Dual PCR identification method for Cryptosporidium bovis and Eimeria bovis

Abstract

The application provides a double-PCR differential detection method for Cryptosporidium parvum and Eimeria bovis, which is used for synchronously differentiating and detecting Cryptosporidium parvum and Eimeria bovis in a to-be-detected sample; the double-PCR differential detection method uses the following primer combination: an upstream primer Cpar-F for Cryptosporidium parvum, a downstream primer Cpar-R for Cryptosporidium parvum, an upstream primer Ebov-F for Eimeria bovis, and a downstream primer Ebov-R for Eimeria bovis. The application also provides application of the double-PCR detection method and a kit for synchronously differentiating Cryptosporidium parvum and Eimeria bovis. The double-PCR differential detection method can simultaneously and specifically amplify two target fragments in the same reaction tube, so that rapid differentiation of two pathogens and mixed infection of the two pathogens can be realized.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a method for simultaneously detecting and identifying bovine Cryptosporidium microsporidium and bovine Eimeria coccidia. Background Technology

[0002] Both *Cryptospora microsporum* and *Eimeria aurea* are common protozoan pathogens that harm the digestive health of cattle, especially in calves, causing diarrhea, dehydration, electrolyte imbalance, growth retardation, and secondary infections. In severe cases, they can lead to death, resulting in economic losses such as decreased feed conversion ratio, increased treatment and care costs, and reduced production performance. Because both pathogens can be transmitted via the fecal-oral route, and their oocysts, eggs, or gametocytes possess a certain degree of resistance in the environment, they can easily create persistent contamination within farms, posing a challenge to biosecurity management in large-scale farms.

[0003] In actual production, the clinical manifestations of the two protozoan infections mentioned above are quite similar, such as watery or pasty diarrhea, lethargy, and decreased feed intake, making accurate differentiation difficult based solely on clinical symptoms. Furthermore, intestinal protozoan infections in calves often coexist with bacteria, viruses, or other parasites; mixed infections can worsen the disease course and affect treatment strategies and control measures. Therefore, establishing a rapid, accurate, and simultaneous detection method to differentiate between the two pathogens is of great significance for early diagnosis, epidemiological monitoring, and precision medicine.

[0004] Currently, routine laboratory testing for *Cryptospora microsporum* and *Eimeria tenella* in bovine protozoa primarily relies on fecal morphology examination, including methods such as flotation, direct smear microscopy, counting, and modified acid-fast staining. While these methods have relatively low instrument requirements, they suffer from limitations such as cumbersome procedures, high dependence on operator experience, insufficient sensitivity in low-parasite-quantity samples, and difficulty in species identification. In fecal samples with mixed infections or significant background contamination, false negatives or misdiagnoses are common when oocyst morphology is similar or their numbers are low. Immunological tests, such as ELISA and rapid immunochromatographic test strips, can improve detection efficiency to some extent, but their results are easily affected by factors such as antigen concentration, sample matrix interference, antibody specificity, and cross-reactivity. Furthermore, their applicability and stability under different regional and prevalent strain backgrounds require further validation. Molecular biological methods, such as conventional PCR, nested PCR, and real-time quantitative PCR, are increasingly used for protozoan pathogen detection and typing due to their high specificity and sensitivity. However, most existing molecular detection methods use single amplification mode. If multiple pathogens need to be screened simultaneously, multiple reaction systems need to be established and multiple amplification and electrophoresis / signal readings are usually performed. This not only increases sample and reagent consumption and prolongs the detection cycle, but also increases the frequency of pipetting operations and the risk of cross-contamination. On the other hand, methods such as real-time quantitative PCR have high requirements for instruments and equipment and operating costs, which limits their widespread application in rapid screening in primary laboratories or on-site.

[0005] Therefore, there is an urgent need to develop a detection technology that is easy to operate, highly specific, highly sensitive, and capable of simultaneously identifying and detecting Cryptosporidium bovis and Eimeria coccidia bovis in the same reaction system, in order to meet the practical needs of rapid clinical screening, identification of mixed infections, and monitoring and early warning in large-scale farms. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a dual PCR method that can simultaneously identify and detect Cryptosporidium bovis and Eimeria bovis in the same reaction system, thereby increasing the throughput of clinical sample testing and reducing testing costs.

[0007] A second objective of this invention is to propose the application of the aforementioned dual PCR method.

[0008] The third objective of this invention is to provide a kit for the simultaneous identification of *Cryptospora microsporidium* and *Eimeria tenella*.

[0009] The technical solution for achieving the above-mentioned objective of this invention is as follows: A dual PCR method for the identification and detection of *Cryptospora microsporum* and *Eimeria aurea* in bovine samples is disclosed, for the simultaneous identification and detection of these two species in test samples. The dual PCR method uses the following two pairs of specific primers as a primer combination: The upstream primer Cpar-F for Cryptosporidium bovis, whose gene sequence is SEQ ID NO.1; The downstream primer Cpar-R for Cryptosporidium bovis, whose gene sequence is SEQ ID NO.2; The upstream primer Ebov-F for Eimeria coccidia bovis, with the gene sequence SEQ ID NO.3; The downstream primer Ebov-R of Eimeria abortus, whose gene sequence is SEQ ID NO.4; After amplification using the primers, the amplified product of *Cryptospora microsporum* was 325 bp, and the amplified product of *Eimeria tenella* was 217 bp.

[0010] Furthermore, the dual PCR identification detection method includes the following steps: 1) Sample preparation: Extract whole genome DNA from the sample to be tested as a template. The sample to be tested is one of the following: the body, oocyst, or sporozoite of Cryptosporidium bovis; and one of the following: the body, oocyst, or sporozoite of Eimeria tenella. 2) Duplex PCR amplification: PCR amplification was performed using the primer composition described above; 3) Result interpretation: The PCR amplification products were detected by agarose gel electrophoresis. The presence of a 325 bp band indicated a positive result for Cryptosporidium bovis, the presence of a 217 bp band indicated a positive result for Eimeria aureus, the presence of two bands indicated a double positive result, and the absence of any bands indicated a negative result.

[0011] Step 1) DNA extraction is preferably performed using oocysts, as oocysts are the most common and standardized material and can be easily obtained from fresh feces.

[0012] Preferably, the total volume of the reaction system for duplex PCR amplification in step 2) is 25 μL, comprising: The mixture consisted of 12.5 μL of Premix Taq, 1 μL of upstream mixed primers, 1 μL of downstream mixed primers, 2 μL of template DNA, and 8.5 μL of ddH2O. The primers were mixed in equal volumes. The template DNA was a mixture of whole-genome DNA extracted from *Cryptospora microsporum* and *Eimeria tenella*.

[0013] The reaction procedure for double PCR amplification is as follows: Pre-denaturation at 94℃ for 5 min; followed by 35 cycles: denaturation at 94℃ for 30 s, annealing for 35 s, extension at 72℃ for 1 min; and finally extension at 72℃ for 7 min, followed by holding at 4℃.

[0014] Gradient annealing was used, and the annealing temperature was optimized between 57℃ and 62℃, with each optimization increasing by 1℃, and the final optimization result was 60℃.

[0015] The annealing temperature is 57°C to 62°C, preferably 60°C.

[0016] More preferably, electrophoresis is performed using a 1.5% agarose gel at 220 V for 30 min.

[0017] The minimum detection concentrations of the method of the present invention are: 0.01 pg / μL for Cryptosporidium bovis and 0.1 pg / μL for Eimeria tenella bovis.

[0018] The dual PCR detection method described in this invention is used in the simultaneous screening of bovine Cryptosporidium microsporidium and bovine Eimeria coccidia and their mixed infections in bovine fecal samples.

[0019] A kit for simultaneously identifying bovine Cryptosporidium microsporidium and bovine Eimeria coccidia, comprising the primer composition described above, PCR amplification reaction mixture, template DNA, and sterile double-distilled water (which also serves as a negative control).

[0020] Preferably, the PCR amplification reaction mixture in the kit is Premix Taq, and the template DNA is positive control template DNA or positive plasmid DNA.

[0021] Duplex PCR is a molecular detection technique that simultaneously amplifies two different target sequences within the same PCR reaction system. This technique offers advantages such as simultaneous detection in a single reaction, saving samples and reagents, and reducing operational errors and contamination risks. The amplified products can be interpreted using conventional agarose gel electrophoresis. It requires minimal equipment and is easily adopted in grassroots laboratories.

[0022] The beneficial effects of this invention are as follows: 1. The present invention proposes a dual PCR method for the identification and detection of Cryptosporidium bovis and Eimeria coccidia bovis. Two pairs of specific primers are designed for the conserved regions of the 18S rRNA genes of the two pathogens, and a dual amplification system is established through system optimization (including annealing temperature and reaction conditions). This method can simultaneously and specifically amplify the two target fragments in the same reaction tube, thereby achieving rapid identification of the two pathogens and their mixed infections.

[0023] 2. This dual PCR detection method uses a conventional DNA polymerase amplification system for amplification. After the reaction, the results are directly interpreted by 1.5% agarose gel electrophoresis: the presence of a 325 bp band indicates a positive result for Cryptosporidium bovis, the presence of a 217 bp band indicates a positive result for Eimeria aureus, the presence of both bands indicates a double positive / mixed infection, and the absence of a corresponding band indicates a negative result. The interpretation is intuitive and clear.

[0024] 3. This dual PCR detection method has high sensitivity: the lowest detection concentration of Bovine Cryptosporidium can reach 0.01 pg / μL, and the lowest detection concentration of Bovine Eimeria coccidia can reach 0.1 pg / μL; compared with traditional fecal microscopy and other methods, it is more conducive to improving the detection rate and identification accuracy in the context of low parasite quantity or mixed infection.

[0025] 4. Rapid, efficient, accurate, and low-cost detection: The preferred annealing temperature for the dual PCR is 60℃ (which can be optimized within the range of 57℃ to 62℃). A single amplification can simultaneously obtain detection results for two pathogens, reducing the time and material costs associated with repeated system construction and multiple amplifications. This method requires only a conventional PCR instrument and electrophoresis system to complete the detection, without relying on expensive real-time quantitative PCR platforms. It is suitable for rapid screening of clinical samples and monitoring applications in livestock farms, helping to promptly determine the infection status of the two pathogens and guide the formulation of prevention and control measures. Attached Figure Description

[0026] Figure 1 This is an agarose gel electrophoresis image of the annealing temperature optimized in Example 5. In the image, M: DL2000 DNA Marker, 1: 57℃, 2: 58℃, 3: 59℃, 4: 60℃, 5: 61℃, 6: 62℃, N: negative control. Figure 2This is an agarose gel electrophoresis image of the double PCR specificity verification in Example 6. In the image, M: DL2000 DNA Marker; 1: *Cryptospora bovis* + *Eimeria tenella*; 2: *Cryptospora bovis* + other Cryptosporidia species (excluding *Cryptospora bovis*); 3: *Cryptospora bovis* + other *Eimeria tenella* species (excluding *Eimeria tenella*); 4: *Cryptospora bovis*; 5: *Haemaphysalis contortus*; 6: *Eimeria tenella*; 7: *C. elegans*; 8: *Cryptospora bovis* + *Cryptospora bovis*; 9: *Cryptospora bovis* + *Haemaphysalis contortus*; 10: *Cryptospora bovis* + *Eimeria tenella*; 11: *Cryptospora bovis* 12: Eimeria tenella + Cryptosporidium elegans; 13: Eimeria tenella + Haemophilus contortus; 14: Eimeria tenella + Eimeria ovis; 15: Eimeria tenella + Cryptosporidium elegans; 16: Cryptosporidium elegans + Haemophilus contortus + Eimeria ovis; 17: Cryptosporidium elegans + Haemophilus contortus + Cryptosporidium elegans; 18: Cryptosporidium elegans + Eimeria ovis + Cryptosporidium elegans; 19: Haemophilus contortus + Eimeria ovis + Cryptosporidium elegans; N: Negative control; Figure 3 The image shows an agarose gel electrophoresis result of the dual PCR sensitivity test in Example 7. In the image, M: DL2000 DNA Marker, 1: 10 pg / μL, 2: 1 pg / μL, 3: 0.1 pg / μL, 4: 0.01 pg / μL, 5: 0.001 pg / μL, 6: 0.0001 pg / μL, and N: negative control. Detailed Implementation

[0027] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0028] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0029] 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.

[0030] Unless otherwise specified, all instruments, reagents and consumables used in this invention are purchased from the market.

[0031] Example 1: Extraction of genomic DNA from test samples (1) Select bovine Cryptosporidium microsporidium ( Cryptosporidium parvum) oocysts, Eimeria coccidia ( Eimeria bovis Oocysts were used as the target pathogen samples; simultaneously, Haemonchus contortus (…) was selected. Haemonchus contortus ) eggs, Eimeria coccidia oocysts, Caenorhabditis elegans ( Caenorhabditis elegans Insect eggs and other samples were used as control samples for subsequent double PCR specificity evaluation.

[0032] (2) Genomic DNA was extracted from the above samples using a commercial genomic DNA extraction kit, and the obtained DNA was used as a template for PCR amplification. For the two pathogen samples, Cryptosporidium bovis and Eimeria aureus, the two DNAs were mixed in equal volumes in the reaction system as template DNA; and sterile double-distilled water (ddH2O) was used as a negative control template.

[0033] Example 2: Extraction of plasmid DNA Plasmid DNA was obtained by purifying positive samples via agarose gel electrophoresis, followed by gel extraction and extraction, and then constructing the target gene fragment into a plasmid vector. There was no difference in detection performance between plasmid DNA and positive sample DNA. The specific experimental method is as follows: 1. Recovery and purification of target DNA fragments The amplified electrophoresis products were purified according to the instructions of the Takara agarose gel purification kit. (1) Locate the target band region under cold light source ultraviolet light, and quickly cut the gel block with a sterile blade. Take care to avoid DNA damage caused by prolonged ultraviolet irradiation.

[0034] (2) Chop the cut gel block and transfer it to a 1.5 mL centrifuge tube.

[0035] (3) Weigh the gel block and add Buffer GM to the gel block. When the gel concentration is 1.0%, the amount of Buffer GM used is 3 gel volumes; when the gel concentration is 1.0%-1.5%, the amount of Buffer GM used is 4 gel volumes; when the gel concentration is 1.5%-2.0%, the amount of Buffer GM used is 5 gel volumes.

[0036] (4) After uniform mixing, dissolve the glue block at 37°C. During this period, the glue block should be shaken intermittently to ensure that it is fully dissolved, which takes about 5-10 minutes. For high-concentration glue blocks, the time can be extended appropriately.

[0037] (5) Place the Spin Column on the Collection Tube.

[0038] (6) Pipette the dissolved solution into a Spin Column, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0039] (7) Add 700 μL Buffer WB, centrifuge at 12000 r / min for 30 s at room temperature, and discard the filtrate.

[0040] (8) Repeat step 7.

[0041] (9) Place the Spin Column on the Collection Tube and centrifuge at 12000 r / min for 1 min at room temperature.

[0042] (10) Place the Spin Column in a new 1.5 mL EP tube, add 30 μL of preheated Elution Buffer to 60 °C to the center of the membrane at the bottom of the Spin Column, and let stand at room temperature for 1 min.

[0043] (11) Elute DNA by centrifuging at 12000 r / min for 1 min at room temperature.

[0044] 2. Ligation and transformation of gel-recovered products with the carrier pMD19-T The target gene fragment was constructed into the pMD19-T vector system using the TA cloning method. The specific experimental steps are as follows: (1) Construct a 10 μL T vector ligation system: 1 μL pMD19-T vector, 4 μL purified product, 5 μL Solution I, vortexed for 5 s, then centrifuged for 10 s, and ligated overnight at 4℃; (2) Take 10 μL of the ligation product and gently suspend it with 50 μL of DH5α competent cells, and equilibrate on ice for 30 min to complete the cell membrane permeability treatment. (3) After being heat-shocked in a 42℃ water bath for 90 s, immediately subjected to an ice bath for 15 min; (4) Add 800 μL of pre-warmed LB liquid medium without antibiotics, and shake at 37℃ and 200 r / min for 1 h to recover; (5) Collect the bacterial cells by centrifugation at 4000×g and resuspend them in 100 μL LB liquid medium; (6) Take 100 μL of the conversion solution and spread it on an ampicillin-LB agar plate. Adsorb at room temperature for 30 min and then incubate at 37℃ upside down for 18±2 h. (7) Pick a single white colony and inoculate it into LB liquid medium containing resistance. Shake at 37°C and 220 r / min. After 14 h, extract plasmids for verification.

[0045] 3. Extraction of bacterial DNA Follow the instructions for the Tiangen bacterial genomic DNA extraction kit.

[0046] (1) The bacterial cell (plasmid was amplified in E. coli) pellet was resuspended in 200 μL of buffer GA and gently shaken until completely dissolved; (2) Add 20 μL of proteinase K solution, mix well, then add 220 μL of buffer GB and shake for 15 s; (3) Add 20 μL of proteinase K solution to the centrifuge tube and mix well; (4) Pyrolyze at 70℃ for 10 min, then remove droplets from the tube wall by instantaneous centrifugation; (5) Inject 220 μL of anhydrous ethanol, shake and mix for 15 s, then centrifuge briefly and transfer the entire mixture to the CB3 adsorption column; (6) Centrifuge at 12000×g for 30 s and discard the filtrate. Wash with 500 μL buffer GD and 600 μL wash buffer PW twice. (7) Centrifuge the empty column at 12000×g for 2 min, and let it stand at room temperature for 5 min to fully dry the washing solution; (8) Add 50-200 μL of TE buffer to elute, let stand for 2 min, and then centrifuge at 12000×g to elute DNA.

[0047] 4. Plasmid identification The extracted bacterial DNA was used as a template for PCR. 5 μL of the PCR product was spotted onto a 1.5% agarose gel, and the control wells of a DL2000 DNA Marker were set. Electrophoresis was performed at a constant voltage of 120 V for 30 min. After electrophoresis, the gel was placed in a blue light imaging system to observe the bands and take photographs.

[0048] 5. Positive plasmid sequencing and phylogenetic analysis Positive plasmids were subjected to bidirectional sequencing. The obtained sequences were compared with reference sequences for Cryptosporidium microsporidium and Eimeria tenella in the GenBank database.

[0049] Example 3: Design and synthesis of specific primers for duplex PCR (1) Using the conserved regions of the bovine Cryptosporidium microsporidium 18S rRNA gene and the bovine Eimeria coccidia 18S rRNA gene as targets, two pairs of specific amplification primers were designed and determined to achieve simultaneous amplification of dual targets in the same reaction system.

[0050] 18S rRNA is not the only conserved gene segment, but rRNA genes usually exist in multiple copies, making them easier to detect in samples with low insect populations, thus improving detection sensitivity. Moreover, 18S rRNA is generally conserved among closely related species, and its structure is suitable for use as species-specific primers.

[0051] (2) Send the primer information to the biotechnology company for synthesis; dilute each primer to 10 μmol / L with sterile ddH2O before the experiment.

[0052] (3) The specific product size was obtained by gel extraction and Sanger sequencing after agarose gel electrophoresis.

[0053] The sequences of the double PCR primers used and the sizes of the amplified fragments (the amplified fragments refer to the fragments amplified by specific primers for Cryptosporidium bovis and Eimeria bovis, with sizes of 325 bp and 217 bp, respectively) are shown in Table 1.

[0054] Table 1. Primer sequences for double PCR

[0055] Example 4: Establishment of Duplex PCR Reaction System and Procedure (1) Positive template DNA (positive plasmid DNA or positive sample DNA, with no difference in detection effect between plasmid DNA and positive sample DNA; positive sample DNA was used in this embodiment) of *Cryptospora bovis* and *Eimeria tenella* was used as the template. The template in the duplex PCR reaction system was obtained by mixing equal amounts of positive DNA from *Cryptospora bovis* and *Eimeria tenella*, and the duplex PCR reaction system was prepared according to the following reaction system.

[0056] (2) The dual PCR reaction system is shown in Table 2.

[0057] Table 2 Dual PCR Reaction System Element Volume / μL Premix Taq 12.5 μL Upstream primers (mixed) 1 μL Downstream primers (mixed) 1 μL Template DNA 2 μL <![CDATA[ddH2O]]> 8.5 μL Total volume 25 μL Note: The "upstream primer (mixed)" is a mixture of upstream primers for two pathogens; the "downstream primer (mixed)" is an equal volume mixture of downstream primers for two pathogens.

[0058] (3) The reaction procedure for double PCR is shown in Table 3. In this example, the annealing temperature was initially selected as 57°C, and this parameter was optimized in Example 5.

[0059] Table 3 Duplex PCR Reaction Procedure Reaction steps Temperature (°C) reaction time Loop count Pre-variation 94 5 min 1 transsexual 94 30 s annealing 57 35 s 35 extend 72 1 min Post-extension 72 7 min 1 Insulation 4 — — (4) PCR product detection: After amplification, the amplification products are detected by agarose gel electrophoresis; preferably, 1.5% agarose gel is used, and the electrophoresis conditions are preferably 220 V for 30 min. The bands are observed and recorded using an ultraviolet imaging system.

[0060] Example 5: Optimization of Annealing Temperature (1) Using positive template DNA from bovine Cryptosporidium microsporidium and Eimeria coccidia bovine, a double PCR reaction was prepared according to the reaction system described in Example 4.

[0061] (2) Set gradient annealing temperatures (57℃, 58℃, 59℃, 60℃, 61℃, 62℃), keep other amplification conditions unchanged, and perform double PCR amplification; do not add template DNA to the negative control; preferably set 3 replicates for each treatment.

[0062] (3) Electrophoresis was performed after amplification to detect and compare the brightness and clarity of the bands.

[0063] See Figure 1 Electrophoresis results showed that the two target bands (325 bp and 217 bp) were clearest and brightest when the annealing temperature was 60℃. Therefore, 60℃ was determined to be the optimal annealing temperature for duplex PCR.

[0064] Example 6: Duplex PCR Specificity Assay (1) The dual PCR method established in Example 3 and optimized in Example 5 (annealing temperature of 60℃) was used to amplify DNA templates as follows: 1) Single DNA from *Cryptospora bovis*; 2) Single DNA from Eimeria tenella var. bovis; 3) Mixed DNA of *Cryptospora microsporum* and *Eimeria tenella*; 4) Mixed DNA of *Cryptospora microsporum* and control parasites (*Haemaphysalis contortus*, *Eimeria tenella*, and *C. elegans*); 5) Mixed DNA of Eimeria coccidia caudatum and control parasites; 6) Only parasite DNA was compared; 7) Negative control (ddH2O).

[0065] (2) Electrophoresis detection was performed after amplification.

[0066] See Figure 2 Electrophoresis results showed that the target band (325 bp and / or 217 bp) was amplified only in the reaction containing bovine Cryptosporidium microsporidium and / or bovine Eimeria coccidia template, while no corresponding band appeared in the other control templates and negative controls, indicating that the doublet PCR method has good specificity.

[0067] Example 7 Dual PCR Sensitivity Test (1) The positive plasmid DNA of *Cryptospora microsporum* and *Eimeria tenella* was serially diluted using a 10-fold dilution method, starting at a concentration of 10 ng / μL, and then successively diluted to obtain concentrations of 1 ng / μL, 0.1 ng / μL, 0.01 ng / μL, etc. DNA was still detectable at a concentration of 0.01 ng / μL. This was followed by further serial dilutions to obtain concentrations of 1 pg / μL, 0.1 pg / μL, 0.01 pg / μL, 0.001 pg / μL, 0.0001 pg / μL, etc.

[0068] (2) In the same dual PCR reaction system, 0.5 μL of each of the bovine Cryptosporidium microsporidium and Eimeria coccidia DNA templates were added and amplified under the optimal conditions determined in Examples 3-4; no template DNA was added to the negative control; and three replicates were preferably set for each gradient.

[0069] (3) Electrophoresis was performed after amplification and the lowest detectable concentration was recorded.

[0070] See Figure 3 The results showed that the lowest detectable concentration of *Cryptospora microsporidium* in bovine tissue was 0.01 pg / μL, and the lowest detectable concentration of *Eimeria tenella* in bovine tissue was 0.1 pg / μL, indicating that the doublet PCR method has high sensitivity.

[0071] Example 8 Duplex PCR Repeatability Test (1) Select bovine Cryptosporidium microsporidium DNA, bovine Eimeria coccidia DNA, and control parasite DNA (Haemaphysalis contortus, Eimeria coccidia foetida, Caenorhabditis elegans, etc.) and randomly combine them, while setting up a negative control.

[0072] (2) Perform double PCR amplification according to the optimal reaction system and procedure determined in Example 4; each sample group should be repeated at least 3 times.

[0073] (3) Electrophoresis was performed on each repeat amplification result and the band consistency was compared.

[0074] The results showed that the position and intensity of the target band were stable and consistent in the repeated experiments, and no band was observed in the negative control, indicating that the doublet PCR method has good repeatability and high stability.

[0075] For example, a sample containing a combination of Cryptosporidium bovis and Haemonchus contortus showed only a 325 bp band.

[0076] Example 9: Clinical validation and comparison of the duplex PCR method (1) Eighty samples were randomly selected from bovine fecal samples obtained from the epidemiological survey. The infection status of the two pathogens in the above 80 samples was pre-determined, with a total of 23 positive samples, including 2 single positive samples of Cryptosporidium bovis, 14 single positive samples of Eimeria tenella bovis, and 7 double positive or mixed infection samples, and 57 negative samples. The samples were tested and compared with the dual PCR method of this embodiment using microscopic methods such as acid-fast staining or McMaster counting.

[0077] (2) Double PCR interpretation criteria: The presence of a 325 bp band indicates a positive result for Cryptosporidium bovis; the presence of a 217 bp band indicates a positive result for Eimeria tenella; the presence of both bands indicates a double positive result or mixed infection; the absence of the corresponding band indicates a negative result.

[0078] (3) The positive rates, single positive rates and double positive rates of duplex PCR and microscopic examination were statistically analyzed and compared. The results are shown in Table 4.

[0079] Table 4. Clinical validation results of dual PCR method quantity Positive rate Cryptosporidium microsporidium positivity rate Positive rate of Eimeria coccidia in bovine cells Double positive rate Known state 80 28.75%（23 / 80） 11.25%（9 / 80） 26.25%（21 / 80） 8.75%（7 / 80） PCR 80 28.75%（23 / 80） 11.25%（9 / 80） 26.25%（21 / 80） 8.75%（7 / 80） Microscopic examination 80 20.00%（16 / 80） 5.00%（4 / 80） 15.00%（12 / 80） 1.25%（1 / 80） The results showed that in 80 clinical samples with known infection states, the dual PCR detection results of this embodiment were completely consistent with the known results, enabling simultaneous differentiation and detection of Cryptosporidium bovis, Eimeria aureus, and their mixed infections. Compared with traditional microscopy, microscopy is insufficient in detecting known positive samples, indicating a high risk of false negatives. The dual PCR of this embodiment has more reliable clinical screening and differentiation application value.

[0080] Although the present invention has been described above through embodiments, those skilled in the art should understand that any improvements and modifications made to the present invention without departing from its spirit and essence should fall within the protection scope of the present invention.

Claims

1. A dual PCR method for the identification and detection of *Cryptospora bovis* and *Eimeria aurea* in bovine samples, used for the simultaneous identification and detection of *Cryptospora bovis* and *Eimeria aurea* in test samples; characterized in that... The dual PCR identification detection method uses the following two pairs of specific primers as primer combinations: The upstream primer Cpar-F for Cryptosporidium bovis, whose gene sequence is SEQ ID NO.1; The downstream primer Cpar-R for Cryptosporidium bovis, whose gene sequence is SEQ ID NO.2; The upstream primer Ebov-F for Eimeria coccidia bovis, with the gene sequence SEQ ID NO.3; The downstream primer Ebov-R of Eimeria abortus, whose gene sequence is SEQ ID NO.4; After amplification using the primer combination, the amplified product of Cryptosporidium bovis was 325 bp, and the amplified product of Eimeria tenella bovis was 217 bp.

2. The dual PCR identification and detection method according to claim 1, characterized in that, Including the following steps: 1) Sample preparation: Extract whole genome DNA from the sample to be tested as a template. The sample to be tested is one of the following: the body, oocyst, or sporozoite of Cryptosporidium bovis; and one of the following: the body, oocyst, or sporozoite of Eimeria tenella. 2) Duplex PCR amplification: PCR amplification was performed using the primer composition described above; 3) Result interpretation: The PCR amplification products were detected by agarose gel electrophoresis. The presence of a 325 bp band indicated a positive result for Cryptosporidium bovis, the presence of a 217 bp band indicated a positive result for Eimeria aureus, the presence of two bands indicated a double positive result, and the absence of any bands indicated a negative result.

3. The dual PCR identification and detection method according to claim 2, characterized in that, The total volume of the reaction system for duplex PCR amplification in step 2) is 25 μL, containing: Premix Taq 12.5 μL, upstream mixed primer 1 μL, downstream mixed primer 1 μL, template DNA 2 μL, ddH2O 8.5 μL; the primers are mixed in equal volumes, and the template DNA is an equal volume mixture of whole genome DNA extracted from Bovine Cryptosporidium microsporidium and Bovine Eimeria coccidia.

4. The dual PCR identification and detection method according to claim 1, characterized in that, The reaction procedure for double PCR amplification is as follows: Pre-denaturation at 94℃ for 5 min; followed by 35 cycles: denaturation at 94℃ for 30 s, annealing for 35 s, extension at 72℃ for 1 min; and finally extension at 72℃ for 7 min, followed by holding at 4℃.

5. The dual PCR identification and detection method according to claim 4, characterized in that, The annealing temperature is 57°C to 62°C.

6. The dual PCR identification and detection method according to claim 5, characterized in that, The annealing temperature is 60℃.

7. The dual PCR identification and detection method according to claim 2, characterized in that, Electrophoresis was performed using a 1.5% agarose gel at 220 V for 30 min.

8. The application of the dual PCR identification detection method according to any one of claims 1 to 7 in the simultaneous screening of bovine Cryptosporidium microsporidium and Eimeria coccidia bovine infection and their mixed infection in bovine fecal samples.

9. A kit for simultaneously identifying *Cryptospora microsporum* and *Eimeria tenella*, characterized in that, The mixture contains the primer composition of claim 1, a PCR amplification reaction mixture, template DNA, and sterile double-distilled water.

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