Five-plex real-time fluorescent quantitative PCR detection kit and detection method for simultaneous detection of five parasites

Abstract

The present invention belongs to the technical field of biological detection. Provided are a five-plex real-time fluorescent quantitative PCR detection kit and a detection method for the simultaneous detection of five parasites. The method and the kit can be used for simultaneously detecting Toxoplasma gondii, Neospora caninum, Eimeria stiedai, Giardia lamblia and Trypanosoma evansi in the same system; and have the characteristics of qualitative and quantitative detection capability, high sensitivity, strong specificity, good repeatability and good stability.

Description

A five-fold real-time quantitative PCR detection kit and method for simultaneously detecting five parasites. Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a five-fold real-time fluorescence quantitative PCR detection kit and detection method for simultaneously detecting five parasites, and particularly relating to a five-fold probe method real-time fluorescence quantitative PCR detection kit, detection method, and dedicated detection primer and probe combination for simultaneously detecting Toxoplasma gondii, Neosporidium, Eimeria skelli, Giardia lamblia, and Trypanosoma eeris in the same system. Background Technology

[0002] *Toxoplasma gondii*, *Neospora caninum*, *Eimeria stiedai*, *Giardia lamblia*, and *Trypanosoma evansi* are globally prevalent parasitic pathogens, with sheep, cattle, pigs, and rabbits considered intermediate hosts. *Toxoplasma gondii* can cause human diseases, with clinical outcomes ranging from asymptomatic presentation to eye diseases, reproductive disorders, and neurological symptoms. *Neospora* infection is a significant cause of abortion. *Eimeria stiedai* infection can cause hepatic coccidiosis. *Giardia lamblia* enteritis is gastroenteritis caused by the pathogenic protozoan *Giardia lamblia*. *Trypanosoma evansi* is a pathogenic protozoan found in animal plasma and hematopoietic organs, causing Surra disease, one of the most important animal diseases in Asia and South America. Parasitic infections are a significant factor affecting sheep, cattle, pig, and rabbit farming, causing substantial economic losses. Effective management and control of farm parasite infections depends on early detection.

[0003] Despite its low sensitivity, microscopic examination remains a primary diagnostic method for parasites in resource-constrained environments. Serological diagnostic tests for parasitic infections are characterized by low specificity. A common nucleic acid-based detection method is polymerase chain reaction (PCR), which has become the gold standard for parasite monitoring due to its high sensitivity and the availability of commercial kits. However, traditional PCR amplification products undergo time-consuming, labor-intensive, and cumbersome agarose gel electrophoresis and screening. Furthermore, traditional PCR methods are susceptible to contamination from tube opening and are easily affected by inhibitors that may be present in sheep, cattle, pig, and rabbit samples.

[0004] With the development of molecular biology techniques, real-time fluorescence PCR has become the preferred tool for laboratory diagnosis of parasitic infections. In particular, TaqMan probe-based real-time quantitative PCR is an accurate, sensitive, and rapid method for detecting and quantifying target genomes. This method, based on the continuous measurement of fluorescence signals during the amplification reaction, has been used to detect many parasites, including Toxoplasma gondii, Neosporidia, Giardia lamblia, and Trypanosoma eerilys. For example, methods or kits for detecting Toxoplasma gondii, Neospora, Giardia lamblia, and Trypanosoma eeris using singlet real-time quantitative PCR based on TaqMan probes have been reported (Mousavi P, et al. (2018). Detection of Toxoplasma gondiiin acute and chronic phases of infection in immunocompromised patients and pregnant women with real-time PCR assay using TaqMan fluorescent probe. Iran J Parasitol. 13(3):373-381. Bandelj P, et al. (2023). First molecular detection of Neospora caninumin feces of Grey Wolf (Canis lupus) and Golden Jackal (Canis aureus) populations in Slovenia. Animals (Basel). 13(19):3089. doi:10.3390 / ani13193089). However, traditional TaqMan probe-based singlet real-time quantitative PCR methods for detecting multiple parasites in the same sample are time-consuming and expensive. With the development of this technology, TaqMan probe-based multiplex real-time quantitative PCR (qPCR) utilizes target gene-specific TaqMan probes to simultaneously detect multiple target genes in a single reaction. TaqMan probe-based multiplex real-time quantitative PCR technology can simultaneously detect multiple parasites in a closed tube, offering advantages such as high throughput, fast detection speed, simple operation, low risk of contamination, and low detection cost. For example, existing technologies have reported methods for detecting *Toxoplasma gondii* and *Neospora* using dual-phase qPCR based on TaqMan probes (Truong M, J. (2023). Analytical sensitivity of a multiplex quantitative PCR for Toxoplasma gondii and Neospora caninum. Parasitol Res. 122(4):1043-1047.doi:10.1007 / s00436-023-07796-5).

[0005] However, there is currently no five-fold TaqMan probe-based real-time quantitative PCR detection method or related detection products that can simultaneously detect Toxoplasma gondii, Neosporidium, Eimeria skelli, Giardia lamblia, and Trypanosoma eeris in the same detection system. Summary of the Invention

[0006] To address one or more problems existing in the prior art, this invention analyzes the published gene structure sequences of *Toxoplasma gondii*, *Neospora*, *Eimeria skemsenii*, *Giardia lamblia*, and *Trypanosoma eeris* reference strains, designs specific primers and probes for each, and performs systematic optimization and validation. Ultimately, a novel TaqMan probe-based five-fold real-time quantitative PCR detection method is established, capable of simultaneously detecting these five parasites in a single closed test tube (i.e., in the same reaction system). This method exhibits high sensitivity and specificity, and shows no cross-reactivity with other parasite genomes. Furthermore, this method can accurately identify *Toxoplasma gondii*, *Neospora*, *Eimeria skemsenii*, *Giardia lamblia*, and *Trypanosoma eeris* in different matrices. Therefore, this method can be applied to diagnosis, surveillance, and epidemiology, and also provides a novel TaqMan probe-based five-fold real-time quantitative PCR detection kit for these five parasites, along with its dedicated primer and probe sets. This invention is mainly achieved through the following technical solutions.

[0007] In a first aspect, the present invention provides a five-fold real-time quantitative PCR primer and TaqMan probe combination for simultaneously detecting *Toxoplasma gondii*, *Neospora*, *Eimeria skelli*, *Giardia lamblia*, and *Trypanosoma eeris* in the same system, wherein the five-fold real-time quantitative PCR primer and TaqMan probe combination comprises:

[0008] Real-time quantitative PCR primers and TaqMan probes for detecting Toxoplasma gondii include: a forward primer GZQTgG3PDH-F with a nucleotide sequence as shown in SEQ ID NO:1, a reverse primer GZQTgG3PDH-R with a nucleotide sequence as shown in SEQ ID NO:2, and a TaqMan probe GZQTgG3PDH-P with a nucleotide sequence as shown in SEQ ID NO:3;

[0009] Primers and TaqMan probes for real-time quantitative PCR detection of Neosporidia include: a forward primer GZQNcNC5-F with a nucleotide sequence as shown in SEQ ID NO:4, a reverse primer GZQNcNC5-R with a nucleotide sequence as shown in SEQ ID NO:5, and a TaqMan probe GZQNcNC5-P with a nucleotide sequence as shown in SEQ ID NO:6;

[0010] Real-time quantitative PCR primers and TaqMan probes for detecting Eimeria stearothermiae include: forward primer GZQEsADF-F with nucleotide sequence as shown in SEQ ID NO:7, reverse primer GZQEsADF-R with nucleotide sequence as shown in SEQ ID NO:8, and TaqMan probe GZQEsADF-P with nucleotide sequence as shown in SEQ ID NO:9.

[0011] The real-time quantitative PCR primers and TaqMan probes for detecting Giardia lamblia include: a forward primer GZQGlGDH-F with the nucleotide sequence shown in SEQ ID NO:10, a reverse primer GZQGlGDH-R with the nucleotide sequence shown in SEQ ID NO:11, and a TaqMan probe GZQGlGDH-P with the nucleotide sequence shown in SEQ ID NO:12.

[0012] Real-time quantitative PCR primers and TaqMan probes for detecting Trypanosoma eerilyum include: forward primer GZQTeCOX1-F with nucleotide sequence as shown in SEQ ID NO:13, reverse primer GZQTeCOX1-R with nucleotide sequence as shown in SEQ ID NO:14, and TaqMan probe GZQTeCOX1-P with nucleotide sequence as shown in SEQ ID NO:15.

[0013] The TaqMan probe is fluorescently labeled, with a fluorescent emitting group at its 5' end and a fluorescent quenching group at its 3' end.

[0014] In some embodiments, the 5' end of the TaqMan probe GZQTgG3PDH-P is labeled with a FAM fluorescent emitting group, and the 3' end is labeled with a QSY fluorescent quenching group; the 5' end of the TaqMan probe GZQNcNC5-P is labeled with a VIC fluorescent emitting group, and the 3' end is labeled with a QSY fluorescent quenching group; the 5' end of the TaqMan probe GZQEsADF-P is labeled with an ABY fluorescent emitting group, and the 3' end is labeled with a QSY fluorescent quenching group; the 5' end of the TaqMan probe GZQGlGDH-P is labeled with a JUN fluorescent emitting group, and the 3' end is labeled with a QSY fluorescent quenching group; the 5' end of the TaqMan probe GZQTeCOX1-P is labeled with a CY5 fluorescent emitting group, and the 3' end is labeled with an MGB-NFQ fluorescent quenching group.

[0015] In a second aspect, the present invention provides a five-fold real-time quantitative PCR detection kit for the simultaneous detection of Toxoplasma gondii, Neosporidium, Eimeria skelli, Giardia lamblia, and Trypanosoma eeris, comprising the five-fold real-time quantitative PCR primers and TaqMan probe combination provided in the first aspect of the present invention.

[0016] In some embodiments, the kit further includes positive controls, wherein the positive controls include recombinant plasmids containing the Toxoplasma gondii G3PDH gene (pG3PDH), recombinant plasmids containing the Neosporidium NC5 gene (pNcNC5), recombinant plasmids containing the Eimeria skelliae ADF gene (pEsADF), recombinant plasmids containing the Giardia lamblia GDH gene (pGlGDH), and recombinant plasmids containing the Trypanosoma eeris COX1 gene (pTeCOX1).

[0017] In some embodiments, the kit further includes a negative control, wherein the negative control includes a system that does not contain the Toxoplasma gondii G3PDH gene, Neosporidium NC5 gene, Eimeria skelli ADF gene, Giardia lamblia GDH gene, and Trypanosoma eeris COX1 gene, and may further be nuclease-free water.

[0018] In a third aspect, the present invention provides the application of the five-fold real-time quantitative PCR primer and TaqMan probe combination provided in the first aspect of the present invention or the five-fold real-time quantitative PCR detection kit provided in the second aspect of the present invention to simultaneously detect Toxoplasma gondii, Neosporidium, Eimeria stearothermiae, Giardia lambliae and Trypanosoma eeris in the same system.

[0019] In a fourth aspect, the present invention provides a five-fold real-time quantitative PCR detection method for simultaneously detecting *Toxoplasma gondii*, *Neospora*, *Eimeria skrvii*, *Giardia lamblia*, and *Trypanosoma eeris*. This method includes performing five-fold real-time quantitative PCR amplification on genomic DNA extracted from the sample to be tested using the five-fold real-time quantitative PCR primers and TaqMan probe combination provided in the first aspect of the present invention or the five-fold real-time quantitative PCR detection kit provided in the second aspect of the present invention, and collecting fluorescence signals to qualitatively detect *Toxoplasma gondii*, *Neospora*, *Eimeria skrvii*, *Giardia lamblia*, and *Trypanosoma eeris*, and / or quantitatively detecting the identified parasitic nucleic acids.

[0020] In some implementations, the method includes the following steps:

[0021] 1) Establishing a standard curve: Plasmid standards of varying concentrations were prepared using positive controls of *Toxoplasma gondii*, *Neospora*, *Eimeria sk.*, *Giardia lamblia*, and *Trypanosoma eeris* (each plasmid standard contained equal concentrations of positive controls of *Toxoplasma gondii*, *Neospora*, *Eimeria sk.*, *Giardia lamblia*, and *Trypanosoma eeris*). Using plasmid standards of different concentrations as templates, five-fold real-time quantitative PCR detection was performed using the five-fold real-time quantitative PCR primers and TaqMan probe combination provided in the first aspect of this invention. After the detection, the Log10 value (X-axis) of each plasmid standard concentration was plotted against its corresponding Ct value (Y-axis) to obtain the standard curve.

[0022] 2) Extract genomic nucleic acid from the sample to be tested, and use the extracted genomic nucleic acid as a template to perform five-fold real-time quantitative PCR detection using the five-fold real-time quantitative PCR primers and TaqMan probe combination provided in the first aspect of the present invention;

[0023] 3) Use the specific amplification curves and the obtained Ct values ​​to perform qualitative detection of Toxoplasma gondii, Neosporidium, Eimeria skeletalis, Giardia lamblia, and Trypanosoma eeris, and / or use the standard curves obtained in step 1) to perform quantitative detection of the parasitic nucleic acids that are identified.

[0024] In some embodiments, the reaction system for the fivefold real-time quantitative PCR detection in steps 1) and 2) includes: GZQTgG3PDH-F, GZQTgG3PDH-R, GZQNcNC5-F, GZQNcNC5-R, GZQEsADF-F, GZQEsADF-R, GZQGlGDH-F and GZQGlGDH-R, all with a final concentration of 200-300 nM; GZQTeCOX1-F and GZQTeCOX1-R, all with a final concentration of 400-500 nM; GZQTgG3PDH-P, GZQTgG3PDH-P, GZQEsADF-P and GZQGlGDH-P, all with a final concentration of 50-100 nM; and GZQTeCOX1-P, with a final concentration of 100-200 nM.

[0025] In some implementations, the reaction procedure for the five-fold real-time quantitative PCR detection in steps 1) and 2) includes: 50°C for 2 min, 1 cycle; 95°C for 10 min, 1 cycle; 95°C for 15 s, 60°C for 1 min, 40 cycles.

[0026] In some implementations, the criteria for qualitative detection are:

[0027] In the channel corresponding to the fluorescent emitting group (e.g., FAM) labeled at the 5′ end of GZQTgG3PDH-P, if a specific S-shaped curve appears and the Ct value is ≤35, the result is judged as positive for Toxoplasma gondii nucleic acid; if there is no Ct value and no specific fluorescent amplification curve, the result is judged as negative for Toxoplasma gondii nucleic acid; if 35 < Ct value < 38 and a specific fluorescent amplification curve appears, the result is judged as suspicious for Toxoplasma gondii nucleic acid. Suspicious samples need to be resampled and DNA extracted for retesting. If the retest Ct value is <38, it is judged as positive; otherwise, it is judged as negative.

[0028] In the channel corresponding to the fluorescent emitting group (e.g., VIC) labeled at the 5′ end of GZQNcNC5-P, if a specific S-shaped curve appears and the Ct value is ≤35, the result is judged as positive for Neosporidium nucleic acid; if there is no Ct value and no specific fluorescence amplification curve, the result is judged as negative for Neosporidium nucleic acid; if 35 < Ct value < 38 and a specific fluorescence amplification curve appears, the result is judged as suspicious for Neosporidium nucleic acid. Suspicious samples need to be resampled and DNA extracted for retesting. If the retest Ct value is <38, it is judged as positive; otherwise, it is judged as negative.

[0029] In the channel corresponding to the fluorescent emitting group (e.g., ABY) labeled at the 5′ end of GZQEsADF-P, if a specific S-shaped curve appears and the Ct value is ≤35, the result is judged as positive for Eimeria stegeriae nucleic acid; if there is no Ct value and no specific fluorescent amplification curve, the result is judged as negative for Eimeria stegeriae nucleic acid; if 35 < Ct value < 38 and a specific fluorescent amplification curve appears, the result is judged as suspicious for Eimeria stegeriae nucleic acid. Suspicious samples need to be resampled and DNA extracted for retesting. If the retest Ct value is <38, it is judged as positive; otherwise, it is judged as negative.

[0030] In the channel corresponding to the fluorescent emitting group (e.g., JUN) labeled at the 5′ end of GZQGlGDH-P, if a specific S-shaped curve appears and the Ct value is ≤35, the result is judged as positive for Giardia lamblia nucleic acid; if there is no Ct value and no specific fluorescent amplification curve, the result is judged as negative for Giardia lamblia nucleic acid; if 35 < Ct value < 38 and a specific fluorescent amplification curve appears, the result is judged as suspicious for Giardia lamblia nucleic acid. Suspicious samples need to be resampled and DNA extracted for retesting. If the retest Ct value is <38, it is judged as positive; otherwise, it is judged as negative.

[0031] In the channel corresponding to the fluorescent emitting group (e.g., CY5) labeled at the 5′ end of GZQTeCOX1-P, if a specific S-shaped curve appears and the Ct value is ≤35, the result is judged as positive for Trypanosoma eeris nucleic acid; if there is no Ct value and no specific fluorescent amplification curve, the result is judged as negative for Trypanosoma eeris nucleic acid; if 35 < Ct value < 38 and a specific fluorescent amplification curve appears, the result is judged as suspicious for Trypanosoma eeris nucleic acid. Suspicious samples need to be resampled and DNA extracted for retesting. If the retest Ct value is <38, it is judged as positive; otherwise, it is judged as negative.

[0032] In some embodiments, the detection limits of the method for Toxoplasma gondii, Neosporidium, Eimeria stes, Giardia lamblia, and Trypanosoma eeris are 2.0 copies / μL, 1.5 copies / μL, 1.5 copies / μL, 1.8 copies / μL, and 1.1 copies / μL, respectively.

[0033] Compared with the prior art, the present invention has the following characteristics:

[0034] 1. This invention targets prevalent strains of *Toxoplasma gondii*, *Neospora*, *Eimeria skelli*, *Giardia lamblia*, and *Trypanosoma eerilys*, both domestically and internationally. The G3PDH, NC5, ADF, GDH, and COX1 genes are selected as target genes. After multi-gene sequence alignment, regions with low homology and numerous mutation sites are eliminated, ultimately selecting highly conserved regions from the G3PDH, NC5, ADF, GDH, and COX1 gene sequences for primer and probe design. However, since the purpose of this invention is to simultaneously detect the above five parasite target genes in the same detection system (i.e., using five fluorescence channels), if the detection system for each target gene is incompatible, the Ct values ​​differ significantly, and / or interference occurs between primers and probes, fluorescence inhibition may occur, thus affecting the detection rate during the detection process. In view of this, through creative efforts, the inventors further designed and determined primer and probe sets with similar amplification efficiencies for detecting the nucleic acids of the above five parasites in the shorter, highly conserved sequences of the above five target detection genes. At the same time, by adjusting the ratio of primers to fluorescent probes in the detection system during PCR amplification, the amplification efficiency for nucleic acids of different parasites was further kept consistent.

[0035] 2. When co-infection with Toxoplasma gondii, Neosporidium, Eimeria skrvii, Giardia lamblia, and Trypanosoma eeris is present, relying solely on single-cell fluorescent PCR to determine the presence of multiple parasites is time-consuming, labor-intensive, and costly. This invention provides a method and kit for simultaneously detecting Toxoplasma gondii, Neosporidium, Eimeria skrvii, Giardia lamblia, and Trypanosoma eeris in the same detection system. It utilizes specific probes labeled with fluorescent emitting groups targeting the G3PDH gene of Toxoplasma gondii, the NC5 gene of Neosporidium, the ADF gene of Eimeria skrvii, the GDH gene of Giardia lamblia, and the COX1 gene of Trypanosoma eeris, respectively. This allows for a single detection to determine the presence of multiple parasites, saving time and effort and significantly reducing detection costs.

[0036] 3. The method and kit provided by this invention have high specificity. For related parasites such as Strongyloides stercoralis, Nematoda, Cynodon tatarsus, Cynodon tatarsus, and Balacellus spp., there are no nonspecific amplification curves, which can ensure the accuracy of detection.

[0037] 4. This invention provides a method and kit for rapid detection of Toxoplasma gondii, Neosporidium, Eimeria skrvii, Giardia lamblia, and Trypanosoma eeris. The method and kit are characterized by qualitative and quantitative detection capabilities, high sensitivity (detection limits for Toxoplasma gondii, Neosporidium, Eimeria skrvii, Giardia lamblia, and Trypanosoma eeris are 2.0 copies / μL, 1.5 copies / μL, 1.5 copies / μL, 1.8 copies / μL, and 1.1 copies / μL, respectively), high specificity, good repeatability, and excellent stability (coefficients of variation (CV) for both intra- and inter-batch repeatability tests are less than 3%). Attached Figure Description

[0038] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 shows the fluorescence amplification curve of Toxoplasma gondii detected by the five-fold real-time quantitative PCR detection method provided by the present invention.

[0040] Figure 2 shows the standard curve for detecting Toxoplasma gondii using the five-fold real-time quantitative PCR detection method provided by this invention.

[0041] Figure 3 shows the fluorescence amplification curve of Neosporidium detected by the five-fold real-time quantitative PCR detection method provided by the present invention.

[0042] Figure 4 shows the standard curve for detecting Neosporidia using the five-fold real-time quantitative PCR detection method provided by this invention.

[0043] Figure 5 shows the fluorescence amplification curve of Eimeria stearothermiae detected by the five-fold real-time quantitative PCR detection method provided by the present invention.

[0044] Figure 6 shows the standard curve for detecting Eimeria stearothermiae using the five-fold real-time quantitative PCR detection method provided by this invention.

[0045] Figure 7 shows the fluorescence amplification curve of Giardia lamblia detected by the five-fold real-time quantitative PCR detection method provided by the present invention.

[0046] Figure 8 shows the standard curve for detecting Giardia lamblia using the five-fold real-time quantitative PCR detection method provided by this invention.

[0047] Figure 9 shows the fluorescence amplification curve of Trypanosoma eeris in the five-fold real-time quantitative PCR detection method provided by the present invention.

[0048] Figure 10 shows the standard curve for detecting Trypanosoma eerilyum using the five-fold real-time quantitative PCR detection method provided by this invention.

[0049] Figure 11 shows the fluorescence amplification curves of the specificity test of the five-fold real-time quantitative PCR detection method provided by the present invention. Detailed Implementation

[0050] This invention targets the G3PDH gene of *Toxoplasma gondii*, the NC5 gene of *Neospora*, the ADF gene of *Eimeria skrvii*, the GDH gene of *Giardia lamblia*, and the COX1 gene sequence of *Trypanosoma eeris*. Specific primers and probes targeting these five target genes were designed and synthesized. A five-probe real-time quantitative PCR method was established to simultaneously detect these five parasites in the same system, and a convenient detection kit is provided. The kit and detection method can simultaneously detect the target genes of *Toxoplasma gondii*, *Neospora*, *Eimeria skrvii*, *Giardia lamblia*, and *Trypanosoma eeris*. The primers and probes targeting different target genes do not interfere with each other and exhibit qualitative and quantitative capabilities, high sensitivity, strong specificity, good repeatability, and excellent stability.

[0051] The technical solution of the present invention will be clearly and completely described below with reference to embodiments. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other. Obviously, the described embodiments are only some embodiments of the present invention, 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.

[0052] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials used in this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0053] Both the primers and probes used can be synthesized using existing techniques.

[0054] Example 1: Design and determination of primers and TaqMan probes for five-fold real-time quantitative PCR for simultaneous detection of Toxoplasma gondii, Neospora, Eimeria skelli, Giardia lamblia, and Trypanosoma eeris.

[0055] This invention targets prevalent strains of *Toxoplasma gondii*, *Neospora*, *Eimeria skrvii*, *Giardia lamblia*, and *Trypanosoma eeris* both domestically and internationally. The invention selects the *G3PDH* gene from *Toxoplasma gondii*, the *NC5* gene from *Neospora*, the *ADF* gene from *Eimeria skrvii*, the *GDH* gene from *Giardia lamblia*, and the *COX1* gene from *Trypanosoma eeris* as target genes. After multi-gene sequence alignment, regions with low gene homology and numerous mutation sites are eliminated. Highly conserved regions within the *G3PDH*, *NC5*, *ADF*, *GDH*, and *COX1* gene sequences are then selected for primer and probe design. Furthermore, the amplification efficiency for nucleic acids from different parasites is made similar, and interference between primers and probes is avoided, thus preventing fluorescence inhibition. The resulting primer and probe combinations for detecting *Toxoplasma gondii*, *Neospora*, *Eimeria skrvii*, *Giardia lamblia*, and *Trypanosoma eeris* are shown below.

[0056] The primer and probe combination used to detect the G3PDH gene of Toxoplasma gondii includes:

[0057] Forward primer GZQTgG3PDH-F: 5'-TTGCATAGGTTGCAGTCACTGA-3' (SEQ ID NO:1);

[0058] Reverse primer GZQTgG3PDH-R: 5'-ACCAAAGTTGCACAGATACTCATGA-3' (SEQ ID NO:2);

[0059] TaqMan probe GZQTgG3PDH-P: 5'-(FAM)-CCCCTCTGCTGGCGAAAAGTGAAA-(QSY)-3' (SEQ ID NO: 3).

[0060] The detection region for the G3PDH gene of *Toxoplasma gondii* is:

[0061] The primer-probe combination used to detect the Neosporidium NC5 gene includes:

[0062] Forward primer GZQNcNC5-F: 5'-GCGGACGGGTCGTTGTT-3' (SEQ ID NO:4);

[0063] Reverse primer GZQNcNC5-R: 5'-CCGTTCACACACTATAGCCACAAA-3' (SEQ ID NO:5);

[0064] TaqMan probe GZQNcNC5-P: 5'-(VIC)-CCTGCGGCAGCAAGGCTCCTT-(QSY)-3'(SEQ ID NO:6).

[0065] The detection region for the Neosporidium NC5 gene is:

[0066] The primer-probe combination used to detect the ADF gene in Eimeria stearothermia includes:

[0067] Forward primer GZQEsADF-F: 5'-TCATCCTCAAGATTGACCACGAT-3' (SEQ ID NO:7);

[0068] Reverse primer GZQEsADF-R: 5'-TCACTAGCTGGAAGTTCCTTTGTG-3' (SEQ ID NO:8);

[0069] TaqMan probe GZQEsADF-P: 5'-(ABY)-AAGGGCACTGGTGACGCCTCAACTC-(QSY)-3'(SEQ ID NO:9).

[0070] The detection region for the ADF gene of *Eimeria stevidii* is:

[0071] The primer-probe combination used to detect the GDH gene in Giardia lamblia includes:

[0072] Forward primer GZQGlGDH-F: 5'-GGGCGGCTCCGACTTT-3' (SEQ ID NO: 10);

[0073] Reverse primer GZQGlGDH-R: 5'-ACTGGCAGAAGCGCATGAC-3' (SEQ ID NO:11);

[0074] TaqMan probe GZQGlGDH-P: 5'-(JUN)-ACCCAAAGGGCAAGTCCGACAACG-(QSY)-3' (SEQ ID NO: 12).

[0075] The detection region for the GDH gene of Giardia lamblia is as follows:

[0076] The primer-probe combination used to detect the COX1 gene in Trypanosoma eeris includes:

[0077] Forward primer GZQTeCOX1-F: 5'-GGGTGACTTAGCCCATGCA-3' (SEQ ID NO:13);

[0078] Reverse primer GZQTeCOX1-R: 5'-CCCACACAATTGAAGAGAGAAAATAG-3' (SEQ ID NO:14);

[0079] TaqMan probe GZQTeCOX1-P: 5'-(CY5)-AGCCTCGGGTGATC-MGB-NFQ-3'(SEQ ID NO:15).

[0080] The detection region for the COX1 gene of Trypanosoma eeris is:

[0081] Example 2: A method for five-fold real-time quantitative PCR detection

[0082] The method for simultaneously detecting five parasites in a sample using the primer and probe combination designed in Example 1 in the same detection system via fivefold real-time quantitative PCR includes the following steps:

[0083] 1. Extract total DNA from the sample to be tested for later use;

[0084] 2. Preparation of the reaction system: For example, a 20 μL reaction system may include: 10 μL TaqMan Mix (purchased from Thermo Fisher Scientific), 3 μL primers and TaqMan probe Mix (GZQTgG3PDH-F concentration of 220 nM (final concentration, the same below), GZQTgG3PDH-R concentration of 220 nM, GZQTgG3PDH-P concentration of 60 nM, GZQNcNC5-F concentration of 220 nM, GZQNcNC5-R concentration of 220 nM, GZQNcNC5-P concentration of 60 nM, GZQEsADF-F concentration of 220 nM). M, GZQEsADF-R concentration of 220 nM, GZQEsADF-P concentration of 60 nM, GZQGlGDH-F concentration of 220 nM, GZQGlGDH-R concentration of 220 nM, GZQGlGDH-P concentration of 60 nM, GZQTeCOX1-F concentration of 440 nM, GZQTeCOX1-R concentration of 440 nM, GZQTeCOX1-P concentration of 120 nM), 5 μL template DNA and 2 μL nuclease-free water.

[0085] 3. Amplification program: 50℃ for 2 min, 1 cycle; 95℃ for 10 min, 1 cycle; 95℃ for 15 s, 60℃ for 1 min, 40 cycles. Five fluorescence channels were used: FAM fluorescence channel: fluorescent emitting group "FAM", quenching group "QSY"; VIC fluorescence channel: fluorescent emitting group "VIC", quenching group "QSY"; ABY fluorescence channel: fluorescent emitting group "ABY", quenching group "QSY"; JUN fluorescence channel: fluorescent emitting group "JUN", quenching group "QSY"; CY5 fluorescence channel: fluorescent emitting group "CY5", quenching group "MGB-NFQ".

[0086] 4. Result Determination

[0087] The positive control showed a Ct value < 30 in all five channels and exhibited a specific S-shaped fluorescence amplification curve, while the negative control showed no Ct value and no specific fluorescence amplification curve. Both conditions were met for the experimental result to be considered valid. The criteria for determining the test results are shown in Table 1.

[0088] Table 1: Criteria for Judging Test Results

[0089] In the FAM fluorescence channel, if the Ct value of the sample to be tested is ≤35 and a specific S-shaped fluorescence amplification curve appears, it is judged to be positive for Toxoplasma gondii nucleic acid; if there is no Ct value and no specific fluorescence amplification curve, it is judged to be negative for Toxoplasma gondii nucleic acid; if 35 < Ct value < 38 and a specific fluorescence amplification curve appears, it is judged to be suspicious for Toxoplasma gondii nucleic acid. Suspicious samples need to be resampled and DNA extracted for retesting. A Ct value < 38 is judged as positive, otherwise it is judged as negative.

[0090] In the VIC fluorescence channel, if the Ct value of the sample to be tested is ≤35 and a specific S-shaped fluorescence amplification curve appears, it is judged as positive for Neosporidium nucleic acid; if there is no Ct value and no specific fluorescence amplification curve, it is judged as negative for Neosporidium nucleic acid; if 35 < Ct value < 38 and a specific fluorescence amplification curve appears, it is judged as suspicious for Neosporidium nucleic acid. Suspicious samples need to be resampled and DNA extracted for retesting. A Ct value < 38 is judged as positive, otherwise it is judged as negative.

[0091] In the ABY fluorescence channel, if the Ct value of the test sample is ≤35 and a specific S-shaped fluorescence amplification curve appears, it is judged as positive for Eimeria stegeriae nucleic acid; if there is no Ct value and no specific fluorescence amplification curve, it is judged as negative for Eimeria stegeriae nucleic acid; if 35 < Ct value < 38 and a specific fluorescence amplification curve appears, it is judged as suspicious for Eimeria stegeriae nucleic acid. Suspicious samples need to be resampled and DNA extracted for retesting. A Ct value < 38 is judged as positive, otherwise it is judged as negative.

[0092] In the JUN fluorescence channel, if the Ct value of the test sample is ≤35 and a specific S-shaped fluorescence amplification curve appears, it is judged as positive for Giardia lamblia nucleic acid; if there is no Ct value and no specific fluorescence amplification curve, it is judged as negative for Giardia lamblia nucleic acid; if 35 < Ct value < 38 and a specific fluorescence amplification curve appears, it is judged as suspicious for Giardia lamblia nucleic acid. Suspicious samples need to be resampled and DNA extracted for retesting. If the Ct value is <38, it is judged as positive; otherwise, it is judged as negative.

[0093] In the CY5 fluorescence channel, if the Ct value of the sample to be tested is ≤35 and a specific S-shaped fluorescence amplification curve appears, it is judged as positive for Trypanosoma eeris nucleic acid; if there is no Ct value and no specific amplification curve, it is judged as negative for Trypanosoma eeris nucleic acid; if 35 < Ct value < 38 and a specific fluorescence amplification curve appears, it is judged as suspicious for Trypanosoma eeris nucleic acid. Suspicious samples need to be resampled and DNA extracted for retesting. If the Ct value is <38, it is judged as positive; otherwise, it is judged as negative.

[0094] Example 3: Five-fold Real-time Quantitative PCR Detection Kit

[0095] This invention also provides a five-fold real-time quantitative PCR detection kit for simultaneously identifying and detecting *Toxoplasma gondii*, *Neospora*, *Eimeria skelli*, *Giardia lamblia*, and *Trypanosoma eeris* in the same detection system, which may include:

[0096] Example 1 describes the design and determination of a five-fold real-time quantitative PCR primer and TaqMan probe for the simultaneous detection of *Toxoplasma gondii*, *Neospora*, *Eimeria skelli*, *Giardia lamblia*, and *Trypanosoma eeris*. Specifically:

[0097] The real-time quantitative PCR primers and TaqMan probes for detecting Toxoplasma gondii include: the forward primer GZQTgG3PDH-F with the nucleotide sequence shown in SEQ ID NO:1, the reverse primer GZQTgG3PDH-R with the nucleotide sequence shown in SEQ ID NO:2, and the TaqMan probe GZQTgG3PDH-P with the nucleotide sequence shown in SEQ ID NO:3.

[0098] The real-time quantitative PCR primers and TaqMan probes used to detect Neosporidia include: the forward primer GZQNcNC5-F with the nucleotide sequence shown in SEQ ID NO:4, the reverse primer GZQNcNC5-R with the nucleotide sequence shown in SEQ ID NO:5, and the TaqMan probe GZQNcNC5-P with the nucleotide sequence shown in SEQ ID NO:6.

[0099] The real-time quantitative PCR primers and TaqMan probes for detecting Eimeria stearothermiae include: the forward primer GZQEsADF-F with the nucleotide sequence shown in SEQ ID NO:7, the reverse primer GZQEsADF-R with the nucleotide sequence shown in SEQ ID NO:8, and the TaqMan probe GZQEsADF-P with the nucleotide sequence shown in SEQ ID NO:9.

[0100] The real-time quantitative PCR primers and TaqMan probes for detecting Giardia lamblia include: the forward primer GZQGlGDH-F with the nucleotide sequence shown in SEQ ID NO:10, the reverse primer GZQGlGDH-R with the nucleotide sequence shown in SEQ ID NO:11, and the TaqMan probe GZQGlGDH-P with the nucleotide sequence shown in SEQ ID NO:12.

[0101] The real-time quantitative PCR primers and TaqMan probes used to detect Trypanosoma eerilyum include: the forward primer GZQTeCOX1-F with the nucleotide sequence shown in SEQ ID NO:13, the reverse primer GZQTeCOX1-R with the nucleotide sequence shown in SEQ ID NO:14, and the TaqMan probe GZQTeCOX1-P with the nucleotide sequence shown in SEQ ID NO:15.

[0102] The TaqMan probe is fluorescently labeled, with a fluorescent emitting group at its 5' end and a fluorescent quenching group at its 3' end. For example, the fluorescent emitting group can be selected from the group consisting of FAM, VIC, ABY, JUN, and CY5, and the fluorescent quenching group can be selected from the group consisting of QSY and MGB-NFQ. Moreover, the fluorescent emitting group labeled at the 5' end of the TaqMan probe is different for different parasites.

[0103] Specifically, in the kit provided by this invention, the 5′ and 3′ ends of each TaqMan probe can be labeled as follows: the 5′ end of TaqMan probe GZQTgG3PDH-P is labeled with FAM, and the 3′ end is labeled with QSY; the 5′ end of TaqMan probe GZQNcNC5-P is labeled with VIC, and the 3′ end is labeled with QSY; the 5′ end of TaqMan probe GZQEsADF-P is labeled with ABY, and the 3′ end is labeled with QSY; the 5′ end of TaqMan probe GZQGlGDH-P is labeled with JUN, and the 3′ end is labeled with QSY; the 5′ end of TaqMan probe GZQTeCOX1-P is labeled with CY5, and the 3′ end is labeled with MGB-NFQ. The reason why this invention chose the five fluorescent emitting groups FAM, VIC, ABY, JUN, and CY5 is that experiments have shown that the relative emission spectra of these five fluorescent emitting groups have a high degree of matching with the filters of different channels of the instrument, small overlap of spectra, and the intensity of the five fluorescent signals is comparable, so better performance can be obtained in a multiple experimental system.

[0104] For ease of detection, the kit also includes positive controls (as detailed in Example 4 below), which include recombinant plasmids containing the G3PDH gene of Toxoplasma gondii (pTgG3PDH), recombinant plasmids containing the NC5 gene of Neosporidium (pTgNcNC5), recombinant plasmids containing the ADF gene of Eimeria stearothermiae (pEsADF), recombinant plasmids containing the GDH gene of Giardia lamblia (pGlGDH), and recombinant plasmids containing the COX1 gene of Trypanosoma eeris (pTeCOX1).

[0105] For ease of testing, the kit also includes a negative control, which includes a system that does not contain the Toxoplasma gondii G3PDH gene, Neosporidium NC5 gene, Eimeria schlegelii ADF gene, Giardia lamblia GDH gene, and Trypanosoma eeris COX1 gene, and may further be selected as nuclease-free water.

[0106] For ease of testing, the kit may also include the instructions for the five-fold real-time quantitative PCR detection method provided in Example 2.

[0107] Example 4: Characteristics of the five-fold real-time quantitative PCR detection method and kit

[0108] 4.1. Preparation of positive controls

[0109] The genes for *Toxoplasma gondii* G3PDH, *Neospora* NC5, *Eimeria sk.* ADF, *Giardia lamblia* GDH, and *Trypanosoma eeris* COX1 were synthesized using TaKaRa. These synthesized gene fragments were ligated into the pMD19-T vector and transformed into strain JM109. The resulting positive recombinants were used as positive controls and named pTgG3PDH (*Toxoplasma gondii*), pNcNC5 (*Neospora*), pEsADF (*Eimeria sk.*), pGlGDH (*Giardia lamblia*), and pTeCOX1 (*Trypanosoma eeris*), respectively.

[0110] 4.2. Optimization of Conditions for the Five-fold Real-time Quantitative PCR Detection Method

[0111] Plasmid DNA was extracted using the Qiagen Plasmid Maxi Kit (QIAGEN, Germany) (i.e., each positive control). The plasmid DNA concentration was measured using a UV spectrophotometer. The five positive controls were serially diluted 10-fold, and each concentration was recorded as 10⁻⁶. 5 A dilution buffer of 1 copy / μL was prepared and mixed as a template in a 1:1:1:1:1 ratio, with a total volume of 20 μL. Forward and reverse primers were mixed with their corresponding probes at different final primer and probe concentrations. The forward and reverse primers were added to the corresponding probes at concentrations of 0.44 μL, 0.44 μL, and 0.12 μL (final concentrations 440 nM, 440 nM, and 120 nM), and 0.22 μL, 0.22 μL, and 0.06 μL (final concentrations 220 nM, 220 nM), respectively. The primer and probe concentrations were 0.11 μL, 0.11 μL, and 0.03 μL (final concentrations of 110 nM, 110 nM, and 30 nM), respectively. Following the amplification program provided in this invention: 50℃ for 2 min, 1 cycle; 95℃ for 10 min, 1 cycle; 95℃ for 15 s, 60℃ for 1 min, 40 cycles, a five-fold real-time quantitative PCR reaction was performed. The optimal primer and probe concentration ratio was found to obtain the lowest Ct value and the highest fluorescence intensity increase (ΔRn). The results are shown in Table 2.

[0112] Table 2: Results of primer and probe concentration grouping experiments

[0113] Table 2 shows that by using different combinations of probes and primer concentrations to explore the optimal reaction conditions, it was found that the optimal reaction conditions were achieved when the concentrations of GZQTeCOX1-F, GZQTeCOX1-R, GZQTeCOX1-P, GZQTgG3PDH-F, GZQTgG3PDH-R, GZQTgG3PDH-P, GZQNcNC5-F, GZQNcNC5-R, and GZQNcNC5-P were 440 nM, 220 nM, 60 nM, and 220 nM respectively. When the concentrations of ZQEsADF-F, GZQEsADF-R, GZQEsADF-P, GZQGlGDH-F, GZQGlGDH-R, and GZQGlGDH-P are 220 nM, the fluorescence intensity of CY5 and VIC is the highest and the Ct value is the lowest in this five-fold real-time quantitative PCR reaction (because the fluorescence of CY5 and VIC in this five-fold real-time quantitative PCR detection method is relatively weak, the primer-probe combination with the highest fluorescence intensity of CY5 and VIC is selected as the optimal combination).

[0114] 4.3. Preparation of Standard Curve

[0115] Five positive controls were serially diluted 10-fold, and 10 samples were taken. 8 10 7 10 6 10 5 10 4 10 3 A gradient concentration of plasmid standards at concentrations of [copy / μL] was used as templates to construct a standard curve. A five-fold real-time quantitative PCR reaction system was used: 10 μL TaqMan Mix, 2 μL nuclease-free water, 3 μL primer and TaqMan probe mix, and 5 μL DNA template. The amplification program was: 50℃ for 2 min, 1 cycle; 95℃ for 10 min, 1 cycle; 95℃ for 15 s, 60℃ for 1 min, 40 cycles. Fluorescence signals were collected, and the standard curve was analyzed.

[0116] The results are shown in Figures 1-10, which are the amplification curves and standard curves of the target detection genes of five parasites using the five-fold real-time quantitative PCR detection method of the present invention. In Figures 1, 3, 5, 7, and 9, the six curves from left to right represent 10... 8 10 7 10 6 10 5 10 4 10 3Concentration at copies / μL. The results showed that the method of this invention exhibited high amplification efficiencies (E) for the target genes of five parasites (Toxoplasma gondii, Neosporidium, Eimeria skelli, Giardia lamblia, and Trypanosoma eeris), with E values ​​of 95.534%, 96.203%, 107.818%, 100.851%, and 104.487%, respectively. Correlation coefficient R0 2 The correlation coefficients were 0.996, 0.996, 0.996, 0.992, and 0.996, respectively. The correlation coefficients and amplification efficiency indicate that, within the current concentration range of dilutions, the template amount and the corresponding Ct value exhibit a good linear relationship.

[0117] 4.4. Specificity Analysis

[0118] Using DNA from *Strongyloides stercoralis*, *Aspiculuris tetraptera*, *Syphacia obvelata*, *Syphacia muris*, and *Balantidium coli* as templates, and nuclease-free water as a negative control, quantitative real-time PCR amplification was performed using the method and kit of this invention. The results are shown in Figure 11. It can be seen that the method yielded negative results for all of the above parasites, while positive results were obtained for *Toxoplasma gondii*, *Neospora*, *Eimeria spp.*, *Giardia lamblia*, and *Trypanosoma eerilys*, indicating that the method has high specificity and no cross-reactivity with other parasites.

[0119] 4.5. Sensitivity Analysis

[0120] The analytical sensitivity of this method was evaluated using plasmid standards of *Toxoplasma gondii*, *Neospora*, *Eimeria skelli*, *Giardia lamblia*, and *Trypanosoma eeris* at different dilutions. Using plasmid standards of various dilutions as templates, reactions were performed according to the provided reaction system and amplification procedure for the five-fold real-time quantitative PCR detection method. For higher concentrations of plasmid standards, eight replicates were performed per dilution; for lower concentrations, twenty replicates were performed per dilution. The results are shown in Table 3.

[0121] Table 3: Limit of Detection for Five-fold Real-time Quantitative PCR Detection Method

[0122] Table 3 shows that the detection limits for *Toxoplasma gondii*, *Neospora*, *Eimeria stes*, *Giardia lamblia*, and *Trypanosoma evansi* were 2.0 copies / μL, 1.5 copies / μL, 1.5 copies / μL, 1.8 copies / μL, and 1.1 copies / μL, respectively. This indicates that the method of the present invention has high sensitivity.

[0123] 4.6. Stability Analysis

[0124] Using plasmid standards at 5 concentrations and 10-fold serial dilutions as templates, the dilutions were performed sequentially at 10... 7 10 6 10 5 10 4 10 3 The plasmid standard was tested at a concentration of 1 copy / μL according to the provided reaction system and amplification procedure for the five-fold real-time quantitative PCR detection method. In each experiment, three replicates were performed for each dilution of the plasmid standard to analyze intra-batch variability. Then, using the same plasmid standard as a template, one independent replicate experiment was performed on three consecutive days within one week. In each experiment, three replicates were performed for each dilution of the plasmid standard to analyze inter-batch variability. The coefficient of variation (CV) of the Ct value was calculated to verify the repeatability and stability of the method. The results are shown in Table 4.

[0125] Table 4: Repeatability and stability test results of the five-fold real-time quantitative PCR detection method

[0126] Table 4 shows that the intra-assay coefficients of variation (CVs) for the five-fold real-time quantitative PCR method in detecting *Toxoplasma gondii*, *Neospora*, *Eimeria skrvii*, *Giardia lamblia*, and *Trypanosoma eeris* were 0.07%–0.59%, 0.07%–0.75%, 0.19%–2.10%, 0.28%–1.43%, and 0.33%–0.95%, respectively. The inter-assay CVs for the same detection methods were 0.40%–0.75%, 0.22%–0.80%, 0.46%–1.49%, 0.50%–2.13%, and 0.26%–0.89%, respectively. It is evident that the CV values ​​for both intra-assay and inter-assay repeatability tests were less than 3%, indicating that the method of this invention exhibits good repeatability and stability.

[0127] Example 5: Clinical diagnostic performance of the five-fold real-time quantitative PCR detection method

[0128] DNA extracted from 600 clinical specimens (including blood, serum, and rectal swabs) of Hu sheep collected in Yanji area, Yanbian Prefecture, Jilin Province in September 2023 was analyzed using the five-fold real-time quantitative PCR detection method and kit established in this invention to detect *Toxoplasma gondii*, *Neospora*, *Eimeria sk.*, *Giardia lamblia*, and *Trypanosoma eeris*. Epidemiological survey data were also analyzed. Positive controls (DNA from *Toxoplasma gondii*, *Neospora*, *Eimeria sk.*, *Giardia lamblia*, and *Trypanosoma eeris*) and nuclease-free water (as a negative control) were included in the clinical sample testing to rule out false negatives and false positives.

[0129] The test results showed that among 600 clinical samples, 5 were positive for *Toxoplasma gondii*, 5 were positive for *Neospora*, 11 were positive for *Eimeria skrvii*, 66 were positive for *Giardia lamblia*, and 0 were positive for *Trypanosoma eeris*. In one sealed test tube, 7 samples were found to be double-positive for *Eimeria skrvii* and *Giardia lamblia* using a five-fold real-time quantitative PCR method, meaning these 7 clinical samples contained both *Eimeria skrvii* and *Giardia lamblia*. These results indicate that the five-fold real-time quantitative PCR detection method and kit provided by this invention can be used to detect the presence of *Toxoplasma gondii*, *Neospora*, *Eimeria skrvii*, *Giardia lamblia*, and *Trypanosoma eeris* in clinical samples, and can also identify whether clinical samples are infected with multiple parasites.

[0130] The embodiments described herein are for illustrative purposes only, and various modifications or alterations made by those skilled in the art based on the embodiments should also be included within the substantive scope of the patent application.

[0131] Industrial application

[0132] This invention provides a five-fold real-time quantitative PCR detection kit and detection method for the simultaneous detection of five parasites. The kit and detection method are characterized by qualitative and quantitative detection, high sensitivity, strong specificity, good repeatability, and good stability, making them suitable for industrial applications.

Claims

1. A five-fold real-time quantitative PCR primer and TaqMan probe combination, used for the simultaneous detection of *Toxoplasma gondii*, *Neospora*, *Eimeria skelli*, *Giardia lamblia*, and *Trypanosoma eeris* in the same system, characterized in that... The five-fold real-time quantitative PCR primer and TaqMan probe combination includes: Real-time quantitative PCR primers and TaqMan probes for detecting Toxoplasma gondii include: a forward primer GZQTgG3PDH-F with a nucleotide sequence as shown in SEQ ID NO:1, a reverse primer GZQTgG3PDH-R with a nucleotide sequence as shown in SEQ ID NO:2, and a TaqMan probe GZQTgG3PDH-P with a nucleotide sequence as shown in SEQ ID NO:3; Primers and TaqMan probes for real-time quantitative PCR detection of Neosporidia include: a forward primer GZQNcNC5-F with a nucleotide sequence as shown in SEQ ID NO:4, a reverse primer GZQNcNC5-R with a nucleotide sequence as shown in SEQ ID NO:5, and a TaqMan probe GZQNcNC5-P with a nucleotide sequence as shown in SEQ ID NO:6; Real-time quantitative PCR primers and TaqMan probes for detecting Eimeria stearothermiae include: forward primer GZQEsADF-F with nucleotide sequence as shown in SEQ ID NO:7, reverse primer GZQEsADF-R with nucleotide sequence as shown in SEQ ID NO:8, and TaqMan probe GZQEsADF-P with nucleotide sequence as shown in SEQ ID NO:

9. The real-time quantitative PCR primers and TaqMan probes for detecting Giardia lamblia include: a forward primer GZQGlGDH-F with the nucleotide sequence shown in SEQ ID NO:10, a reverse primer GZQGlGDH-R with the nucleotide sequence shown in SEQ ID NO:11, and a TaqMan probe GZQGlGDH-P with the nucleotide sequence shown in SEQ ID NO:

12. Real-time quantitative PCR primers and TaqMan probes for detecting Trypanosoma eerilyum include: forward primer GZQTeCOX1-F with nucleotide sequence as shown in SEQ ID NO:13, reverse primer GZQTeCOX1-R with nucleotide sequence as shown in SEQ ID NO:14, and TaqMan probe GZQTeCOX1-P with nucleotide sequence as shown in SEQ ID NO:

15. The TaqMan probe is fluorescently labeled, with a fluorescent emitting group at its 5′ end and a fluorescent quenching group at its 3′ end.

2. The five-fold real-time quantitative PCR primer and TaqMan probe combination according to claim 1, characterized in that: The TaqMan probe GZQTgG3PDH-P has a FAM fluorescent emitting group labeled at its 5′ end and a QSY fluorescent quenching group labeled at its 3′ end. The TaqMan probe GZQNcNC5-P is labeled with a VIC fluorescent emitting group at its 5′ end and a QSY fluorescent quenching group at its 3′ end. The TaqMan probe GZQEsADF-P is labeled with an ABY fluorescent emitting group at its 5′ end and a QSY fluorescent quenching group at its 3′ end. The TaqMan probe GZQGlGDH-P is labeled with a JUN fluorescent emitting group at its 5′ end and a QSY fluorescent quenching group at its 3′ end. The TaqMan probe GZQTeCOX1-P is labeled with a CY5 fluorescent emitting group at its 5′ end and an MGB-NFQ fluorescent quenching group at its 3′ end.

3. A five-fold real-time quantitative PCR detection kit for simultaneous detection of Toxoplasma gondii, Neospora, Eimeria skearensis, Giardia lamblia, and Trypanosoma eeris, characterized in that, The kit includes the five-fold real-time quantitative PCR primers and TaqMan probe combination as described in claim 1 or 2.

4. The five-fold real-time quantitative PCR detection kit according to claim 3, characterized in that, The kit also includes positive controls, which include recombinant plasmids containing the G3PDH gene of Toxoplasma gondii, recombinant plasmids containing the NC5 gene of Neosporidium, recombinant plasmids containing the ADF gene of Eimeria skelli, recombinant plasmids containing the GDH gene of Giardia lamblia, and recombinant plasmids containing the COX1 gene of Trypanosoma eeris.

5. The five-fold real-time quantitative PCR detection kit according to claim 4, characterized in that, The kit also includes a negative control, wherein the negative control includes a system that does not contain the Toxoplasma gondii G3PDH gene, Neosporidium NC5 gene, Eimeria schlegelii ADF gene, Giardia lamblia GDH gene, and Trypanosoma eeris COX1 gene.

6. The five-fold real-time quantitative PCR detection kit according to claim 5, characterized in that, The negative control was nuclease-free water.

7. The application of the five-fold real-time quantitative PCR primer and TaqMan probe combination as described in claim 1 or 2, or the five-fold real-time quantitative PCR detection kit as described in any one of claims 3-6, to simultaneously detect Toxoplasma gondii, Neosporidium, Eimeria stearothermiae, Giardia lamblia, and Trypanosoma eeris in the same system.

8. A five-fold real-time quantitative PCR detection method for simultaneously detecting Toxoplasma gondii, Neosporidium, Eimeria skearensis, Giardia lamblia, and Trypanosoma eeris, characterized in that, The method includes performing five-fold real-time quantitative PCR amplification on genomic DNA extracted from the sample to be tested using the five-fold real-time quantitative PCR primer and TaqMan probe combination as described in claim 1 or 2, or the five-fold real-time quantitative PCR detection kit as described in any one of claims 3-6, and collecting fluorescence signals to qualitatively detect Toxoplasma gondii, Neosporidium, Eimeria skelli, Giardia lamblia, and Trypanosoma eeris, and / or quantitatively detect the parasitic nucleic acids identified.

9. The five-fold real-time quantitative PCR detection method according to claim 8, characterized in that, The method includes the following steps: 1) Establishing a standard curve: Plasmid standards of varying concentrations were prepared using positive controls of Toxoplasma gondii, Neospora, Eimeria stessonii, Giardia lamblia, and Trypanosoma eeris. Using plasmid standards of different concentrations as templates, five-fold real-time quantitative PCR detection was performed using the five-fold real-time quantitative PCR primers and TaqMan probe combination described in claim 1 or 2. After the detection, the Log10 value (X-axis) of each plasmid standard concentration was plotted against its corresponding Ct value (Y-axis) to obtain the standard curve. 2) Extract genomic nucleic acid from the sample to be tested, and use the extracted genomic nucleic acid as a template to perform five-fold real-time quantitative PCR detection using the five-fold real-time quantitative PCR primers and TaqMan probe combination described in claim 1 or 2; 3) Use the specific amplification curves and the obtained Ct values ​​to perform qualitative detection of Toxoplasma gondii, Neosporidium, Eimeria stessonii, Giardia lamblia, and Trypanosoma eeris, and / or use the standard curves obtained in step 1) to perform quantitative detection of the parasitic nucleic acids that are identified.

10. The five-fold real-time quantitative PCR detection method according to claim 9, characterized in that, The reaction system for the five-fold real-time quantitative PCR detection in steps 1) and 2) includes: GZQTgG3PDH-F, GZQTgG3PDH-R, GZQNcNC5-F, GZQNcNC5-R, GZQEsADF-F, GZQEsADF-R, GZQGlGDH-F and GZQGlGDH-R, all with a final concentration of 200-300 nM; GZQTeCOX1-F and GZQTeCOX1-R, all with a final concentration of 400-500 nM; GZQTgG3PDH-P, GZQTgG3PDH-P, GZQEsADF-P and GZQGlGDH-P, all with a final concentration of 50-100 nM; and GZQTeCOX1-P, with a final concentration of 100-200 nM.

11. The five-fold real-time quantitative PCR detection method according to claim 9, characterized in that, The reaction procedure for the five-fold real-time quantitative PCR detection in steps 1) and 2) includes: 50℃ for 2 min, 1 cycle; 95℃ for 10 min, 1 cycle; 95℃ for 15 s, 60℃ for 1 min, 40 cycles.

12. The five-fold real-time quantitative PCR detection method according to any one of claims 8-11, characterized in that, The criteria for qualitative testing are as follows: If a specific S-shaped curve appears in the channel corresponding to the fluorescent emitting group labeled at the 5′ end of GZQTgG3PDH-P, and the Ct value is ≤35, the result is judged as positive for Toxoplasma gondii nucleic acid; if there is no Ct value and no specific fluorescence amplification curve, the result is judged as negative for Toxoplasma gondii nucleic acid. If 35 < Ct value < 38 and a specific fluorescence amplification curve appears, the result is considered suspicious for Toxoplasma gondii nucleic acid. Suspected samples need to be resampled and DNA extracted for retesting. If the retest Ct value is < 38, it is considered positive; otherwise, it is considered negative. If a specific S-shaped curve appears in the channel corresponding to the fluorescent emitting group labeled at the 5′ end of GZQNcNC5-P, and the Ct value is ≤35, the result is judged as positive for Neosporidium nucleic acid; if there is no Ct value and no specific fluorescence amplification curve, the result is judged as negative for Neosporidium nucleic acid; if 35 < Ct value < 38, and a specific fluorescence amplification curve appears, the result is judged as suspicious for Neosporidium nucleic acid. Suspicious samples need to be resampled and DNA extracted for retesting. If the retest Ct value is <38, it is judged as positive; otherwise, it is judged as negative. If a specific S-shaped curve appears in the channel corresponding to the fluorescent emitting group labeled at the 5′ end of GZQEsADF-P, and the Ct value is ≤35, the result is judged as positive for Eimeria stegeriae nucleic acid; if there is no Ct value and no specific fluorescence amplification curve, the result is judged as negative for Eimeria stegeriae nucleic acid; if 35 < Ct value < 38, and a specific fluorescence amplification curve appears, the result is judged as suspicious for Eimeria stegeriae nucleic acid. Suspicious samples need to be resampled and DNA extracted for retesting. If the retest Ct value is <38, it is judged as positive; otherwise, it is judged as negative. If a specific S-shaped curve appears in the channel corresponding to the fluorescent emitting group labeled at the 5′ end of GZQGlGDH-P, and the Ct value is ≤35, the result is judged as positive for Giardia lamblia nucleic acid; if there is no Ct value and no specific fluorescent amplification curve, the result is judged as negative for Giardia lamblia nucleic acid. If 35 < Ct value < 38 and a specific fluorescence amplification curve appears, the result is considered to be suspicious for Giardia lamblia nucleic acid. Suspicious samples need to be resampled and DNA extracted for retesting. If the retest Ct value is < 38, it is judged as positive; otherwise, it is judged as negative. If a specific S-shaped curve appears in the channel corresponding to the fluorescent emitting group labeled at the 5′ end of GZQTeCOX1-P and the Ct value is ≤35, the result is judged as positive for Trypanosoma eeris nucleic acid; if there is no Ct value and no specific fluorescence amplification curve, the result is judged as negative for Trypanosoma eeris nucleic acid. If 35 < Ct value < 38 and a specific fluorescence amplification curve appears, the result is considered suspicious for Trypanosoma eiri. Suspicious samples need to be resampled and DNA extracted for retesting. If the retest Ct value is < 38, it is considered positive; otherwise, it is considered negative.

13. The five-fold real-time quantitative PCR detection method according to any one of claims 8-12, characterized in that, The detection limits of the method for Toxoplasma gondii, Neosporidium, Eimeria stearothermiae, Giardia lamblia, and Trypanosoma eeris are 2.0 copies / μL, 1.5 copies / μL, 1.5 copies / μL, 1.8 copies / μL, and 1.1 copies / μL, respectively.

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