CPA primer group and kit for detecting seven chicken eimeria coccidiosis and application of CPA primer group and kit

Abstract

The invention discloses a CPA primer group and a kit for detecting seven chicken eimeria coccidiosis and application of the CPA primer group and the kit, and belongs to the technical field of biological detection. Specific primers are designed for conservative COI genes of chicken eimeria, an optimal reaction system and 61 DEG C constant-temperature amplification conditions are optimized and determined, and a CPA detection method for detecting chicken eimeria is established. The method is extremely high in sensitivity, the lower limit of detection on a plasmid standard substance reaches 102 copies / L, extremely low pathogen load equivalent to 0.5 oocyst can be stably detected in practical application, the clinical sample verification coincidence rate reaches 100%, and a powerful technical support is provided for early warning, field monitoring and precise prevention and control of chicken coccidiosis.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a CPA primer set, kit, and application for detecting seven species of Eimeria coccidia in chickens. Background Technology

[0002] Coccidiosis in chickens is a serious protozoan disease caused by Eimeria species, particularly affecting chicks and often leading to high morbidity and mortality. Infected adult chickens frequently become carriers, causing intestinal tissue damage, reduced feed conversion efficiency, hindered weight gain, and a series of complications. Coccidiosis is widespread, impacting both clinical and subclinical infections in chickens, affecting meat and egg production and animal welfare, and resulting in significant economic losses for the poultry industry.

[0003] Traditional detection methods include microscopic examination of fecal samples and pathological autopsy followed by lesion scoring for diagnosis. With advancements in experimental techniques, novel molecular diagnostic technologies have been developed and applied, including polymerase chain reaction (PCR), random amplified polymorphic DNA (RAPD-PCR), and quantitative real-time PCR. While these methods have improved accuracy, they generally require specialized experimental equipment, involve complex procedures, and demand a high level of technical expertise from operators, thus limiting their practical application.

[0004] Current specific molecular detection technologies are mostly designed for single-species Eimeria coccidia. However, in clinical practice, chicken coccidiosis often presents as a mixed infection of multiple Eimeria species. This single-species detection approach cannot simultaneously cover other co-infecting species, resulting in significant blind spots in the detection range, leading to a higher rate of missed diagnoses and ultimately affecting the accuracy of diagnostic results and the effectiveness of subsequent control measures.

[0005] Therefore, there is an urgent need in this field to develop a detection method that can simultaneously detect multiple Eimeria species in chickens, and possesses high sensitivity and ease of operation. Existing technologies, limited by their single detection range, are unable to effectively address the common mixed infections in clinical settings, which has become a major bottleneck restricting the accurate diagnosis and effective control of coccidiosis in chickens. Developing a detection scheme that covers multiple pathogens and is adaptable to field applications is of great significance for improving diagnostic efficiency and optimizing control strategies. Summary of the Invention

[0006] The purpose of this invention is to provide a CPA primer set, kit, and application for detecting seven species of Eimeria tenella in chickens, thereby addressing the problems existing in the prior art. This invention provides a CPA primer set and method for simultaneously detecting seven species of Eimeria tenella in chickens, effectively solving the problem of missed detection in mixed infections. This method, based on the COI gene, possesses excellent specificity and high sensitivity (detection limit 0.5 oocysts), is simple and rapid to operate, requires no complex instruments, and enables rapid, visual detection on-site, providing an efficient technical means for the prevention and control of coccidiosis in chickens.

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

[0008] This invention provides a CPA primer set for detecting seven species of Eimeria coccidia in chickens. The primer set consists of primer COI-4-1 as shown in SEQ ID NO. 16, primer COI-4-2 as shown in SEQ ID NO. 17, primer COI-4-3 as shown in SEQ ID NO. 18, primer COI-4-4 as shown in SEQ ID NO. 19, and primer COI-4-5 as shown in SEQ ID NO. 20.

[0009] Optionally, the 5' end of primer COI-4-1 is labeled with biotin, and the 5' end of primer COI-4-2 is labeled with fluorescein isothiocyanate (FITC).

[0010] Optionally, the seven species of chicken Eimeria tenella are Eimeria tenella, Eimeria necatrix, Eimeria maxima, Eimeria acervuline, Eimeria brunetti, Eimeriamitis, and Eimeria praecox.

[0011] The present invention also provides the application of the CPA primer set described above in the preparation of a kit for detecting Eimeria tenella in chickens.

[0012] The present invention also provides a kit for detecting Eimeria coccidia in chickens, the kit comprising the CPA primer set described above.

[0013] Optionally, the kit is used to detect at least one of Eimeria tenella, Eimeria toxicae, Eimeria giantiformis, Eimeria tuftediformis, Eimeria bryonyri, Eimeria mildis, and Eimeria precociousiformis.

[0014] The present invention also provides a method for using the aforementioned reagent kit, comprising the following steps:

[0015] (1) Extract genomic DNA from the sample to be tested;

[0016] (2) Using the genomic DNA obtained in step (1) as a template, prepare the CPA reaction system and perform isothermal amplification;

[0017] (3) After mixing the amplification product from step (2) with the detection buffer, use the side-flow chromatography test strip to interpret the results; if only the first detection line of the test strip is colored from bottom to top, or if the first detection line and the second detection line are colored at the same time, it is determined to be positive for Eimeria tenella; if only the second detection line is colored, it is determined to be negative.

[0018] Optionally, the CPA reaction system in step (2) consists of 2.5 μL of 10×Bst Buffer, 2.5 μL of 5M Betaine, 1 μL of 100 mM MgSO4, 2.5 μL of 10 mM dNTPs, 1 μL of 10 μM primer COI-4-1, 1 μL of 10 μM primer COI-4-2, 1.5 μL of 10 μM primer COI-4-3, 1.5 μL of 10 μM primer COI-4-4, 1.5 μL of 10 μM primer COI-4-5, 1 μL of 8 U / μL Bst DNA polymerase, 1 μL of DNA template, and 8 μL of ddH2O.

[0019] Optionally, the isothermal amplification reaction temperature in step (2) is 61°C and the reaction time is 1 hour.

[0020] This invention also provides the application of the CPA primer set or the kit described herein in the detection of Eimeria tenella in chickens for non-disease diagnostic purposes, wherein the Eimeria tenella is at least one of Eimeria tenella, Eimeria virulenta, Eimeria gianta, Eimeria tufteda, Eimeria bryonis, Eimeria milda, and Eimeria precociousa.

[0021] The present invention discloses the following technical effects:

[0022] This invention provides a cross-primer amplification (CPA) primer set capable of simultaneously detecting seven major Eimeria species in chickens. Based on this primer set, a CPA detection method for Eimeria in chickens is established. This method effectively solves the problems of detection blind spots and high false negative rates in existing single-species detection schemes in clinical mixed infection scenarios. This invention exhibits excellent specificity while ensuring broad-spectrum conservation, showing no cross-reactivity with other common intestinal parasites.

[0023] Compared to traditional molecular detection techniques such as PCR, the CPA method established in this invention requires no complex and expensive instruments, is simple and quick to operate, and can complete amplification within one hour. Combined with lateral flow chromatography test strips, it enables visual interpretation of the results, greatly reducing the technical threshold and operating costs. This method has extremely high sensitivity, with a detection limit of 10 for plasmid standards. 2 With a viral load of 0.5 copies / µL, it can stably detect extremely low pathogen loads equivalent to 0.5 oocysts in practical applications, and the clinical sample validation rate reaches 100%, providing strong technical support for early warning, on-site monitoring and precise control of coccidiosis in chickens. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 Electrophoresis results of the amplification products of four sets of CPA primers;

[0026] Figure 2 The images show the results of four sets of CPA primer amplification products lateral flow chromatography test strips; where a is the detection result of the blank control and b is the detection result of the positive sample.

[0027] Figure 3 This is a side-flow chromatography analysis diagram of CPA temperature gradient amplification.

[0028] Figure 4 A conservation test diagram for the CPA method established in this invention;

[0029] Figure 5 This is a specificity detection diagram of the CPA method established in this invention;

[0030] Figure 6 Sensitivity detection graph for the CPA method established in this invention;

[0031] Figure 7 CPA detection graphs for seven species of chicken Eimeria coccidia and negative clinical samples;

[0032] Figure 8 This is a graph illustrating the clinical sensitivity of the CPA method established in this invention. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] Example 1: Construction of a positive plasmid standard for the Eimeria coccidia gene in chickens.

[0039] 1. Amplification of the target gene

[0040] Specific primers were designed and synthesized based on the conserved sequence of the Eimeria coccidia mitochondrial cytochrome C oxidase I (COI) gene to amplify a target fragment of approximately 806 bp (as shown in SEQ ID NO.21). The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0041] Upstream primer F: 5'-GGTTCAGGTGTTGGTTGGAC-3', SEQ ID NO.22;

[0042] Downstream primer R: 5'-AATCCAATAACCGCACCAAG-3', SEQ ID NO.23.

[0043] SEQ ID NO.21:

[0044] .

[0045] Using extracted Eimeria coccidia genomic DNA as a template, PCR amplification was performed. The amplified products were separated by 2% agarose gel electrophoresis and observed under a UV imaging system. A specific band of approximately 806 bp was observed, consistent with the expected size. Subsequently, the target band was recovered using the StarPrep DNA purification / gel recovery kit according to the manufacturer's instructions.

[0046] 2. Construction, screening, and plasmid extraction of positive clones

[0047] The recovered PCR products were ligated into the pMD19-T (Simple) vector. All ligation products were transformed into competent cells (DH5α).

[0048] The revived bacterial culture was spread onto a solution containing ampicillin (Amp). + Incubate overnight at 37°C upside down on LB agar plates. Select single colonies for colony PCR verification, choosing those that amplify the expected band size. Extract high-purity plasmids using a plasmid extraction kit; these serve as positive plasmid standards. Further confirmation of the inserted COI sequence is achieved using Sanger sequencing.

[0049] Example 2: Screening of CPA amplification primers and establishment of a CPA-based detection method

[0050] 1. Design of CPA amplification primers

[0051] Based on the gene sequence of the 806 bp target fragment in Example 1, cross-primer amplification (CPA) primers were designed using Primer Explorer V5 (http: / / primerexplorer.jp / e / ) and the NCBI database. Subsequently, the specificity of all designed primers was evaluated using NCBI's BLAST tool, and the four sets of CPA primers with the best specificity were finally selected and synthesized by Qingke Biotechnology Co., Ltd. (Beijing) (primer information is shown in Table 1).

[0052] Table 1. CPA-specific primers

[0053]

[0054] 2. CPA reaction and primer screening

[0055] Using the positive plasmid standard constructed in Example 1 as a template, four sets of candidate primers were added to the CPA reaction system shown in Table 2 for primer screening.

[0056] Table 2 CPA Reaction System

[0057]

[0058] After thoroughly mixing the above reaction system, incubate it in a metal bath at 59°C for 1 hour to complete CPA amplification. The products were analyzed by agarose gel electrophoresis and side-flow chromatography (LFD) strips.

[0059] Agarose gel electrophoresis: 5 μL of amplification product was subjected to 2% agarose gel electrophoresis to compare the brightness and specificity of the amplified bands.

[0060] LFD: Add 75 μL of HybriDetect Assay Buffer to the remaining product, mix well, insert the test strip, and observe the results after 3 minutes. Simultaneously, set up a primer blank control without template. If only the first detection line (F) shows color, or if both the first and second detection lines (S) show color simultaneously, the result is considered positive; this is because residual primers that did not fully participate in the reaction can also cause the detection lines to show color. If only the second detection line (S) shows color, the result is considered negative.

[0061] 3. Results

[0062] After agarose gel electrophoresis, the four groups of CPA amplification products showed that the fourth group of primers had the highest amplification efficiency, the brightest band, and no non-specific bands. Figure 1 ).

[0063] LFD results showed that the four groups of CPA primer blank controls did not interfere with the experimental results. Figure 2 (a); In positive samples, the F line corresponding to primer group 4 showed the strongest color development and the best detection effect. Figure 2 (b).

[0064] Example 3: Optimization of CPA Reaction Conditions

[0065] The optimal primer set (Group 4) selected in Example 2 was used for CPA amplification, with the positive standard plasmid constructed in Example 1 as a template. The reaction system was the same as in Table 2 of Example 2. Five temperature gradients were set: 55℃, 57℃, 59℃, 61℃, and 63℃, and the reactions were carried out in a metal bath for 1 hour each. After the reaction, the results were uniformly detected using side-flow chromatography test strips, and the clarity and intensity of the first detection line (F line) at different temperatures were compared.

[0066] The results showed that, under reaction conditions of 61℃, the first detection line (F line) developed the clearest and fastest color, with the lowest background. Figure 3 Therefore, 61℃ was determined as the optimal reaction temperature for subsequent experiments.

[0067] Example 4 Evaluation of the conservation, specificity, and sensitivity of the CPA method

[0068] To comprehensively evaluate the reliability of the CPA method established in this invention, this embodiment tested the conservatism, specificity, and sensitivity of the established CPA method.

[0069] 1. Conservative testing

[0070] The test materials were genomic DNA from Eimeria tenella, Eimeria toxicaecatrix, Eimeria maxima, Eimeria acervuline, Eimeria brunetti, Eimeria mitis, and Eimeria praecox.

[0071] Genomic DNA from the seven chicken Eimeria coccidia species was used to perform CPA amplification according to the system established in Example 2 and the optimal temperature (61°C) established in Example 3, and the LFD results were interpreted.

[0072] The results are as follows Figure 4 As shown, the first detection line of all samples was clearly visible, indicating that the method has broad-spectrum conservation.

[0073] 2. Specific detection

[0074] The test materials were genomic DNA from Enterocytozoon bieneusi, Blastocystis, and Cryptosporidium.

[0075] Genomic DNA from the other three common parasites in chickens mentioned above, and ddH2O as a negative control, were used for CPA amplification according to the system established in Example 2 and the optimal temperature (61°C) established in Example 3. The LFD results were interpreted.

[0076] The results are as follows Figure 5 As shown, the detection results for the three non-target parasites and the ddH2O control were all negative (only the S line was visible), indicating that the method has good specificity and no cross-reactivity.

[0077] 3. Sensitivity Detection

[0078] The positive standard plasmid constructed in Example 1 was serially diluted 10-fold (10... 6 10 5 10 4 10 3 10 2 10 1 and 10 0 CPA amplification was performed according to the system established in Example 2 and the optimal temperature (61°C) established in Example 3, and the results were interpreted by LFD.

[0079] The results are as follows Figure 6As shown, the detection limit of the CPA method constructed in this invention for plasmid DNA is 10. 2 copies / μL.

[0080] Example 5: Evaluation of the clinical efficacy of CPA testing

[0081] This embodiment aims to verify the accuracy of the established CPA detection method on actual clinical samples and to evaluate its detection sensitivity in simulated clinical infection low-viral-load samples.

[0082] 1. Clinical sample validation

[0083] Sample source: 52 clinical chicken fecal samples were collected. The infection status of these samples had been pre-determined by microscopic examination and routine PCR methods, serving as the gold standard control.

[0084] The samples were divided into four groups: Eimeria tenella (1-7), Eimeria necatrix (8-14), Eimeria maxima (15-21), Eimeria acervuline (22-28), Eimeria brunetti (29-35), Eimeria mitis (36-42), Eimeria praecox (43-49), and a negative control group (no coccidia infection, 50-52).

[0085] DNA extraction: DNA was extracted from all samples using a commercially available fecal genomic DNA extraction kit.

[0086] CPA detection: Take 1 μL of the above DNA extract as a template and perform CPA amplification according to the reaction system determined in Example 2 (using the fourth set of primers) and the optimal reaction conditions (61℃) determined in Example 3. The reaction time is 1 hour. After amplification, the results are interpreted using a lateral flow chromatography test strip.

[0087] The results are as follows Figure 7 The CPA test results of 52 clinical samples were completely consistent with the pre-determined infection status. All positive samples (1-49) showed positive results on the lateral flow chromatography strips (both the first test line, F, and the second test line, S, were visible simultaneously). All negative samples (50-52) showed negative results on the lateral flow chromatography strips (only the second test line, S, was visible).

[0088] The above demonstrates that the CPA method established in this invention has an overall concordance rate of 100% compared with the conventional PCR method, proving that the method has high accuracy and reliability in clinical applications.

[0089] 2. Evaluation of clinical test sensitivity

[0090] Previous studies have indicated that the mitochondria of a single chicken coccidia oocyst carry a large number of genome copies, with unspecified oocysts exceeding 500 copies and sporulated oocysts exceeding 1000 copies. Therefore, although the CPA method established in this study has a sensitivity of 10 in plasmid standard testing... 2 The copy number was 0.1 copies / µL, but given the high copy number of its mitochondrial genome, it is theoretically sufficient to achieve a detection sensitivity as low as 0.1 oocysts. To verify this theoretical hypothesis, this study further detected genomic DNA from serially diluted oocysts.

[0091] Template preparation: Genomic DNA was extracted from oocysts of Eimeria tenella (using Eimeria tenella as an example) at known concentrations. Genomic DNA template solutions equivalent to 1 oocyst / μL, 0.5 oocysts / μL, and 0.1 oocysts / μL were prepared by quantitative and serial dilution.

[0092] CPA assay: Using the serially diluted oocyst genomic DNA as templates, CPA amplification and LFD assay were performed, with three replicates for each gradient. The reaction system and conditions were the same as above.

[0093] CPA assays using serially diluted oocyst genomic DNA as templates showed that samples with a concentration of 1 oocyst / μL were all strongly positive. Samples with a concentration of 0.5 oocysts / μL were positive. Figure 8 ).

[0094] This demonstrates that the CPA method established in this invention has a minimum stable detection limit for genomic DNA equivalent to 0.5 oocysts in actual detection, exhibiting extremely high detection sensitivity, which is sufficient to meet the detection needs of low-dose infections in clinical practice.

[0095] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A CPA primer set for detecting seven species of Eimeria tenella in chickens, characterized in that, The primer set consists of primer COI-4-1 as shown in SEQ ID NO. 16, primer COI-4-2 as shown in SEQ ID NO. 17, primer COI-4-3 as shown in SEQ ID NO. 18, primer COI-4-4 as shown in SEQ ID NO. 19, and primer COI-4-5 as shown in SEQ ID NO.

20.

2. The CPA primer set according to claim 1, characterized in that, The 5' end of primer COI-4-1 is labeled with biotin, and the 5' end of primer COI-4-2 is labeled with fluorescein isothiocyanate (FITC).

3. The CPA primer set according to claim 1, characterized in that, The seven species of chicken Eimeria tenella are Eimeria tenella, Eimeria necatrix, Eimeria maxima, Eimeria acervuline, Eimeria brunetti, Eimeria mitis, and Eimeria praecox.

4. The use of the CPA primer set according to any one of claims 1-3 in the preparation of a kit for detecting Eimeria tenella in chickens.

5. A kit for detecting Eimeria tenella in chickens, characterized in that, The kit comprises the CPA primer set as described in any one of claims 1-3.

6. The reagent kit according to claim 5, characterized in that, The kit is used to detect at least one of Eimeria tenella, Eimeria toxicae, Eimeria giantiformis, Eimeria tuftediformis, Eimeria bryonici, Eimeria mildis, and Eimeria precociousiformis.

7. A method of using the kit according to claim 5 or 6, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the genomic DNA obtained in step (1) as a template, prepare the CPA reaction system and perform isothermal amplification; (3) After mixing the amplification product from step (2) with the detection buffer, use the side-flow chromatography test strip to interpret the results; if only the first detection line of the test strip is colored from bottom to top, or if the first detection line and the second detection line are colored at the same time, it is determined to be positive for Eimeria tenella; if only the second detection line is colored, it is determined to be negative.

8. The method of use according to claim 7, characterized in that, The CPA reaction system described in step (2) consists of 2.5 μL of 10×Bst Buffer, 2.5 μL of 5M Betaine, 1 μL of 100mM MgSO4, 2.5 μL of 10mM dNTPs, 1 μL of 10 μM primer COI-4-1, 1 μL of 10 μM primer COI-4-2, 1.5 μL of 10 μM primer COI-4-3, 1.5 μL of 10 μM primer COI-4-4, 1.5 μL of 10 μM primer COI-4-5, 1 μL of 8 U / μL Bst DNA polymerase, 1 μL of DNA template, and 8 μL of ddH2O.

9. The method of use according to claim 7, characterized in that, The isothermal amplification reaction temperature in step (2) is 61°C and the reaction time is 1 hour.

10. The application of the CPA primer set according to any one of claims 1-3 or the kit according to claim 5 or 6 in the detection of Eimeria tenella in chickens for non-disease diagnostic purposes, characterized in that, The chicken Eimeria coccidia is at least one of Eimeria tenella, Eimeria toxicae, Eimeria giantis, Eimeria tuftedis, Eimeria bryonis, Eimeria mildis, and Eimeria precociousis.

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