CPA primer and kit for detecting chicken eimeria tenella and application of CPA primer and kit

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a CPA primer and a kit for detecting chicken eimeria tenella and application of the CPA primer and the kit. Specific isothermal amplification primers are designed aiming at chicken eimeria tenella genes, target fragments are amplified by utilizing the isothermal amplification primers, and then a lateral flow chromatography test strip is connected to visualize results. The method is easy to operate, high in sensitivity and good in specificity, detection can be completed only through constant-temperature heating equipment, and a new technology and method are provided for detecting chicken eimeria tenella.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and more specifically, relates to CPA primers, kits for detecting Eimeria tenella in chickens, and their applications. Background Art

[0002] Chicken coccidiosis is a parasitic disease caused by Eimeria spp., which poses a serious threat to the chicken farming industry. The morbidity and mortality rates of chicks are high; most adult chickens are carriers, and after infecting chickens, it will cause intestinal damage, reduced feed conversion efficiency, hindered weight gain, and a series of complications. Eimeria spp. shows endemic distribution, and clinical and subclinical infections can affect effective meat and egg production as well as animal welfare. Eimeria tenella poses a great threat to the chicken farming industry.

[0003] Traditional methods for detecting chicken coccidia are to examine fecal or litter samples under a microscope to find the oocyst life cycle stage that is resistant to the environment or to perform corresponding diagnoses after the death of chickens through lesion scoring. With the progress of experimental techniques, new molecular diagnostic techniques have also been developed and applied, including polymerase chain reaction, random amplified polymorphic DNA, and fluorescence quantitative PCR, etc. However, these detection methods all require professional detection equipment, such as fluorescence quantitative PCR instruments and gradient PCR instruments. These professional equipment are not only expensive but also have cumbersome operations and long time consumption. Summary of the Invention

[0004] The purpose of the present invention is to provide CPA primers, kits for detecting Eimeria tenella in chickens, and their applications to solve the above technical problems.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The present invention provides a method for detecting Eimeria tenella in chickens, comprising the following steps:

[0007] (1) Extracting DNA from the sample to be tested;

[0008] (2) Using the DNA of the sample to be tested as a template, and performing amplification with isothermal amplification primers to obtain an amplification product; the sequences of the isothermal amplification primers are as shown in SEQ ID NO.2 - 6;

[0009] (3) Mixing the amplification product with HybriDetect Assay Buffer to obtain a mixture, connecting the mixture to a Milenia HybriDetect lateral flow chromatographic test strip, and making a judgment based on the results of the test strip;

[0010] (4) The judgment method is as follows: when only the first test line on the test strip shows color, it is a positive result; when only the second test line on the test strip shows color, it is a negative result; when both test lines on the test strip show color, it is also a positive result.

[0011] The present invention designs isothermal amplification primers for the genes of Eimeria tenella in chickens, and after amplifying the target fragment by extracting sample DNA and connecting it to a lateral flow chromatography test strip to visualize the result, thereby detecting Eimeria tenella in chickens.

[0012] Furthermore, the sample to be tested is chicken feces.

[0013] Furthermore, the reaction system for amplification includes: 2 - 3 μL of 10×Bst Buffer, 2 - 3 μL of 5M betaine, 0.5 - 1.5 μL of 100 mM MgSO4, 2 - 3 μL of 10 mM dNTPs, 0.5 - 1.5 μL of 8U / μL Bst DNA polymerase, 7 - 9 μL of H2O, 0.5 - 1.5 μL of DNA sample, and 0.5 - 1.5 μL of each isothermal amplification primer.

[0014] Furthermore, the amplification conditions are 58 - 60 °C for 0.5 - 1.5 h.

[0015] Furthermore, the volume ratio of the amplification product to HybriDetect Assay Buffer in the mixture is 0.9 - 1.1:2.9 - 3.1.

[0016] The present invention also provides a kit for detecting Eimeria tenella in chickens, and the kit includes the above isothermal amplification primers.

[0017] Furthermore, the kit also includes 10×Bst Buffer, betaine with a concentration of 5M, MgSO4 with a concentration of 100 mM, dNTPs with a concentration of 10 mM, and Bst DNA polymerase with a concentration of 8U / μL.

[0018] The present invention also provides the application of the kit in detecting chickens infected with Eimeria tenella.

[0019] The present invention has the following beneficial effects:

[0020] The present invention establishes a method for detecting Eimeria tenella in chickens based on isothermal amplification technology and lateral flow chromatography test strips, and has carried out preliminary applications. This method is simple to operate, has high sensitivity and specificity, and only requires a constant temperature device to complete the detection. It is a very promising method for detecting Eimeria tenella in chickens, providing new technologies and methods for the detection of Eimeria tenella in feces. Description of the Drawings

[0021] Figure 1 It is a gradient reaction temperature graph. Among them, 1 is 55 °C, 2 is 57 °C, 3 is 59 °C, 4 is 61 °C, and 5 is 63 °C.

[0022] Figure 2 It is a specific detection graph. Among them, 1 is *Eimeria tenella*, 2 is *Eimeria necatrix*, 3 is *Eimeria maxima*, 4 is *Eimeria acervulina*, 5 is *Eimeria praecox*, 6 is *Eimeria mitis*, and 7 is *Eimeria brunetti*.

[0023] Figure 3 It is a sensitivity analysis graph of isothermal amplification for plasmids. Among them, 1 is 1×10 3 copies / μL; 2 is 1×10 2 copies / μL; 3 is 1×10 1 copies / μL; 4 is 1×10 copies / μL.

[0024] Figure 4 It is a sensitivity analysis result graph of isothermal amplification for sporulated oocysts. Among them, 1 is 1000 oocysts, 2 is 100 oocysts, 3 is 10 oocysts, 4 is 5 oocysts, 5 is 1 oocyst, and 6 is 0 oocysts.

[0025] Figure 5 It is a sensitivity analysis result graph of isothermal amplification for unsporulated oocysts. Among them, 1 is 1000 oocysts, 2 is 100 oocysts, 3 is 10 oocysts, 4 is 5 oocysts, 5 is 1 oocyst, and 6 is 0 oocysts.

[0026] Figure 6 It is a result graph of isothermal amplification for clinical applications. Among them, A is the detection result graph on the fourth day, B is the detection result graph on the fifth day, and C is the detection result graph on the sixth day. 1 is the blank group, 2 is the fecal sample after infecting 100 oocysts, 3 is the fecal sample after infecting 500 oocysts, 4 is the fecal sample after infecting 1000 oocysts, and 5 is the fecal sample after infecting 2000 oocysts. Specific Embodiments

[0027] The present invention will be described in detail below with specific examples, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0028] Example 1: Development of isothermal amplification primers for detecting *Eimeria tenella* in chickens.

[0029] I. Design of isothermal amplification primers and screening of reaction temperature.

[0030] For the 1161 bp gene sequence of Eimeria tenella, as shown in SEQ ID NO.1, isothermal amplification primers were designed using PrimerExplorer V5 (http: / / primerexplorer.jp / e / ) and NCBI. Specificity analysis was performed by Blast on the NCBI website, and the isothermal amplification primer with the best specificity was selected and sent to Tsingke Biotechnology Co., Ltd. (Beijing) for synthesis. The primer sequences are shown in Table 1.

[0031]

[0032]

[0033] Table 1: Primer Sequences

[0034]

[0035] The PCR amplification system is shown in Table 2.

[0036] Table 2: PCR Reaction System

[0037]

[0038]

[0039] After mixing the reaction system in Table 2, to screen the optimal temperature for the isothermal amplification reaction, 5 gradient reaction temperatures were set, namely 55 °C, 57 °C, 59 °C, 61 °C, and 63 °C. The amplification time was 1 h. After the reaction ended, 75 μL of HybriDetect Assay Buffer was added and mixed evenly. Then, the Milenia HybriDetect lateral flow chromatography test strip was connected. After 3 min, the results were observed. The first detection line showing color was a positive result, the second detection line showing color was a negative result, and both lines showing color was also a positive result (not all primers participated in the isothermal amplification reaction). The Milenia HybriDetect lateral flow chromatography test strip is a ready-to-use general test strip based on the lateral flow technology of colloidal gold particles. A biotin ligand is fixed at the first detection line, and a gold-labeled FITC-specific IgG antibody is embedded at the second detection line.

[0040] The results are as Figure 1 shown. At 59 °C, the amplification effect on Eimeria tenella was the best. Therefore, the reaction temperature of the isothermal amplification was set to 59 °C for subsequent experiments.

[0041] II. Primer Specificity Evaluation.

[0042] The primer set in Table 1 was used to amplify seven species of Eimeria. The amplification reaction system was as shown above, and the results are as Figure 2As shown, the primers in Table 1 only have an amplification effect on the gene of Eimeria tenella in chickens.

[0043] III. Sensitivity detection.

[0044] The sequence shown in SEQ ID NO.1 was amplified. After the amplified product was recovered by 1% agarose gel electrophoresis, a tail was added to the recovered product (incubated at 72 °C for 30 min using rTaq enzyme), and then ligated with the T Vector pMD19(Simple) vector through a DNA ligation kit (TaKaRa, 6022) to obtain a positive plasmid.

[0045] The sensitivity of the isothermal amplification method was evaluated by gradient diluting the positive plasmid and oocysts of Eimeria tenella, respectively. The positive plasmid was serially diluted 10-fold (10 - 10 3 copies / μL) and then detected by isothermal amplification. The results are as Figure 3 shown. The detection limit of this method is 10 1 copies / μL. The sporulated oocysts were serially diluted (1000 - 1) and then DNA was extracted for isothermal amplification detection. The results are as Figure 4 shown. This method can detect as few as 1 sporulated oocyst at least. The unsporulated oocysts were serially diluted (1000 - 1) and then DNA was extracted for isothermal amplification detection. The results are as Figure 5 shown. This method can detect as few as 1 unsporulated oocyst at least.

[0046] IV. Clinical application.

[0047] Fourteen-day-old healthy chickens were infected with Eimeria tenella oocysts at four gradients (100, 500, 1000, 2000), with 5 chickens in each group and three replicates. A non-infected group was set as the blank group. Fecal DNA was extracted and detected by isothermal amplification on the 4th, 5th, and 6th days respectively. The detection results are as Figure 6 shown. Eimeria tenella in chickens could be significantly detected in the feces of all 3 groups infected with 100 oocysts on the 4th day, and the blank group was negative. This method can quickly detect whether the chickens infected with 100 oocysts are diseased on the 4th day at the earliest.

[0048] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To avoid repetition, preferred embodiments of the present invention are described.

[0049] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made to these embodiments by those skilled in the art once they learn of the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

[0050] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A method for detecting Eimeria tenella in chicken, characterized in that: The following steps are involved: (1) extracting DNA from the sample to be tested; (2) using the sample DNA to be tested as a template, and using isothermal amplification primers to amplify to obtain an amplification product; the isothermal amplification primer sequences are shown in SEQ ID NOs. 2 to 6; (3) mixing the amplified product with HybriDetect Assay Buffer to obtain a mixed solution, connecting the mixed solution to a Milenia HybriDetect lateral flow chromatography test strip, and performing a judgment based on the test strip result; (4) The judgment method is: when only the first detection line of the test strip is colored, it is a positive result; when only the second detection line of the test strip is colored, it is a negative result; when both detection lines of the test strip are colored, it is also a positive result.

2. A method for detecting Eimeria tenella in chicken according to claim 1, characterized in that: The sample to be tested is chicken feces.

3. A method for detecting Eimeria tenella in chicken according to claim 1, characterized in that: The amplification reaction system includes: 2 to 3 μL of 10×Bst Buffer, 2 to 3 μL of 5M betaine, 0.5 to 1.5 μL of 100mM MgSO4, 2 to 3 μL of 10mM dNTPs, 0.5 to 1.5 μL of 8U / μL Bst DNA polymerase, 7 to 9 μL of H2O, 0.5 to 1.5 μL of DNA sample, and 0.5 to 1.5 μL of each isothermal amplification primer.

4. A method for detecting Eimeria tenella in chicken according to claim 1, characterized in that: The amplification conditions are 58-60° C., 0.5-1.5 h.

5. A method for detecting Eimeria tenella in chicken according to claim 1, characterized in that: The volume ratio of the amplified product to the HybriDetect Assay Buffer in the mixed solution is 0.9-1.1:2.9-3.

1.

6. A kit for detecting Eimeria tenella in chicken, characterized in that: The kit comprises the isothermal amplification primer according to claim 1.

7. A kit for detecting Eimeria tenella in chicken, characterized in that: The kit also includes 10×BstBuffer, betaine at a concentration of 5 M, MgSO4 at a concentration of 100 mM, dNTPs at a concentration of 10 mM, and BstDNA polymerase at a concentration of 8 U / μL.

8. Use of the kit according to claim 6 in detecting Eimeria tenella infection in chickens.

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