuge at 2000 g for 5 min. Discard the supernatant. Repeat twice. Resuspend the precipitate in 1 mL of deionized water and transfer it to a 1.5 mL centrifuge tube and centrifuge at 2000 g for 5 min. (4) Discard the supernatant, add an equal volume of glass beads and 100 μL of oocyst lysis buffer to the precipitate, and vortex for 5 min until the oocysts are completely ruptured. The formula of the oocyst lysis buffer is 10 mM Tris-base, 10 mM Na2EDTA·2H2O, 1.5 mg / mL SDS, 100 mM NaCl, pH = 8.0; (5) Centrifuge at 10700 g for 10 min at room temperature. The supernatant is the DNA sample, which is used as a template for subsequent PCR and RAA amplification.

7. The method according to claim 6, characterized in that The following steps are involved: (1) extracting DNA samples from chicken feces according to the method described in claim 6; (2) Using 1 μL of DNA sample as a template, perform RAA reaction with the primer-probe combination of claim 1 at 37°C for 10 min. The reaction system is as follows: total volume 25 μL, buffer 12 μL, upstream primer F (1 μM) 1 μL, downstream primer R (1 μM) 1 μL, probe 1 μL, DNA template 1 μL, purified water 7 μL, and magnesium acetate 2 μL; (6) Take 10 μL of the reaction product and add it to 90 μL of 1× PBS, mix well, insert the LFD test strip, and read the result after 2 minutes. A negative result is a red strip only on the quality control line, and a positive result is a red strip on both the quality control line and the test line.

8. A RAA-LFD kit for detecting chicken coccidia oocysts in chicken feces, characterized in that: Include: (1) DNA extraction reagents: water, 20% NaClO aqueous solution, 0.4-0.6 mm glass beads, and oocyst lysis buffer, wherein the oocyst lysis buffer is formulated as 10 mM Tris-base, 10 mM Na2EDTA·2H2O, 1.5 mg / mL SDS, 100 mM NaCl, pH = 8.0; (2) The primer-probe combination of claim 1; (3) Other reagents for the RAA reaction; (4)LFD test strips.

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