A TaqMan-MGB multiplex fluorescent quantitative PCR primer probe set, kit and method for detecting three pathogenic Eimeria of goats

Abstract

This invention relates to the field of parasite detection technology, specifically to a TaqMan-MGB multiplex quantitative PCR primer set, probe, kit, and method for detecting three pathogenic Eimeria species in goats. This invention provides a TaqMan-MGB multiplex quantitative PCR primer set and probe for detecting *Eimeria cruzi*, *Eimeria aurea*, and *Eimeria yabrilliosa*, and establishes a TaqMan-MGB multiplex quantitative PCR method for detecting these three species based on this primer set and probe. The primer set, probe, and TaqMan-MGB multiplex quantitative PCR method of this invention can determine the mixed or single infection status of *Eimeria cruzi*, *Eimeria aurea*, and *Eimeria yabrilliosa* in a single TaqMan-MGB multiplex quantitative PCR reaction, offering advantages such as better repeatability, higher sensitivity, stronger specificity, and simpler operation compared to conventional PCR and nano-PCR methods.

Description

Technical fields:

[0001] This invention relates to the field of parasite detection technology, specifically to a TaqMan-MGB multiplex real-time PCR primer and probe set, kit, and method for detecting three pathogenic Eimeria coccidia species in goats. Background technology:

[0002] Goat coccidiosis is a type of parasitic protozoan that lives in the digestive tract of goats. It belongs to the genus *Eimeria* of the family *Eimeriidae*. Infection in goats not only causes damage or necrosis of the host's intestinal tissues, leading to hemorrhagic enteritis, but also causes lesions in other tissues or organs. The main clinical symptoms include lethargy, loss of appetite, rough coat, pale visible mucous membranes, watery diarrhea, and even death. It is one of the more serious parasitic diseases in dairy goat farming. Because goat coccidiosis has a simple development and diverse transmission routes, it is widely prevalent and common in goats under various feeding conditions. Goats of any breed and age can be infected with coccidiosis, with lambs aged 1-3 months having the highest infection rate, almost 100%.

[0003] Currently, numerous species of coccidia have been found to infect goats. Thirteen species of goat coccidia have been reported in the literature and are generally accepted by scholars. These coccidia differ in their parasitic sites, pathogenicity, and degree of harm. Among them, *Eimeria arloingi*, *Eimeria christenseni*, and *Eimeria ninakohlyakimovae* are recognized as the three most common and pathogenic goat coccidia. Mixed infections are common, making it difficult to distinguish different species using conventional detection methods.

[0004] With the development of molecular biology techniques, polymerase chain reaction (PCR) has been widely used in the detection of pathogenic microorganisms due to its advantages such as specificity, speed, and sensitivity. However, traditional conventional PCR can only qualitatively identify the DNA in the sample, not accurately quantify it, and the specificity and sensitivity of this method need further improvement. Summary of the Invention

[0005] In view of this, the present invention provides a TaqMan-MGB multiplex real-time PCR primer and probe set, kit, and method for detecting three pathogenic Eimeria coccidia in goats, providing a faster and more effective detection method for the diagnosis of protozoa parasitic in the goat digestive tract.

[0006] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0007] The TaqMan-MGB multiplex real-time PCR primer and probe set is used to detect three pathogenic Eimeria species in goats. The primer and probe set contains one or more specific primers and probes for detecting Eimeria krusei, Eimeria aurea, and Eimeria argytii.

[0008] The sequence of the upstream primer Ec-F for Eimeria krillinii is: 5'-GCCTGCTGGTGCTTCTTCTT-3';

[0009] The sequence of the downstream primer Ec-R for Eimeria krillinii is: 5'-CCGCGTTGCCTTGTTGTACTT-3';

[0010] The sequence of the Ec-P probe for *Eimeria krill* is as follows:

[0011] FAM-GCCACAAACAAAC-MGB;

[0012] The sequence of the upstream primer Ea-F for Eimeria aureus is: 5'-GCGGCTTTCGCTTCTACACC-3';

[0013] The sequence of the downstream primer Ea-R for Eimeria aureus is: 5'-ACAAATGCGCGACAAGGGG-3';

[0014] The sequence of the Ea-P probe for *Eimeria aureus* is as follows:

[0015] VIC-ATCAGGCGTGCTAGGA-MGB;

[0016] The sequence of the upstream primer En-F for Eimeria japonica is: 5'-CCCTTCAACTACACCGCCATT-3';

[0017] The sequence of the downstream primer En-R for Eimeria japonica is: 5'-TCAACATCCACACAACCAAGTATGG-3';

[0018] The sequence of the Eimeria tenella probe En-P is as follows:

[0019] Cy5-ACGTCTTGTAAACAACATAA-MGB.

[0020] A kit for detecting three pathogenic Eimeria coccidia species in goats contains the aforementioned TaqMan-MGB multiplex real-time PCR primer and probe set.

[0021] The above kit is used for the detection of one or more of Eimeria krusei, Eimeria aurea, and Eimeria argyroderma.

[0022] The steps of a TaqMan-MGB multiplex quantitative PCR method for detecting three pathogenic Eimeria coccidia species in goats are as follows:

[0023] 1) Extract DNA from the sample to be tested;

[0024] 2) TaqMan-MGB multiplex real-time PCR amplification was performed using primer and probe sets. The reaction conditions were: 37℃ digestion for 2 min, 95℃ pre-denaturation for 30 s, 95℃ denaturation for 10 s, 60℃ annealing for 30 s, for 45 cycles.

[0025] 3) After amplification, the results are analyzed based on the Ct value and amplification curve of each sample.

[0026] The TaqMan-MGB multiplex quantitative PCR reaction system includes:

[0027] 2×Taq Pro U + Multiple Probe qPCR Mix 10μL,

[0028] 0.3 μL each of the Eimeria kurlensis-specific primers Ec-F (10 μmol / L) and Ec-R (10 μmol / L), and 0.7 μL of the probe Ec-P (10 μmol / L) were used.

[0029] 0.7 μL each of the Eimeria aureus specific primers Ea-F (10 μmol / L) and Ea-R (10 μmol / L), and 0.5 μL of the probe Ec-P (10 μmol / L) were added.

[0030] 0.5 μL each of the Eimeria jaundice-specific primers En-F (10 μmol / L) and En-R (10 μmol / L), and 0.3 μL of the probe Ec-P (10 μmol / L) were used.

[0031] Add 3.0 μL of DNA template to be tested and bring the DEPC to a total of 20 μL.

[0032] Compared with the prior art, the present invention has the following advantages and effects:

[0033] 1. This invention designs specific primers and TaqMan MGB probes based on the ITS1-ITS2 genes of Eimeria cruzi, Eimeria aurea, and Eimeria yabrillia. After screening different primer and probe combinations, the primer and probe combinations with the best amplification effect are obtained. By optimizing the annealing temperature, primer concentration, probe concentration, and reaction system conditions, a positive recombinant plasmid is constructed as a standard to plot a standard curve. This establishes a TaqMan-MGB multiplex quantitative PCR method that can simultaneously detect the three major pathogenic coccidia species of Eimeria cruzi, Eimeria aurea, and Eimeria yabrillia, or a single pathogenic coccidia species.

[0034] 2. The primer set, probe, and TaqMan-MGB multiplex quantitative PCR method of this invention can determine the mixed infection and single infection status of Eimeria krusei, Eimeria aurea, and Eimeria argenti in the sample by a single TaqMan-MGB multiplex quantitative PCR reaction. It has the advantages of better repeatability, higher sensitivity, stronger specificity, and higher work efficiency than ordinary PCR.

[0035] 3. The method of the present invention has the characteristics of high specificity, high detection sensitivity and simple operation, and is suitable for clinical detection, providing technical support for the diagnosis and control of parasitic protozoa in the digestive tract of goats. Attached Figure Description

[0036] Figure 1 Figure 1 shows the optimized annealing temperature results for TaqMan-MGB multiplex quantitative PCR of Eimeria clarithii, Eimeria aurea, and Eimeria argenti; where (a) annealing temperature is 60℃; (b) annealing temperature is 62℃; and (c) annealing temperature is 64℃.

[0037] Figure 2 Figure showing the results of primer concentration optimization for TaqMan-MGB multiplex quantitative PCR of Eimeria clarithrophoenicia, Eimeria aurea, and Eimeria argytii.

[0038] Figure 3 Figure showing the results of probe concentration optimization for TaqMan-MGB multiplex quantitative PCR of Eimeria krusei, Eimeria argenti, and Eimeria argyrti.

[0039] Figure 4 Figure showing the specificity test results of TaqMan-MGB multiplex quantitative PCR for Eimeria krusei, Eimeria aurea, and Eimeria argyr.

[0040] Figure 5Figure 1 shows the results of sensitivity tests for TaqMan-MGB single-color quantitative PCR of Eimeria clarithii, Eimeria aurea, and Eimeria yakini; (a) Eimeria clarithii; (b) Eimeria aurea; (c) Eimeria yakini.

[0041] Figure 6 Eimeria plasmid DNA TaqMan-MGB fluorescence quantitative standard curve; (a) Eimeria kearnii; (b) Eimeria aureus; (c) Eimeria yakini Detailed Implementation

[0042] The technical solution of the present invention will be clearly and thoroughly described below with reference to specific embodiments. All other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0043] Quantitative real-time polymerase chain reaction (qPCR) refers to a method that adds fluorescent labels to the PCR reaction system, uses the accumulation of fluorescence signals to monitor the PCR products in real time during the amplification reaction, and finally uses a standard curve to quantify unknown templates. It has advantages such as good reproducibility, high sensitivity, accurate quantification, and high efficiency. TaqMan-MGB multiplex quantitative real-time PCR is a method that combines TaqMan probes labeled with different fluorescent groups with MGB (Minor Groove Binder) in the PCR reaction. MGB is a molecule that can bind to the minor groove of DNA, which can increase the binding ability of the probe to the target sequence and improve the specificity, sensitivity, and accuracy of detection. Therefore, by screening target gene sites, a set of specific qPCR primers and probes were designed, and a multiplex quantitative real-time PCR detection method with good reproducibility, high sensitivity, accurate quantification, and high efficiency was established, which is of great significance for the differential diagnosis and control of major pathogenic coccidiosis species in goats.

[0044] This embodiment provides a primer set and probe for detecting Eimeria crassirhizoma, Eimeria aurea, and Eimeria argyrella via TaqMan-MGB multiplex real-time PCR, including primer Ec-F, primer Ec-R, probe Ec-P, primer Ea-F, primer Ea-R, probe Ea-P, primer En-F, primer En-R, and probe En-P;

[0045] The sequence of the upstream primer Ec-F for *Eimeria crassirhizoma* is as follows:

[0046] 5'-GCCTGCTGGTGCTTCTTCTT-3';

[0047] The sequence of the downstream primer Ec-R for *Eimeria krill* is as follows:

[0048] 5'-CCGCGTTGCTTGTTGTACTT-3';

[0049] The sequence of the Ec-P probe for *Eimeria krill* is as follows:

[0050] FAM-GCCACAAACAAAC-MGB;

[0051] The sequence of the upstream primer Ea-F for *Eimeria aureus* is as follows:

[0052] 5'-GCGGCTTTCGCTTCTACACC-3';

[0053] The sequence of the downstream primer Ea-R for *Eimeria aureus* is as follows:

[0054] 5'-ACAAATGCGCGACAAGGGG-3';

[0055] The sequence of the Ea-P probe for *Eimeria aureus* is as follows:

[0056] VIC-ATCAGGCGTGCTAGGA-MGB;

[0057] The sequence of the upstream primer En-F for *Eimeria jaundice* is as follows:

[0058] 5'-CCCTTCAACTACACCGCCATT-3';

[0059] The sequence of the downstream primer En-R for Eimeria japonica is as follows:

[0060] 5'-TCAACATCCACACAACCAAGTATGG-3';

[0061] The sequence of the Eimeria tenella probe En-P is as follows:

[0062] Cy5-ACGTCTTGTAAACAACATAA-MGB.

[0063] The primer sets and probes mentioned above contain one or more specific primers and probes for detecting Eimeria krusei, Eimeria aurea, and Eimeria argytii, respectively.

[0064] The present invention also provides a kit for detecting three pathogenic Eimeria species in goats, the kit being used in the detection of one or more of Eimeria krusei, Eimeria aurea, and Eimeria argyroderma.

[0065] The specific steps of a TaqMan-MGB multiplex quantitative PCR method for detecting three pathogenic Eimeria species in goats are as follows:

[0066] (1) Extract DNA from the sample to be tested;

[0067] (2) Using the above primer set and probe, TaqMan-MGB multiplex quantitative PCR amplification was performed. The TaqMan-MGB multiplex quantitative PCR amplification system was: 2×Taq Pro U + Multiple Probe qPCR Mix 10 μL; 0.3 μL each of Ec-F (10 μmol / L) and Ec-R (10 μmol / L) specific primers for Eimeria krusei, and 0.7 μL of probe Ec-P (10 μmol / L); 0.7 μL each of Ea-F (10 μmol / L) and Ea-R (10 μmol / L) specific primers for Eimeria aureus, and 0.5 μL of probe Ec-P (10 μmol / L); 0.5 μL each of En-F (10 μmol / L) and En-R (10 μmol / L) specific primers for Eimeria esculenta, and 0.3 μL of probe Ec-P (10 μmol / L); 3.0 μL of DNA template to be tested, and bring the DEPC to 20 μL. The optimized reaction conditions for TaqMan-MGB multiplex real-time PCR were determined as follows: 37℃ digestion for 2 min, 95℃ pre-denaturation for 30 s, 95℃ denaturation for 10 s, 64℃ annealing for 30 s, for 50 cycles.

[0068] 3) After amplification, the results are analyzed based on the Ct value and amplification curve of each sample.

[0069] Experimental example:

[0070] 1. Design and synthesis of primers and probes

[0071] Based on the ITS1-ITS2 gene sequences of *Eimeria clarithii* (accession numbers: MN914085, MH059800), *Eimeria aurea* (accession numbers: MK861046, KX857469, KX857468, MF356556.1), and *Eimeria esculenta* (accession numbers: KY655480, KX787063, KX813711) published in GenBank, specific primers and probes were designed using Primer Premier 6 and Beacon Designer 8 software. The designed primers and probes were preliminarily validated using BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). The designed primer and probe sequences are as follows:

[0072] The sequence of the upstream primer Ec-F for *Eimeria crassirhizoma* is as follows:

[0073] 5'-GCCTGCTGGTGCTTCTTCTT-3';

[0074] The sequence of the downstream primer Ec-R for *Eimeria krill* is as follows:

[0075] 5'-CCGCGTTGCTTGTTGTACTT-3';

[0076] The sequence of the Ec-P probe for *Eimeria krill* is as follows:

[0077] FAM-GCCACAAACAAAC-MGB;

[0078] The sequence of the upstream primer Ea-F for *Eimeria aureus* is as follows:

[0079] 5'-GCGGCTTTCGCTTCTACACC-3';

[0080] The sequence of the downstream primer Ea-R for *Eimeria aureus* is as follows:

[0081] 5'-ACAAATGCGCGACAAGGGG-3';

[0082] The sequence of the Ea-P probe for *Eimeria aureus* is as follows:

[0083] VIC-ATCAGGCGTGCTAGGA-MGB;

[0084] The sequence of the upstream primer En-F for *Eimeria jaundice* is as follows:

[0085] 5'-CCCTTCAACTACACCGCCATT-3';

[0086] The sequence of the downstream primer En-R for Eimeria japonica is as follows:

[0087] 5'-TCAACATCCACACAACCAAGTATGG-3';

[0088] The sequence of the Eimeria tenella probe En-P is as follows:

[0089] Cy5-ACGTCTTGTAAACAACATAA-MGB;

[0090] Primers and probes were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0091] 2. Establishment and optimization of the TaqMan-MGB multiplex real-time PCR detection method

[0092] 2.1 Sample

[0093] Genomic DNA samples of pathogens such as Cryptosporidium microsporum, Cryptosporidium shøi, Giardia duodenalis, Microsporidium bithii, Haemonchus contortus, Orophytum esophagitis, Monizbeckia orientalis, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Eimeria krusei, Eimeria albendi, and Eimeria yamlek were extracted, identified, and preserved by the Animal Parasitology Laboratory of Northwest A&F University.

[0094] 2.2 Main Reagents and Instruments

[0095] Taq Pro U + Multiple Probe qPCR Mix was purchased from Novizan Technologies; E. coli JM109 Competent Cells and pMD were also used. TMThe 19-T Vector Cloning Kit was purchased from Takara Bio Inc., Dalian, China; the DNA purification and recovery kit and the plasmid mini-extraction kit were purchased from Tiangen Biotech Beijing Co., Ltd.; the real-time fluorescence quantitative PCR instrument was purchased from Xi'an Tianlong Technology Co., Ltd.; the digital display constant temperature metal water bath was purchased from Guohua Electric Co., Ltd., China; the SimpliAmp Thermal Cycler was purchased from Thermo Fisher Scientific, USA; the benchtop high-speed centrifuge was purchased from Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd., China; the vortex mixer was purchased from Qilin Bell Instrument Manufacturing Co., Ltd., Haimen, China; the vertical pressure steam sterilizer was purchased from the Medical Equipment Factory of Shanghai Boxun Industrial Co., Ltd., China; the constant temperature shaker was purchased from Shanghai Tiancheng Experimental Instrument Manufacturing Co., Ltd., China; and the ultra-clean workbench was purchased from Suzhou Antai Air Technology Co., Ltd., a subsidiary of Sujing Group, China.

[0096] 2.3 Construction of Standard Plasmids

[0097] Using DNA from *Eimeria cruzi*, *Eimeria aurea*, and *Eimeria argyrhea* as templates, the ITS gene fragments of these three species were amplified by PCR. The purified PCR products were then ligated into the pMD19-T vector to obtain standard plasmids pMD19-T-Ec, pMD19-T-Ea, and pMD19-T-En. After appropriate dilution, the plasmid template concentrations were determined using a micro-spectrophotometer, and their DNA copy numbers were calculated.

[0098] 2.4 Establishment and optimization of the TaqMan-MGB multiplex real-time PCR detection method

[0099] First, a TaqMan-MGB singlet real-time PCR method was established, and the annealing temperature, primer concentration, and probe concentration were optimized. Based on the optimized TaqMan-MGB singlet real-time PCR optimal annealing temperature and primer concentration, ITS gene-specific primers and probes for Eimeria crelli, Eimeria aurea, and Eimeria esculenta were added to the system sequentially to optimize the annealing temperature, primer concentration, and probe concentration.

[0100] The initial TaqMan-MGB real-time PCR reaction system consisted of 10 μL of 2×Taq Pro U + Multiple ProbeqPCR Mix; 0.4 μL each of forward and reverse primers (10 μmol / L); 0.4 μL of probe (10 μmol / L); 2.0 μL of DNA template to be detected; and DEPC to a final volume of 20 μL. Initial reaction conditions were: 37℃ digestion for 2 min, 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 64℃ annealing for 30 s, for 45 cycles.

[0101] The final optimized annealing temperature results: Multiplex quantitative PCR amplification was performed at annealing temperature gradients of 60℃, 62℃, and 64℃, with other conditions remaining unchanged. The results are as follows: Figure 1 As shown, the optimal annealing temperature is 62℃.

[0102] Final primer concentration optimization results: 0.30 μL, 0.50 μL, and 0.70 μL of forward and reverse primers (10 μmol / L) were added to the PCR system for *Eimeria crelli*, *Eimeria aurea*, and *Eimeria esculenta* ITS gene-specific primers, respectively (resulting in primer concentrations of 0.15 μmol / L, 0.25 μmol / L, and 0.35 μmol / L). Amplification was performed at the optimal annealing temperature, with other conditions remaining constant. The results are as follows: Figure 2 As shown.

[0103] Final probe concentration optimization results: 0.30 μL, 0.50 μL, and 0.70 μL of upstream and downstream probes (10 μmol / L) were added to the PCR system for *Eimeria crelli*, *Eimeria aurea*, and *Eimeria esculenta* ITS gene-specific probes, respectively (resulting in primer concentrations of 0.15 μmol / L, 0.25 μmol / L, and 0.35 μmol / L). Amplification was performed at the optimal annealing temperature and primer concentration, with other conditions remaining constant. The results are as follows: Figure 3 As shown.

[0104] After optimizing the annealing temperature, primer concentration, and probe concentration, the optimal system for the TaqMan-MGB multiplex quantitative PCR reaction was determined to be: 2×Taq Pro U + Multiple Probe qPCR Mix. 10 μL; 0.3 μL each of Ec-F (10 μmol / L) and Ec-R (10 μmol / L) specific primers for Eimeria krusei, and 0.7 μL of probe Ec-P (10 μmol / L); 0.7 μL each of Ea-F (10 μmol / L) and Ea-R (10 μmol / L) specific primers for Eimeria aureus, and 0.5 μL of probe Ec-P (10 μmol / L); 0.5 μL each of En-F (10 μmol / L) and En-R (10 μmol / L) specific primers for Eimeria esculenta, and 0.3 μL of probe Ec-P (10 μmol / L); 3.0 μL of DNA template to be tested, and bring the DEPC to 20 μL. The optimized reaction conditions were determined as follows: 37℃ contaminant digestion for 2 min, 95℃ pre-denaturation for 30 s, 95℃ denaturation for 10 s, 64℃ annealing for 30 s, for 45 reaction cycles.

[0105] 2.5 Specificity Test of TaqMan-MGB Multiplex Quantitative PCR Detection Method The TaqMan-MGB multiplex quantitative PCR detection method established above was used to detect DNA samples of *Eimeria krusei*, *Eimeria albinocerca*, *Eimeria yamsii*, *Cryptospora microsporum*, *Cryptospora schlegelii*, *Giardia duodenalis*, *Microsporidium bitrichii*, *Haemaphysema contortus*, *Oesophagostomum*, *Moniezia melanogaster*, *Listeria*, *Escherichia coli*, and *Staphylococcus aureus* to verify the specificity of the established method. The results are as follows: Figure 4 As shown in the figure (1. Mixed positive sample; 2-9. *Microsporidium bitarteriosus*, *Cryptospora microsporidium*, *Cryptospora schlegelii*, *Giardia duodenalis*, *Haemaphysema contortus*, *Ostomyces esophagni*, *Listeria*, *Escherichia coli*, *Staphylococcus aureus*; 10. Negative control), it can be seen that the TaqMan-MGB multiplex quantitative PCR detection method established in this invention can detect *Eimeria krusei*, *Eimeria aurea*, and *Eimeria argyrhoeae*, while no amplification bands were observed for other pathogen DNA, indicating that the method has good specificity.

[0106] 2.6 Sensitivity Test of TaqMan-MGB Quantitative Real-Time PCR Detection Method for Eimeria tenella: Positive standard plasmids for Eimeria krusei, Eimeria aurea, and Eimeria argentiformis, namely pMD19-T-Ec, pMD19-T-Ea, and pMD19-T-En, were initially set at concentrations of 4.01 × 10⁻⁶. 11 copies, 2.62×10 8 copies and 2.04×10 11 Copies. The standard plasmid was serially diluted 10-fold using sterile ddH2O and then detected using the optimized TaqMan-MGB real-time PCR method described above. The results are as follows: Figure 5 As shown in the figure (the concentrations of recombinant plasmids for *Eimeria clarithii*, *Eimeria aurea*, and *Eimeria argyrhea* are 1–10: 4.01 × 10⁻⁶), the ratio of *Eimeria clarithii* to *Eimeria argyrhea* is 4.01 × 10⁻⁶. 11 4.01 x 10 copies 2 copies; 11. Negative control; 1–8: 2.62 × 10⁻⁸ 8 26.2 copies; 9. Negative control; 1–10: 2.04 × 10⁻⁶ 11 The results showed that the minimum copy numbers of the ITS gene for Eimeria clarithrophoenicia, Eimeria aurea, and Eimeria argentina, as determined by the TaqMan-MGB quantitative PCR method, were 4010 copies, 262 copies, and 2040 copies, respectively.

[0107] 2.7 Establishment of the standard curve for the TaqMan-MGB method for the detection of Eimeria coccidia using real-time PCR

[0108] The TaqMan-MGB quantitative real-time PCR method can amplify the ITS genes of *Eimeria crestedii*, *Eimeria aurea*, and *Eimeria esculenta*, obtaining S-shaped fluorescence amplification curves and Ct values. The standard equations obtained are: y = -0.5928x + 19.947, and the correlation coefficient is R. 2 =0.9918; y = -0.6912x + 31.619, correlation coefficient: R 2 =0.9882; y = -0.596x + 21.308, correlation coefficient: R 2 =0.9917; indicating that the Ct value of the established method has a good linear relationship with the standard plasmid diluted 10-fold, as shown in the results. Figure 6 As shown.

[0109] The above experiments fully demonstrate that this invention, as a highly sensitive, specific, and easy-to-operate TaqMan-MGB real-time PCR detection method, can efficiently detect Eimeria krusei, Eimeria auriculi, and Eimeria argyrculi in goat fecal samples. It has certain development, utilization, and promotion value and can provide technical support for the clinical diagnosis of goat digestive tract parasitic diseases.

[0110] The above embodiments are merely illustrative of the principles and effects of the present invention. Any modifications or alterations made based on the present invention shall fall within the scope of patent protection of the present invention.

Claims

1. A TaqMan-MGB multiplex real-time PCR primer and probe set for detecting three pathogenic Eimeria coccidia species in goats, characterized in that: The primer-probe set contains specific primers and probes for detecting Eimeria krusei, Eimeria aurea, and Eimeria argyroderma, respectively. The sequence of the upstream primer Ec-F for *Eimeria crassirhizoma* is as follows: 5'-GCCTGCTGGTGCTTCTTCTT -3'; The sequence of the downstream primer Ec-R for *Eimeria krillensis* is as follows: 5'-CCGCGTTGCTTGTTGTACTT -3'; The sequence of the Ec-P probe of *Eimeria crassirhizoma* is as follows: FAM- GCCACAAACAAAC-MGB; The sequence of the upstream primer Ea-F for *Eimeria aureus* is as follows: 5'-GCGGCTTTCGCTTCTACACC-3'; The sequence of the downstream primer Ea-R for Eimeria auriculata is as follows: 5'-ACAAATGCGCGACAAGGGG -3'; The sequence of the Ea-P probe of Eimeria aureus is as follows: VIC- ATCAGGCGTGCTAGGA -MGB; The sequence of the upstream primer En-F for Eimeria japonica is as follows: 5'-CCCTTCAACTACACCGCCATT-3'; The sequence of the downstream primer En-R for Eimeria japonica is as follows: 5'-TCAACATCCACACAACCAAGTATGG -3'; The sequence of the probe En-P of Eimeria japonica is as follows: Cy5-ACGTCTGTAAACAACATAA-MGB.

2. A kit for detecting three pathogenic Eimeria coccidia species in goats, characterized in that: It includes the TaqMan-MGB multiplex real-time PCR primer and probe set as described in claim 1.

3. A TaqMan-MGB multiplex quantitative PCR method for detecting three pathogenic Eimeria coccidia species in goats for non-disease diagnosis and treatment purposes, characterized in that: The steps are as follows: 1) Extract DNA from the sample to be tested; 2) TaqMan-MGB multiplex real-time PCR amplification was performed using the primer and probe set of claim 1. The reaction conditions were: 37℃ contamination digestion for 2 min, 95℃ pre-denaturation for 30 s, 95℃ denaturation for 10 s, 60℃ annealing for 30 s, for 45 cycles. 3) After amplification, the results are analyzed based on the Ct value and amplification curve of each sample.

4. The TaqMan-MGB multiplex quantitative PCR method for detecting three pathogenic Eimeria coccidia species in goats for non-disease diagnosis and treatment purposes, as described in claim 3, is characterized in that... The TaqMan-MGB multiplex quantitative PCR reaction system includes: 2 × Taq Pro U + Multiple Probe qPCR Mix 10 µL、 0.3 μL each of the Eimeria krilli specific primers Ec-F and Ec-R, and 0.7 μL of the probe Ec-P were used. 0.7 μL each of the Ea-F and Ea-R primers specific to Eimeria aureus, and 0.5 μL of the probe Ea-P. 0.5 μL each of the Eimeria jaundice-specific primers En-F and En-R, and 0.3 μL of the probe En-P. 3.0 μL of DNA template to be tested, add DEPC to bring the total volume to 20 μL.

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