A quadruplex PCR primer set and kit for detecting chicken coccidia

Abstract

This invention discloses a quadruple PCR primer set and kit for detecting chicken coccidiosis, belonging to the field of chicken coccidiosis biodetection technology. Compared with the prior art, this invention can simultaneously obtain detection results for four types of chicken coccidiosis, greatly shortening the detection and identification time and providing a guarantee for timely prevention of the spread of chicken coccidiosis. Furthermore, the coccidia oocyst lysis buffer provided by this invention can quickly obtain DNA templates for quadruple PCR detection. The quadruple PCR primers provided by this invention have high detection specificity, low detection limit, good repeatability, and reliable results.

Description

Technical Field

[0001] This invention relates to the field of chicken coccidiosis biological detection technology, and more specifically to a quadruple PCR primer set and kit for detecting chicken coccidiosis. Background Technology

[0002] Coccidiosis in chickens is a parasitic disease caused by a mixed infection of one or more species of Eimeria, severely impacting the poultry industry and causing significant economic losses globally. Clinically, the oocysts excreted by infected chickens are the primary source of infection. Infected chickens exhibit loss of appetite, lethargy, ruffled feathers, and pale combs and visible mucous membranes. The most prominent symptom is bloody diarrhea, and in severe cases, death can occur within 5 days of infection. There are seven species of coccidia in chickens, with *Eimeria tenella* and *Eimeria virulence* being the most pathogenic, followed by *Eimeria spp.* and *Eimeria giantiformis*. The remaining three species are less pathogenic. Infections with *Eimeria tenella*, *Eimeria spp.*, and *Eimeria giantiformis* are the most common, and mixed infections of two or more species are frequently observed.

[0003] In the diagnosis and epidemiological investigation of this disease, traditional methods have always relied on the morphology, size, incubation period, parasitic intestinal segment, intestinal lesions, and shortest sporulation time of oocysts as differential diagnostic indicators. This method is still used in the differential diagnosis of coccidiosis in chickens. However, this method requires single oocyst isolation and purification, oocyst propagation, and case replication, taking at least one month, which is time-consuming and labor-intensive. Currently, another commonly used method is PCR identification. This method also requires the collection and purification of coccidia oocysts, extraction and purification of DNA, before further PCR identification of oocyst species. A complete purification and detection process takes about a day, which is still not conducive to timely detection and rapid prevention of the spread of coccidiosis. Furthermore, current PCR identification methods for chicken coccidiosis are mostly single PCR or quadruple PCR (up to triplet), and there is no four-PCR method that can simultaneously identify four different coccidia species.

[0004] On the other hand, chicken coccidia possess a unique protective structure—the oocyst wall. The oocyst wall structure is relatively consistent across different species, mostly a double-layered structure. The oocyst wall is formed by the release of type I and type II wall-forming bodies from the coccidia's macrogametocytes. The outer layer of the oocyst is electron-dense, with a rough outer surface and a dense inner surface. The thickness of the outer oocyst wall varies among different species; for example, the outer wall thickness of *Eimeria gianti* is 200 nm, while that of *Eimeria tenuifolia* is 90 nm. The inner oocyst wall is electron-transparent, composed of particles from macrogamet development. The thickness of the inner wall is generally consistent across different species, around 40 nm. Due to this unique structure of chicken coccidia, direct extraction of oocyst DNA after lysis using ordinary lysis buffers or kits is ineffective. DNA extraction efficiency is related to the degree of cell lysis, and DNA breakage must be prevented during cell lysis. Therefore, this has limited a range of studies based on coccidia oocyst DNA to some extent.

[0005] Therefore, establishing a rapid detection and identification method for coccidia oocysts is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a quadruple PCR primer set and kit for detecting chicken coccidiosis, which can rapidly identify common types of chicken coccidiosis, and has high detection specificity, low detection limit, good repeatability, and reliable results.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A quadruple PCR primer set for detecting chicken coccidiosis, comprising the following primer pairs:

[0009] A1: 5'-GGGCTTGGATGATGTTTGCT-3', SEQ ID NO.1,

[0010] A2: 5'-TGCTCATCATAGACAGCCGT-3', SEQ ID NO.2;

[0011] T1: 5'-TTAGTCCATCGCAACCCT-3', SEQ ID NO.3,

[0012] T2: 5'-TCCGCTTTCTACCTATTCTT-3', SEQ ID NO.4;

[0013] M1: 5'-GCCAGAGAACTAGCCTAACC-3', SEQ ID NO.5,

[0014] M2: 5'-GAGGACATCCACCCTTCTA-3', SEQ ID NO.6;

[0015] N1: 5'-GGTACTGACTTCATTCATATTGCG-3', SEQ ID NO.7,

[0016] N2: 5'-ACAACGCCTCATAACCCCAA-3', SEQ ID NO. 8.

[0017] Another object of the present invention is to provide a quadruple PCR detection kit for detecting chicken coccidia, comprising the above-mentioned primer set.

[0018] As a preferred embodiment of the present invention, the quadruple PCR detection kit further includes 2×MightyAmpBuffer, MightyAmp DNA Polymerase and ddH2O.

[0019] As a preferred embodiment of the present invention, the total volume of the detection kit is 25 μL, including a 10 μM primer set, with each primer in the primer set being 0.75 μL, and also including 12.5 μL of 2×MightyAmp Buffer, 0.5 μL of MightyAmp DNAPolymeras, and 5 μL of ddH2O.

[0020] More preferably, the quadruple PCR detection kit for detecting chicken coccidia further includes coccidia oocyst lysis buffer: the coccidia oocyst lysis buffer includes lysis buffer 1, lysis buffer 2, lysis buffer 3 and lysis buffer 4; lysis buffer 1 includes bile, trypsin, Triton X-100 and EDTA, lysis buffer 2 is NaOH, lysis buffer 3 is acetic acid and lysis buffer 4 is proteinase K.

[0021] More preferably, the concentrations of each component in the coccidia oocyst lysis buffer are as follows: lysis buffer 1 includes 5% bile, 0.375% trypsin, 1% Triton X-100 and 10 mmol / L EDTA; lysis buffer 2 is 0.25 mol / L NaOH; lysis buffer 3 is 0.25 mol / L acetic acid; and lysis buffer 4 is 20 mg / mL proteinase K.

[0022] More preferably, the quadruple PCR detection kit for detecting chicken coccidia further includes glass beads used in conjunction with coccidia oocyst lysis buffer, wherein the glass beads have a diameter of 0.5 mm and a weight of 0.15 g.

[0023] Another objective of this invention is to provide a method for extracting DNA from chicken coccidia oocysts, using the aforementioned detection kit, comprising the following steps: adding 100 μL of coccidia lysis buffer 1 and 0.15 g of 0.05 mm glass beads to the oocyst precipitate and shaking at 200 r / min for 45 min; then adding 5 μL of lysis buffer 2, immediately inverting to mix, and allowing to stand for digestion for 2 min; next, adding 10 μL of lysis buffer 3, immediately inverting to mix, and briefly centrifuging; adding 1 μL of lysis buffer 4, vortexing to mix, digesting in a 60°C water bath for 20 min, shaking every 5 min, boiling for 2 min, and ice bath for 5 min; transferring the supernatant to a new PCR tube, briefly centrifuging for 60 s, and collecting the supernatant to obtain the DNA template.

[0024] This invention discloses a quadruple PCR primer set and kit for detecting chicken coccidiosis. Compared with the prior art, this invention can simultaneously obtain detection results for four types of chicken coccidiosis, greatly shortening the detection and identification time and providing a guarantee for timely prevention of the spread of chicken coccidiosis. In addition, the coccidia oocyst lysis buffer provided by this invention can quickly obtain DNA templates for quadruple PCR detection. The quadruple PCR primers provided by this invention have high detection specificity, low detection limit, good repeatability, and reliable results. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0026] Figure 1 The results are shown in single PCR amplification of four chicken coccidia using four primer pairs.

[0027] Figure 2 The results are preliminary quadruple PCR amplification of four chicken coccidia using four primer pairs.

[0028] Figure 3 The results of screening for the amount of primers added in quadruple PCR;

[0029] Figure 4 The results of annealing temperature screening in the quadruple PCR reaction program;

[0030] Figure 5 The results of the extension time screening in the quadruple PCR reaction procedure;

[0031] Figure 6 The results of the cycle number screening in the quadruple PCR reaction program;

[0032] Figure 7The result is the first result of a quadruple PCR specific detection.

[0033] Figure 8 The results of the quadruple PCR specific detection are shown in Part II.

[0034] Figure 9 This is the result of the quadruple PCR detection limit;

[0035] Figure 10 Preliminary screening results for coccidia oocyst lysate;

[0036] Figure 11 Screening results for Triton-100 and NaOH in coccidia oocyst lysis buffer;

[0037] Figure 12 The results of screening for NaOH concentration in coccidia oocyst lysis buffer;

[0038] Figure 13 EDTA screening results for coccidia oocyst lysate

[0039] Figure 14 Results of an orthogonal experiment on the final concentration of EDTA added to coccidia DNA extraction, digestion time of proteinase K, and boiling time.

[0040] Figure 15 Verification experiments were conducted to determine the final concentration of EDTA added, the digestion time of proteinase K, and the boiling time in coccidia DNA extraction. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] This invention discloses a quadruple PCR primer set and kit for detecting chicken coccidia. All reagents involved are commercially available, and methods not mentioned are conventional methods, which will not be described in detail.

[0043] Example 1

[0044] 1. The following genes from GenBank were selected as characteristic target genes: *Eimeria tenella* ITS-1 (AF446055), *Eimeria velutipes* ITS-1 (AF446074), *Eimeria giantii* ITS-1 (AF446060), and *Eimeria virulentii* RAPD-SCAR marker selection gene (AY571579.1). Quadruple PCR primer sets were designed and synthesized. The nucleotide sequences and target fragment lengths of the primer sets are detailed in Table 1, and the primer set characteristics are shown in Table 2. The primers were synthesized by Sangon Biotech. According to the primer synthesis order, they were diluted to 100 μM with ddH2O and stored at -20℃ for later use.

[0045] Table 1

[0046]

[0047] Table 2

[0048]

[0049]

[0050] 2. Preparation of DNA template from mixed coccidia oocysts

[0051] Chicken Eimeria coccidia oocysts preserved in 2.5% potassium dichromate solution were counted using a hemocytometer. 100,000 E. acervulina, E. tenella, E. maxima, and E. necatrix oocysts were mixed in a centrifuge tube and centrifuged at 2500 rpm for 10 min to remove the potassium dichromate. The precipitate was retained, and TaKaRa MightyAmp was added to the precipitate. TM In the Genotyping Kit (TMG), add 100 μL of Extraction Buffer and 1 μL of Proteinase K (20 mg / mL), along with 0.15 g of 0.5 mm diameter glass beads. Vortex at 100 rpm for 45 min to break up the coccidia oocyst walls. Extract coccidia oocyst DNA according to the TMG instructions, as follows:

[0052] (1) Digest at 60℃ for 5 minutes, heat at 98℃ for 2 minutes, and then cool to room temperature.

[0053] (2) Centrifuge briefly at room temperature for 30 seconds to precipitate the tissue material, and use the supernatant as a template for PCR.

[0054] (3) Transfer the supernatant to a new PCR tube and store it temporarily at 4°C.

[0055] 3. Using the primer pairs in Table 1, singleton PCR amplification was performed on the templates of *Eimeria tenella*, *Eimeria velutina*, *Eimeria giant*, and *Eimeria virulence*, respectively. See the attached gel electrophoresis diagram for the PCR products. Figure 1 .

[0056] Reaction system: 12.5 μL of 2×MightyAmp Buffer, 0.75 μL each of 10 μM upstream and downstream primers, 1 μL of coccidia mixed oocyst DNA template, 0.5 μL of MightyAmp DNA Polymerase and 9.5 μL of ddH2O.

[0057] Reaction procedure: ① Pre-denaturation 98℃ 2min; ② Denaturation 98℃ 10s; ③ Annealing 60℃ 15s; ④ Extension 68℃ 30s. Repeat steps ② to ④ for 29 cycles until the reaction ends.

[0058] The obtained PCR products were sequenced by BGI Genomics Co., Ltd., and the sequencing results of four Eimeria coccidia strains on NCBI BLAST all met the design results.

[0059] Example 2

[0060] Establishment and screening of quadruple PCR

[0061] 1. Preliminary establishment of a quadruple PCR system:

[0062] Using the primer sets in Table 1, mixed oocyst DNA templates from four chicken coccidia species (Eimeria tenella, Eimeria tenella, Eimeria giantiformis, and Eimeria toxicae) were amplified. Specific target fragments of the expected size were successfully amplified, and no non-specific target fragments were observed. (See Appendix) Figure 2 .

[0063] 2. Optimization of primer addition amount:

[0064] The quadruple PCR reaction system consisted of a total volume of 25 μL, including 12.5 μL of 2×MightyAmp Buffer, 0.5 μL of MightyAmp DNA Polymeras, 1 μL of mixed DNA template from four types of chicken coccidia oocysts, 2 μL, 4 μL, 6 μL, and 8 μL of 10 μM primer sets, with each primer in an equal volume (equivalent to 0.25 μL, 0.50 μL, 0.75 μL, and 1.00 μL of each primer), and the remainder being ddH2O.

[0065] Quadruple PCR amplification was performed using the PCR reaction procedure described in Example 1, and the gel electrophoresis results are shown in the attached figure. Figure 3As shown, the amount of PCR products in all four-fold PCR strains increased with increasing primer concentration. However, when the primer concentration was ≥0.75 μL (0.3 μmol / L), the amount of PCR products in each strain no longer increased. Therefore, 0.75 μL was selected as the optimal primer addition amount for four-fold PCR.

[0066] 3. Optimization of the reaction process:

[0067] (1) Annealing temperature: Prepare the PCR reaction system according to the optimization results in the previous step; set 10 gradient annealing temperatures between 45-63℃, and perform gradient PCR according to the following PCR reaction program. ① Pre-denaturation 98℃ 2min; ② Denaturation 98℃ 10s; ③ Gradient annealing 15s; ④ Extension 68℃ 30s, 29 cycles from step ② to ④.

[0068] The gel electrophoresis results are attached. Figure 4 As shown, the specific fragments of the four Eimeria coccidia species in gradient PCR first gradually brightened and then gradually darkened with increasing annealing temperature. A non-specific band of approximately 400 bp gradually faded or even disappeared with increasing annealing temperature. The target fragments of the four Eimeria coccidia species were brightest at 60.2℃ and 61.6℃, and no non-specific bands were observed. Therefore, 61.6℃, with its higher annealing temperature, was selected as the optimal annealing temperature for quadruple PCR.

[0069] (2) Extension time: Prepare the PCR reaction system according to the optimization results of the previous step; set seven extension time groups, namely 30, 40, 50, 60, 70, 80 and 90s, respectively, annealing temperature is 61.6℃, 29 cycles, and perform quadruple PCR amplification.

[0070] The gel electrophoresis results are attached. Figure 5 As shown, the quadruple PCR products of the four Eimeria coccidia species all increased with the extension time. When the extension time was ≥60s, the target band no longer became brighter, so the optimal extension time was selected as 60s.

[0071] (3) Number of cycles: Prepare the PCR reaction system according to the optimization results of the previous step; and set 61.6℃ as the annealing temperature and 60s as the extension time, and set 25, 30, 35 and 40 cycles respectively for quadruple PCR amplification.

[0072] The gel electrophoresis results are attached. Figure 6 As shown, within the range of 30 to 40 cycles, the quadruple PCR products of the four Eimeria coccidia species all increased with the increase of the number of cycles, and no nonspecific bands appeared. Therefore, the PCR products can be amplified within the range of 30 to 40 cycles during the experiment.

[0073] 4. Quadruple PCR reaction system and reaction procedure

[0074] The established quadruple PCR reaction system was as follows: total volume 25 μL, including 12.5 μL of 2×MightyAmp Buffer, 0.5 μL of MightyAmp DNA Polymeras, 1 μL of mixed DNA template of four types of chicken coccidia, 0.75 μL of each primer in the 10 μM primer set, and 5 μL of ddH2O.

[0075] Quadruple PCR reaction procedure: ① Pre-denaturation at 98℃ for 2 min; ② Denaturation at 98℃ for 10 s; ③ Annealing at 61.6℃ for 15 s; ④ Extension at 68℃ for 60 s. Repeat steps ② to ④ for 30 to 40 cycles.

[0076] Example 3

[0077] Specificity and detection limit of quadruple PCR

[0078] 1. Specificity: DNA templates were extracted from single and mixed oocysts of *Eimeria tenella*, *Eimeria velutipes*, *Eimeria giant*, and *Eimeria toxicae*. DNA templates were also extracted from *Eimeria sclerotium*, three negative fecal samples, and common intestinal bacteria *Escherichia coli* and *Salmonella*. PCR amplification was performed (triple replicates) according to the PCR reaction system and procedure established in Example 2. The negative rate of known negative samples was determined (number of negative samples / number of known negative samples * 100%). The results are shown in the appendix. Figure 7 and attached Figure 8 As shown.

[0079] It was found that when four types of target Eimeria tenella were amplified using single and mixed templates via quadruple PCR, specific target bands of corresponding sizes were amplified, with no other non-target bands. Furthermore, the amplification results for Eimeria tenella, Escherichia coli, Salmonella, and fecal samples were all negative, and the results were consistent across three repeated tests, indicating a 100% negative rate for known negative samples.

[0080] 2. Limit of Detection: Take 100,000 oocysts of each of the four purified *Eimeria acervulina* species (*E. acervulina*, *E. tenella*, *E. maxima*, and *E. necatrix*) in a 2 mL centrifuge tube, centrifuge at 2500 rpm for 10 min, remove potassium dichromate, add dH₂O to the precipitate to adjust the oocyst concentration to 400,000 / mL, and then serially dilute to achieve mixed oocyst concentrations of 40,000, 4,000, 400, and 40 oocysts / mL. Take four 2 mL test tubes, number them H1-H4 according to the mixed coccidia concentration from highest to lowest, add 1 mL of the corresponding dilution of oocyst solution, centrifuge at 2500 rpm for 10 min, discard the supernatant, retain the precipitate, and add chicken feces sample to a final concentration of 100 mg / tube. DNA was extracted from coccidia oocysts of various gradients. Using four different coccidia mixed primers as specific primers and different concentrations of mixed coccidia as templates, quadruple PCR amplification was performed according to the PCR reaction system and procedure established in Example 2. The results were observed by gel electrophoresis (see Appendix). Figure 9 The detection limit of quadruple PCR amplification of coccidia oocyst DNA is determined based on whether the target fragment is present.

[0081] It was found that the intensity of the specific bands in the PCR products decreased with decreasing E. acervulina, E. tenella, and E. maxima DNA template concentrations ≥1 oocyst / μL; and E. necatrix DNA template concentrations ≥10 oocysts / μL. Therefore, the detection limit for the quadruple PCR of E. acervulina, E. tenella, and E. maxima was determined to be 1 oocyst / μL, and the detection limit for the quadruple PCR of E. necatrix was determined to be 10 oocysts / μL.

[0082] Example 4

[0083] The method for extracting coccidia DNA used in the above embodiments

[0084] 1. Collection and purification of coccidia oocysts

[0085] 15-day-old SPF chickens, fasted for 12 hours, were inoculated by gavage with 10,000 oocysts of a virulent strain of *Eimeria tenella*. They were then fed standard chick feed (sterilized at 80℃ for 30 min) and cooled boiled water in a coccidiotropic environment. The plastic sheeting was replaced 144 hours post-inoculation, and fecal samples were collected from 144 to 192 hours post-inoculation. 20g of chicken fecal sample was directly crushed and mixed in a centrifuge tube. 1g of this fecal sample was homogenized, counted using a hemocytometer, and diluted to 100,000 oocysts / g.

[0086] Take 1g of fresh fecal sample into a centrifuge tube, add 5mL of saturated saline to each tube of feces, mix well, centrifuge at 1200r / min for 5min, transfer the supernatant to a new centrifuge tube, add ten times the amount of dH2O, mix well, centrifuge at 2500r / min for 10min, and collect the precipitate.

[0087] 2. Mechanical cell wall disruption

[0088] Purify and precipitate 9 groups of coccidia oocysts (100,000 per tube) according to step 1. Add 1 mL of dH2O to each tube and mix well. Transfer the mixture to 9 2 mL test tubes (T1 to T9 groups). Centrifuge at 2500 r / min for 10 min, discard the supernatant, add 100 μL of Extraction Buffer and 1 μL of Proteinase K to the precipitate (mix by pipetting), add glass beads according to Tables 3 and 4, and vortex to break up the coccidia oocyst walls.

[0089] Table 3 Factor Levels in Orthogonal Experiments

[0090]

[0091] Note: A 1:1 mixture refers to a 1:1 mixture of 0.5mm and 1mm glass beads.

[0092] Table 4 Grouping of Orthogonal Experiments

[0093]

[0094] After the coccidia oocyst walls of the above groups were broken, coccidia oocyst DNA was extracted according to the TMG instructions. (1) Digested at 60℃ for 5 min, heated at 98℃ for 2 min, and then cooled to room temperature. (2) Centrifuged briefly at room temperature for 30 s to precipitate the tissue material. The supernatant was used as the template for PCR. (3) The supernatant was transferred to a new PCR tube and stored temporarily at 4℃.

[0095] The PCR system was prepared, PCR amplification and gel electrophoresis were performed using Eimeria tenella-specific primers T1 and T2. The brightness of the PCR product bands was measured using ImageJ. Based on the measured gray values, the optimal conditions were screened, as shown in Table 5.

[0096] Table 5. Effects of glass beads on coccidia oocysts

[0097]

[0098]

[0099] Comparing the ranges (R) of the four factors revealed that grinding speed (C) > grinding time (D) > glass bead quantity (A) > glass bead size (B), indicating that grinding speed has the greatest impact on cyst cell disruption, followed by grinding time, glass bead quantity, and finally glass bead size. Comparing the k values ​​in each column, the maximum values ​​for each factor were A2, B1, C3, and D3, respectively. Therefore, the optimal level is A2B1C3D3. Thus, using 0.15g glass beads with a diameter of 0.5mm and grinding at 200r / min for 45min yields the highest DNA yield.

[0100] 3. Screening of coccidia lysate

[0101] (1) Determination of trypsin and bile components in coccidia lysate

[0102] Purify and precipitate 3 groups of Eimeria tenella oocysts (100,000 per tube) according to the method in step 1. Add 1 mL of dH2O to each tube and mix well. Transfer the mixture to 3 2 mL centrifuge tubes (T1 to T3 groups) and centrifuge at 2500 r / min for 10 min. Discard the supernatant and retain the precipitate.

[0103] Group T1 was the TMG group, with 100 μL Extraction Buffer and 1 μL Proteinase K added. Group T2 was given 100 μL SNET lysis buffer (components: 20 mmol / L Tris-HCl, 5 mmol / L EDTA, 1% SDS, 400 mmol / L NaCl, dH2O as solvent) and 1 μL Proteinase K added. Group T3 was given 100 μL sporangium digestion solution (components: 10% chicken bile, 0.75% trypsin, dH2O as solvent) and 1 μL Proteinase K (20 mg / mL).

[0104] The T1-T3 groups were broken up according to the optimal method of orthogonal experiment in step 2, then digested at 60℃ for 5 min, boiled for 2 min, ice bath for 5 min, and centrifuged briefly for 30 s. The supernatant was collected to obtain the DNA template.

[0105] Using Eimeria tenella-specific primers T1 and T2 as primers, PCR system preparation, PCR amplification, and gel electrophoresis were performed according to the PCR system and procedure established in Example 2. By comparing the band brightness of T2, T3, and T1, the composition of the sporangium digestion fluid from lysed coccidia oocysts was preliminarily determined (see Appendix). Figure 10 ).

[0106] The results showed that both the sporangium digestion fluid group (trypsin bile) and the TMG group amplified relatively bright target fragments, while the SNET lysis buffer did not amplify the target fragments. The sporangium digestion fluid composition of the coccidia lysis buffer was preliminarily determined to be 5% bile and 0.375% trypsin.

[0107] (2) Determination of Triton X-100 and NaOH content in coccidia lysate

[0108] Purify and precipitate 10 groups of Eimeria tenella oocysts (100,000 per tube) according to the method in step 1. Add 1 mL of dH2O to each tube and mix well. Transfer the mixture to 10 2 mL centrifuge tubes (T1 to T10 groups), centrifuge at 2500 r / min for 10 min, discard the supernatant and retain the precipitate.

[0109] Group T1 served as the TMG control group, with 100 μL of Extraction Buffer added. Groups T2 through T10 received 100 μL of different coccidia lysate solutions according to Table 6. After disrupting the coccidia oocyst walls using the optimal method from the orthogonal experiment in step 2, 5 μL of NaOH at different concentrations was added for digestion for 2 min. The mixture was then neutralized with 10 μL of acetic acid of the same concentration as the NaOH, followed by the addition of 1 μL of proteinase K. The digestion was continued at 60°C for 5 min, followed by boiling for 2 min, ice bath for 5 min, and brief centrifugation for 30 s. The supernatant was then collected.

[0110] Table 6 Screening of Triton X-100 and NaOH components

[0111]

[0112] Note: Triton X-100 is the final concentration, NaOH and acetic acid are the added concentrations.

[0113] Using Eimeria tenella-specific primers T1 and T2 as primers, PCR systems were prepared, PCR amplification was performed, and gel electrophoresis was conducted on 10 groups of crude DNA extracts from coccidia oocysts according to the PCR system and procedure established in Example 2. Triton X-100, NaOH, and acetic acid were screened by comparing the brightness of the target bands (see Appendix). Figure 11 ).

[0114] The results showed that the lysis of coccidia was significantly better with the addition of 5 μL 2M NaOH (T8-T10 groups) than with the addition of 5 μL 10M and 6M NaOH (T2-T7 groups). Furthermore, the PCR product band of the 2M NaOH + 1% Triton X-100 group (T9) was the brightest. Therefore, the components of coccidia lysis buffer 1 were further determined to be 5% bile, 0.375% trypsin, 1% Triton X-100, and lysis buffer 2 was NaOH.

[0115] (3) Further screening of NaOH concentration in coccidia lysate

[0116] Purify and precipitate 18 groups of *Eimeria tenella* oocysts (100,000 oocysts / tube) according to the method in step 1, numbered T1 to T6, with 3 replicates for each group. Add 1 mL of dH2O to each tube and mix well. Transfer the mixture to 18 2 mL centrifuge tubes (T1 to T6 groups), centrifuge at 2500 rpm for 10 min, discard the supernatant, and retain the precipitate.

[0117] Group T1 was the TMG control group, with 100 μL of Extraction Buffer added. Groups T2 to T6 were the experimental groups, with 5 μL each of 2M, 1M, 0.5M, 0.25M, and 0M NaOH added. In addition to NaOH and acetic acid of the same concentration as NaOH, the experimental groups were given coccidia lysate 1 (5% bile, 0.375% trypsin, and 1% Triton X-100), followed by 1 μL of proteinase K. The mixture was digested at 60°C for 5 min, boiled for 2 min, placed on ice for 5 min, and then centrifuged briefly for 30 s. The supernatant was collected.

[0118] Using Eimeria tenella-specific primers T1 and T2 as primers, PCR systems were prepared, PCR amplification was performed, and gel electrophoresis was conducted on 18 groups of crude coccidia oocyst DNA extracts according to the PCR system and procedure established in Example 2. The brightness of the PCR product bands was measured using ImageJ, and based on the grayscale value, the group with the brightest amplified band from T2 to T6 was selected to determine the optimal concentration of NaOH (see Table 7 and Appendix). Figure 12 ).

[0119] Table 7. Screening Results of NaOH Concentration

[0120]

[0121] The results showed that the electrophoretic bands of the PCR products from group T5 (0.25M NaOH) were the brightest, comparable to those of the TMG control group, with no significant difference between the two groups (P>0.05). Therefore, the composition of coccidia lysis buffer 2 was further determined to be 0.25 mol / L NaOH and acetic acid with the same concentration as NaOH, which was then used as lysis buffer 3.

[0122] (4) Screening of EDTA in coccidia lysate

[0123] Purify and precipitate 15 *Eimeria tenella* oocysts (100,000 per tube) according to step 1, and number them as groups T1 to T5, with 3 replicates per group. Add 1 mL of dH2O to each tube and mix well. Transfer the mixture to 15 2 mL centrifuge tubes (groups T1 to T5), centrifuge at 2500 rpm for 10 min, discard the supernatant, and retain the precipitate.

[0124] Group T1 was the TMG control group, with 100 μL of Extraction Buffer added. Groups T2 to T5 were the experimental groups, with 100 μL of Triton X-100 coccidia lysis buffer, Tris-HCl coccidia lysis buffer, EDTA coccidia lysis buffer, and Tris-HCl+EDTA coccidia lysis buffer added to each group, and coccidia oocyst DNA was crudely extracted based on the results of step (3) (0.25 mol / L NaOH and 0.25 mol / L acetic acid were added respectively, followed by 1 μL of proteinase K, digestion at 60℃ for 5 min, boiling for 2 min, ice bath for 5 min, and instantaneous centrifugation for 30 s, and supernatant was collected).

[0125] Table 13

[0126]

[0127]

[0128] Using Eimeria tenella-specific primers T1 and T2 as primers, PCR systems were prepared, PCR amplification was performed, and gel electrophoresis was conducted on crude DNA extracts from 15 coccidia oocysts in 5 groups, following the PCR system and procedure established in Example 2. The brightness of the PCR product bands was measured using ImageJ, and based on the measured grayscale values, it was determined whether Tris-HCl or EDTA needed to be added to the coccidia lysate (see Table 8 and Appendix). Figure 13 ).

[0129] Table 8

[0130]

[0131] The results showed that the PCR product gray value of the EDTA coccidia lysate (T4) group was the highest, significantly higher than that of the experimental groups with Tris-HCl (T3 and T5 groups), and there was no significant difference in the gray value of the electrophoretic bands between the T4 and T1 groups (P > 0.05). Therefore, the composition of coccidia lysate 1 was further determined to be 5% bile, 0.375% trypsin, 1% Triton X-100, and EDTA.

[0132] (5) Orthogonal experiment to screen the amount of EDTA, proteinase K digestion time, and boiling time in coccidia lysate.

[0133] Purify the precipitate of 9 groups of Eimeria tenella oocysts (100,000 per tube) according to the method in step 1. Add 1 mL of dH2O to each tube and mix well. Transfer the mixture to 9 2 mL centrifuge tubes (T1 to T9 groups), centrifuge at 2500 r / min for 10 min, discard the supernatant and retain the precipitate.

[0134] The final concentration of EDTA, digestion time of proteinase K (20 mg / mL), and boiling time in the coccidia oocyst DNA extraction protocol were used as three factors. The coccidia oocyst DNA extraction and screening were carried out according to the three-factor three-level, i.e., L9(33) orthogonal design table, as shown in Table 9-10.

[0135] Table 9

[0136]

[0137] Table 10

[0138]

[0139] Using Eimeria tenella-specific primers T1 and T2 as primers, PCR systems were prepared, PCR amplification was performed, and gel electrophoresis was conducted on nine groups of crude DNA extracts from coccidia oocysts according to the PCR system and procedure established in Example 2. The brightness of the PCR product bands was measured using ImageJ, and the optimal levels of each factor were selected based on the measured grayscale values ​​(see Table 11 and Appendix). Figure 14 ).

[0140] Table 11

[0141]

[0142] The results, by comparing the ranges (R) of the three groups of factors, showed that the proteinase K digestion time (B) > EDTA addition amount (A) > boiling time (C), indicating that the proteinase K digestion time had the greatest impact on the extraction efficiency of coccidia oocyst DNA, followed by the EDTA addition amount, and finally the boiling time. The k values ​​in columns A3, B3, and C3 were the largest, therefore the optimal levels for each factor were A3B3C3, i.e., the orthogonal optimal scheme was to add 10 mmol / L EDTA, digest with proteinase K for 20 min, and boil for 10 min.

[0143] (6) Orthogonal verification

[0144] Boiling during the extraction of coccidia oocyst DNA can release DNA from the tissue, but excessive boiling time can easily cause DNA breakage. The optimal scheme (5) selected (adding 10 mmol / L EDTA, digesting with proteinase K for 20 min, boiling for 10 min) and the group with boiling time shortened to 2 min (adding 10 mmol / L EDTA, digesting with proteinase K for 20 min, boiling for 2 min), and the group with the most PCR products among the nine orthogonal groups (adding 5 mmol / L EDTA, digesting with proteinase K for 20 min, boiling for 2 min) were repeated for the experiment. At the same time, a control group of TMG kit was set up, numbered T1 to T4, and each group was repeated three times. Using Eimeria tenella-specific primers T1 and T2 as primers, the PCR system was prepared, PCR amplification and gel electrophoresis were performed on the crude DNA extracts of 12 coccidia oocysts according to the PCR system and procedure established in Example 2. The optimal level of each factor was determined by measuring the brightness of the PCR product bands using ImageJ and based on the measured gray value (see Table 12 and Appendix). Figure 15 ).

[0145] Table 12

[0146]

[0147] The validation results showed that the gray value of the PCR product band in the group with 10 mmol / L EDTA, proteinase K digestion for 20 min, and boiling for 2 min (T3) was slightly higher than that of the TMG control group, significantly higher than the optimal group among the nine orthogonal groups (T1), and also significantly higher than the gray value of the optimal orthogonal scheme group. Therefore, the optimal EDTA addition amount, proteinase K digestion time, and boiling time are: 10 mmol / L EDTA, proteinase K digestion for 20 min, and boiling for 2 min.

[0148] Therefore, the final determination of the coccidia oocyst lysis buffers included: lysis buffer 1 (5% bile, 0.375% trypsin, 1% Triton X-100, 10 mmol / L EDTA); lysis buffer 2 (0.25 mol / L NaOH); lysis buffer 3 (0.25 mol / L acetic acid); and lysis buffer 4 (20 mg / mL proteinase K).

[0149] (7) Method for extracting DNA from chicken coccidia oocysts

[0150] Take 1g of fresh fecal sample into a centrifuge tube, add 5mL of saturated saline to each tube of feces, mix well, centrifuge at 1200r / min for 5min, transfer the supernatant to a new centrifuge tube, add ten times the amount of dH2O, mix well, centrifuge at 2500r / min for 10min, and collect the precipitate.

[0151] Add 100 μL of coccidia lysis buffer 1 and 0.15 g of 0.05 mm glass beads to the oocyst precipitate and shake at 200 rpm for 45 min. Then add 5 μL of lysis buffer 2, immediately invert to mix, and let stand for 2 min to digest. Next, add 10 μL of lysis buffer 3, immediately invert to mix, and centrifuge briefly. Add 1 μL of lysis buffer 4, vortex to mix, and digest in a 60 °C water bath for 20 min, shaking every 5 min. Boil for 2 min, incubate on ice for 5 min, transfer the supernatant to a new PCR tube, centrifuge briefly for 60 s, and collect the supernatant to obtain the DNA template.

[0152] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0153] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A quadruple PCR detection kit for detecting chicken coccidia, characterized by, The detection kit comprises detection primers and coccidian oocyst lysate; The detection primers comprise the following primer pairs: A1: 5'-GGGCTTGGATGATGTTTGCT-3', SEQ ID NO. 1, A2: 5'-TGCTCATCATAGACAGCCGT-3', SEQ ID NO. 2; T1: 5'-TTAGTCCATCGCAACCCT-3', SEQ ID NO. 3, T2: 5'-TCCGCTTTCTACCTATTCTT-3', SEQ ID NO. 4; M1: 5'-GCCAGAGAACTAGCCTAACC-3', SEQ ID NO. 5, M2: 5'-GAGGACATCCACCCTTCTA-3', SEQ ID NO. 6; N1: 5'-GGTACTGACTTCATTCATATTGCG-3', SEQ ID NO. 7, N2: 5'-ACAACGCCTCATAACCCCAA-3', SEQ ID NO. 8; The coccidian oocyst lysate comprises lysate 1, lysate 2, lysate 3 and lysate 4, and the lysate 1 is 5% bile, 0.375% trypsin, 1% Triton X-100 and 10 mmol / L EDTA; the lysate 2 is 0.25 mol / L NaOH; the lysate 3 is 0.25 mol / L acetic acid, and the lysate 4 is 20 mg / mL protease K.

2. The quadruplex PCR detection kit according to claim 1, characterized in that, 2xMightyAmp Buffer, MightyAmp DNA Polymerase and ddH2O are further included.

3. The quadruplex PCR detection kit according to claim 2, characterized in that, The total system of the detection kit is 25 μL, which comprises 10 μM primer group, and the amount of each primer in the primer group is 0.75 μL, further comprising 2xMightyAmp Buffer 12.5 μL, MightyAmp DNA Polymerase 0.5 μL, four kinds of chicken coccidian mixed oocyst DNA templates 1 μL and ddH2O 5 μL.

4. The quadruple PCR detection kit for detecting chicken coccidia according to claim 3, characterized by, Glass beads matched with the coccidian oocyst lysate are further included, and the diameter of the glass beads is 0.5 mm and the weight is 0.15 g.

5. A method for extracting DNA from chicken coccidial oocysts, characterized by, Extraction is carried out by using the detection kit of claim 4, comprising the following steps: 100 μL of coccidian lysate 1 and 0.15 g of 0.5 mm glass beads are added to the oocyst precipitate, and oscillation is carried out at 200 r / min for 45 min on a shaker; then 5 μL of lysate 2 is added, and immediately after, up-down inversion is carried out to mix well, and digestion is carried out for 2 min; then 10 μL of lysate 3 is added, and immediately after, up-down inversion is carried out to mix well, and instantaneous centrifugation is carried out; 1 μL of lysate 4 is added, and oscillation is carried out to mix well, and digestion is carried out at 60℃ water bath for 20 min, and the oocyst is shaken every 5 min, and boiling is carried out for 2 min, and ice bath is carried out for 5 min, and the supernatant is transferred to a new PCR tube, and instantaneous centrifugation is carried out for 60 s, and the supernatant is taken to obtain the DNA template.

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