Trichinella spiralis and toxoplasma gondii duplex PCR detection kit and application thereof

Abstract

The invention discloses a double PCR (Polymerase Chain Reaction) detection kit for trichina and toxoplasma gondii and application of the double PCR detection kit. The kit comprises upstream and downstream primer pairs for detecting trichina and toxoplasma gondii respectively, 2 * Taq Master Mix and non-enzyme water, the primer pair for detecting the trichina specifically aims at a complete sequence of a trichina T1 isolate ISS 003 ribosome small subunit gene, and the primer pair for detecting the toxoplasma gondii specifically aims at a toxoplasma gondii H66R isolate 529REP repetitive sequence region. The duplex PCR detection system disclosed by the invention is good in specificity, and does not generate cross reaction with other common polypide bodies; corresponding target fragments can be amplified when trichina and toxoplasma gondii are detected, and no amplified fragment appears when common pig parasites are detected, so that the specificity is high; the detection limit is 0.10 ng / mu L, so that the kit has relatively high sensitivity. In conclusion, the detection kit disclosed by the invention is high in sensitivity, strong in specificity, mild in reaction condition, short in reaction period and high in detection flux, and can be used for rapidly screening the toxoplasma gondii and trichina in meat products and live pigs in various scenes.

Description

Technical Field

[0001] This invention relates to the field of parasite detection reagents, specifically to a dual PCR detection kit for Trichinella spiralis and Toxoplasma gondii and its application. Background Technology

[0002] Toxoplasma gondii is an obligate intracellular parasitic protozoan that can infect almost all mammals, with felines being the only definitive host. The main harm of Toxoplasma gondii to livestock is causing reproductive disorders such as abortion and stillbirth in pigs and sheep, which can lead to mass mortality and significant economic losses to the livestock industry. Human toxoplasmosis is mainly transmitted through the consumption of undercooked meat or meat products containing cysts. Immunocompromised individuals are mostly asymptomatic, with the acute phase presenting only with self-limiting flu-like symptoms such as fever and swollen lymph nodes. Immunosuppressed individuals may experience central nervous system damage such as encephalitis and retinochoroiditis. Approximately 30%-40% of AIDS patients develop acute toxoplasmic encephalitis due to immunodeficiency, which can even lead to death. Infection with Toxoplasma gondii in pregnant women can cause miscarriage, premature birth, stillbirth, and other pregnancy complications.

[0003] Currently, routine testing for Toxoplasma gondii primarily involves microscopic examination of feces, water sources, and the environment. However, relying solely on optical microscopy results in low sensitivity and inaccurate findings. Oocysts present in feces, water, and the environment can be enriched through filtration and centrifugation before detection. When examining cysts within tissues, staining methods can be used to differentiate between parasites and host cells. Giemsa and hematoxylin-eosin staining techniques are simple and inexpensive, and are commonly used in clinical practice. However, all three methods are time-consuming and require specialized personnel to ensure reliable results. In 1948, Sabin and Feldman first developed the DT staining test, which is now widely recognized as the gold standard for detecting Toxoplasma gondii antibodies in humans. However, its application is significantly limited by its dependence on both the parasite and healthy serum; furthermore, the DT test is a hazardous procedure and should only be performed in a laboratory by qualified laboratory personnel. The indirect hemagglutination assay (IHA) is a diagnostic method proposed by Jacob and Lunde in 1957. In 1979, Cui Junzhao et al. used the IHA method to detect toxoplasmosis in 70 porcine serum samples and conducted preliminary research. In 1982, the Lanzhou Veterinary Research Institute successfully developed an IHA diagnostic reagent. In 1989, the first PCR detection method for Toxoplasma gondii targeting the B1 gene was established. This method has been widely used for the prenatal diagnosis of congenital toxoplasmosis and toxoplasmosis infection in immunocompromised patients.

[0004] Trichinella is a zoonotic parasite with a worldwide distribution. Humans become infected by consuming raw or undercooked meat containing Trichinella cysts. Data shows that from 1964 to 2004, my country experienced over 600 outbreaks of trichinosis, reporting over 26,000 cases and over 250 deaths. 95% of these cases were caused by pork, making it the leading cause of infection among the three major foodborne zoonotic parasites. It is also the first and mandatory zoonotic parasite to be inspected in meat import / export and animal slaughter. Due to the strong survivability and broad host spectrum of trichinosis, and the lack of specific clinical manifestations, current detection methods are not yet perfect. In 1932, Augustine et al. discovered the value of the individual skin test (IDT) used by Bachman in diagnosing human trichinosis. This method is simple to operate, rapid, requires no special equipment, and provides results in 15 minutes. However, due to factors such as antigen impurity, the detection rate is only 60%, and therefore it is rarely used. In the mid-1980s, Klassen et al. used the restriction endonuclease EcoRI to digest the genomic DNA of Trichinella spiralis and discovered a repetitive sequence with a copy number of 2800 and a length of 1.7 kb, directly tandemly arranged within the genome, accounting for 2% of the total genomic DNA. This fragment was specific to Trichinella spiralis. Subsequently, a pair of PCR primers was designed based on this sequence. The results showed that both samples could amplify 602 bp of specific DNA, with a detection limit of 0.02 muscle larvae per gram. The World Organisation for Animal Health (WOAH) primarily diagnoses trichinosis using the digestion method. This method utilizes hydrochloric acid-pepsin digestion to completely digest the muscle fibers and cysts in the sample, releasing live Trichinella spiralis into the digestive fluid. This method is characterized by its cumbersome operation, time-consuming nature, high equipment requirements, and low sensitivity.

[0005] Currently, the prevalence of Trichinella spiralis and Toxoplasma gondii in pig farms is easily overlooked. In 2024, Li Siwei et al. collected 960 serum samples from commercial pig farms in different regions of Guizhou Province for serological testing of Toxoplasma gondii and Trichinella spiralis. The results showed that the total positive rate for Trichinella spiralis in Guizhou was 1.77%, the total positive rate for Toxoplasma gondii was 17.39%, and the co-positive rate for Trichinella spiralis and Toxoplasma gondii was 1.67%, indicating that there is a clear phenomenon of co-infection with Toxoplasma gondii and Trichinella spiralis. Furthermore, the large number of individual pig farmers and various small and medium-sized pig farms in my country increases the difficulty of detecting, controlling, and preventing the spread of Trichinella spiralis and Toxoplasma gondii.

[0006] Multiplex PCR is a PCR detection technique that inserts multiple primers into the same reaction system for simultaneous detection of two or more pathogenic microorganisms. It is characterized by its simplicity, speed, economy, and high efficiency. Achieving specific amplification of multiple target genes within a single multiplex PCR reaction system requires careful consideration of factors such as target gene sequence selection, primer specificity, amplification efficiency, and concentration ratio. Summary of the Invention

[0007] The purpose of this invention is to develop a simple dual PCR detection kit based on polymerase chain reaction (PCR) that can simultaneously detect Toxoplasma gondii and Trichinella spiralis, in order to provide technical support for the prevention and control of the spread of Toxoplasma gondii and Trichinella spiralis.

[0008] To achieve its purpose, the present invention adopts the following technical solution: This invention provides a dual PCR detection kit for Trichinella spiralis and Toxoplasma gondii, comprising upstream and downstream primer pairs for detecting Trichinella spiralis, upstream and downstream primer pairs for detecting Toxoplasma gondii, 2×Taq Master Mix, and enzyme-free water; the primer pairs for detecting Trichinella spiralis specifically target the complete sequence of the small ribosomal subunit gene of Trichinella spiralis T1 isolate ISS 003, with GenBank accession number JQ823166.1; the primer pairs for detecting Toxoplasma gondii specifically target the 529REP repetitive sequence region of Toxoplasma gondii H66R isolate, with GenBank accession number PP712056.1; The nucleotide sequences of the upstream primer for detecting Trichinella spiralis are shown in Ts-F, and the nucleotide sequences of the downstream primers are shown in Ts-R; the nucleotide sequences of the upstream primer for detecting Toxoplasma gondii are shown in Tg-F, and the nucleotide sequences of the downstream primers are shown in Tg-R.

[0009] As a further preferred embodiment of the technical solution of the present invention, the kit further includes a DNA template; the total reaction volume of the kit is 20 μl, including: 9 μl of 2×Taq Master Mix, 0.5 μl each of the two upstream and downstream primers, 2 μl each of the two parasite DNA templates, and 5 μl of enzyme-free water.

[0010] Furthermore, the concentrations of both upstream and downstream primers are 10 μmol / L.

[0011] Furthermore, the reaction conditions of the PCR detection kit are as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 30 s followed by 60℃ for 30 s, for 20 cycles; extension at 72℃ for 10 min.

[0012] The aforementioned dual PCR detection kit for Trichinella spiralis and Toxoplasma gondii can detect both parasites simultaneously, which is of great significance for accurately preventing and controlling the spread of these two parasites.

[0013] The beneficial effects of this invention are as follows: Based on sequence-specific alignment analysis, this invention utilizes PCR technology to establish a dual PCR detection kit for two common foodborne parasites, Trichinella spiralis and Toxoplasma gondii, enabling rapid detection of these two parasites. The dual PCR detection system established in this invention exhibits high specificity, does not cross-react with other common parasites, and has a detection limit of 0.1 ng / μL for whole-genome DNA. Specificity and sensitivity experiments show that the dual PCR detection system established in this invention amplifies the corresponding target fragments when detecting Trichinella spiralis and Toxoplasma gondii, while no amplified fragments are observed when detecting common swine parasites, indicating high specificity. Furthermore, the detection system has a detection limit of 0.10 ng / μL of parasite DNA concentration, indicating high sensitivity.

[0014] In summary, the detection kit of this invention has high sensitivity and specificity, stable reaction conditions, short reaction cycle and high detection throughput, and can be applied to the rapid screening of Toxoplasma gondii and Trichinella spiralis in meat products and pigs in various scenarios. Attached Figure Description

[0015] Figure 1 Results of establishing a multiplex PCR system for Toxoplasma gondii and Trichinella spiralis: M: DL 2000 DNA Marker; 1: Toxoplasma gondii; 2: Trichinella spiralis; 3: Mixture; Figure 2 For the optimization of double PCR annealing temperature, M: DL 2000 DNA Marker; 1: 54; 2: 56; 3: 58; 4: 60; 5: 62; 6: 64; 7: 66; 8: 68 (°C); Figure 3 The results are for dual PCR sensitivity testing. M: DL 2000 DNA Marker; 1: 2 ng / ul; 2: 1 ng / ul; 3: 0.1 ng / ul; 4: 0.01 ng / ul. Figure 4 For specific detection results using dual PCR, M: DL 2000 DNA Marker; 1: Taenia vesicularis DNA; 2: Trematode DNA; 3: Taenia multicephalus DNA; 4: Sarcocystis jirovecii DNA from goats; 5: Sarcocystis jirovecii DNA from sheep; 6: Positive control; Figure 5 Clinical sample detection results of Toxoplasma gondii and Trichinella spiralis DNA in mice. A: PCR nucleic acid gel electrophoresis band intensity of different tissue samples on day 14 after infection; B: Intensity of PCR nucleic acid gel electrophoresis bands in different tissue samples on day 21 after infection C: Intensity of PCR nucleic acid gel electrophoresis bands in different tissue samples on day 35 after infection. E: PCR nucleic acid gel electrophoresis band intensity of muscle tissue with different treatment methods on day 14 after infection; F: PCR nucleic acid gel electrophoresis band intensity of muscle tissue with different treatment methods on day 21 after infection; G: PCR nucleic acid gel electrophoresis band intensity of muscle tissue with different treatment methods on day 35 after infection; M: DL 2000 DNA Marker; 1: Toxoplasma gondii meat was processed immediately after extraction; 2: Toxoplasma gondii meat was processed after being left at room temperature for 24 hours after extraction; 3: Toxoplasma gondii meat was frozen and then thawed after extraction; 4: Trichinella spiralis meat was processed immediately after extraction; 5: Trichinella spiralis meat was processed after being left at room temperature for 24 hours after extraction; 6: Trichinella spiralis meat was frozen and then thawed after extraction; 7: Positive control. Detailed Implementation

[0016] The present invention will now be described in detail with reference to the accompanying drawings.

[0017] 1. Materials and Methods 1.1 Insect strains and experimental animals Trichinella ( Trichinella spiralis The Jilin isolate and the Toxoplasma gondii GJS strain were both passaged and cultured in Kunming rats by the Important Zoonotic Diseases Team of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Large-scale trematode ( Fasciola gigantica ), multicephalic tapeworm ( Taenia mulliceps ), Vesicular tapeworm ( Taenia hydatigena The DNA was preserved by our team. *Salmonella goatensis* (…) Sarcocystis capracanis ), sheep sarcocystis ( Sarcocystis tenella ), pig roundworms ( Ascaris suum The DNA samples were generously donated by Sichuan Agricultural University and Shanxi Agricultural University, respectively.

[0018] Six-week-old SPF female Kunming mice were purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. All animal experiments were conducted in strict accordance with the approval documents of the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, and in accordance with relevant national animal ethics regulations (License No.: LVRIAEC-2024-010).

[0019] 1.2 Toxoplasma gondii and Trichinella spiralis sample acquisition and DNA extraction The *Trichinella spiralis* isolate from Jilin and *Toxoplasma gondii* GJS isolate standards, preserved in mice and stored at -20°C, were removed from the freezer, thawed, and thoroughly resuspended by shaking. 200 μL of the suspension was aspirated, centrifuged, and the supernatant was discarded. Genomic DNA was extracted according to the instructions of the blood / cell / tissue genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.), and then aliquoted and stored.

[0020] Mice infected with Trichinella spiralis for 30 days were sacrificed, and their muscle and diaphragm were collected, minced, and placed in digestive fluid. After digestion, the precipitate was collected and kept for later use. Mice infected with Toxoplasma gondii for 30 days were sacrificed, and their brain tissue was collected, ground, and kept for later use. Genomic DNA was extracted according to the procedure of the tissue genomic DNA extraction kit, with a Trichinella spiralis DNA concentration of 87.3 ng / μL and a Toxoplasma gondii DNA concentration of 53 ng / μL. The extracted DNA was used as a template for amplification with validation primers, and positive samples were aliquoted and stored.

[0021] 1.3 PCR Primer Design Based on the gene sequences of Trichinella spiralis and Toxoplasma gondii in GenBank, relatively conserved sequences were selected. The complete sequence of the small ribosomal subunit ISS 003 from the Trichinella spiralis T1 isolate (GenBank: JQ823166.1) was selected and named Ts; the 529REP repetitive sequence region from the Toxoplasma gondii H66R isolate (GenBank: PP712056.1) was named Tg. Primers were designed using Premier 5 software to amplify positive samples from Trichinella spiralis and Toxoplasma gondii. Specific primers with fast reaction speeds (short reaction times) were screened, as shown in Table 1.

[0022] Table 1 PCR primer sequences 1.4 Establishment and optimization of PCR amplification system PCR reactions were performed using TB Green Premix Ex Taq reagent, purchased from Baorige Biotechnology (Beijing) Co., Ltd. A single-reaction PCR method for detecting Toxoplasma gondii was established using Tg-F and Tg-R primers. The reaction mixture consisted of 20 μl: 10 μl of 2×TaqMaster Mix, 0.5 μl each of forward and reverse primers (10 μmol / L), 1 μl of DNA template, and 8 μl of enzyme-free water. The reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 66℃ annealing for 30 s, and 72℃ extension for 1 min, for a total of 20 cycles; followed by a final extension at 72℃ for 10 min.

[0023] A single-reaction PCR method for detecting Trichinella spiralis was established using Ts-F and Ts-R primers. The reaction system consisted of 20 μl: 10 μl of 2×TaqMaster Mix, 1 μl each of forward and reverse primers (10 μmol / L), 1 μl of DNA template, and 7 μl of enzyme-free water. The reaction conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 62℃ annealing for 30 s, and 72℃ extension for 1 min, for a total of 20 cycles; and 72℃ extension for 10 min.

[0024] Based on the single PCR reaction data of the two pathogens above, a dual PCR detection method for Toxoplasma gondii and Trichinella spiralis was established. After optimization, the reaction system was 20 μl: 9 μl of 2×Taq Master Mix, 0.5 μl each of the two upstream and downstream primers (10 μmol / L), 2 μl each of the two DNA templates, and 5 μl of enzyme-free water; the reaction conditions were: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 30 s followed by 60℃ for 30 s, for 20 cycles; and 72℃ extension for 10 min.

[0025] 1.5 Sensitivity Test Using an optimized dual PCR method for Toxoplasma gondii and Trichinella spiralis, the DNA of the two pathogens was serially diluted to 1 ng / μL, 0.1 ng / μL, 0.05 ng / μL, and 0.01 ng / μL, respectively, and the above quantitative samples were measured to detect the sensitivity of the reaction system.

[0026] 1.6 Specificity Experiment The established detection methods were used to detect DNA from Taenia vesicularis, Trematodea macrophylla, Taenia multicephala, Sarcocystis suis, Sarcocystis ovis, and Ascaris suis, and the specificity of the established detection methods was observed and evaluated.

[0027] 1.7 Experimental Simulation Sample Preparation and Detection To test the ability of a duplex PCR method to detect Trichinella spiralis DNA in different tissues of mice at different time points, six Kunming mice were randomly divided into three groups of two mice each. Each mouse was inoculated with 20 Toxoplasma gondii brain cysts via oral gavage. Sampling was performed at 14 dpi, 21 dpi, and 35 dpi. Mice were euthanized by cervical dislocation. Brain tissue was collected from the mice. DNA was extracted using the Tiangen Tissue Genomic DNA Extraction Kit. Separately, three Kunming mice were randomly divided into three groups of two mice each. Each mouse was inoculated with 600 Trichinella spiralis cysts via oral gavage. Sampling was performed at 14 dpi, 21 dpi, and 35 dpi. Mice were euthanized by cervical dislocation. Leg skeletal muscle, diaphragm, small intestine, and myocardium were collected from the mice. DNA was extracted using the Tiangen Tissue Genomic DNA Extraction Kit. After DNA extraction, duplex PCR was performed for detection.

[0028] To simulate the detection capability of this dual PCR method for livestock meat products under on-site slaughter, fresh preservation, and cold storage conditions, six Kunming rats were randomly divided into three groups. Each group was inoculated with 20 Toxoplasma gondii brain cysts via oral gavage. Sampling was performed at 14 dpi, 21 dpi, and 35 dpi. Sampling included euthanasia by cervical dislocation. Mice were dissected and muscle tissue samples were collected, divided into three equal parts, and DNA was extracted immediately after meat removal, after 24 hours at room temperature, and after thawing at -20°C for 24 hours. Additionally, three Kunming rats were randomly divided into three groups, each inoculated with 600 Trichinella spiralis via oral gavage. Sampling was performed at 14 dpi, 21 dpi, and 35 dpi. Euthanasia was performed by cervical dislocation. Mice were dissected and muscle tissue was collected. The samples were divided into three equal parts, and each part was processed in three ways: DNA was extracted immediately after meat harvesting, DNA was extracted after the meat was placed at room temperature for 24 hours after harvesting, and DNA was extracted after the meat was thawed at -20°C for 24 hours. All DNA extraction methods used the Tiangen Blood / Cell / Tissue Genomic DNA Extraction Kit. After DNA extraction, double PCR was performed.

[0029] 2. Results 2.1 Optimization of PCR amplification system and reaction conditions Referring to the TaKaRa Taq DNA Polymerase instructions, I experimented with PCR systems, annealing temperatures, and other conditions, keeping all other duplex PCR amplification reactions constant except for the annealing temperature. Annealing temperatures were set at 68, 66, 64, 62, 60, 58, 56, and 54℃, and the amplification effects were observed to ultimately determine the amplification system and the optimal annealing temperature. Figure 1 The genomic DNA of one and two insect species was detected. Single and multiple amplification systems were established sequentially, and the template and primer amounts of the two foodborne pathogens were adjusted to make the band brightness basically consistent for better interpretation. The PCR products were identified by 1% agarose gel electrophoresis. The amplified Toxoplasma gondii DNA band size was about 180 bp, and the amplified Trichinella spiralis DNA band size was about 300 bp. like Figure 2 As shown, as the temperature decreases, the brightness of the bands is similar between 68 and 64℃. The bands also gradually darken between 62 and 58℃, until only one band appears between 56 and 54℃. Therefore, 64℃ was selected for subsequent experiments.

[0030] 2.2 Sensitivity Test The results are as follows Figure 3As shown, the bands gradually darken as the concentration of genomic DNA decreases. Even when the initial mass concentration of the genomic DNA template is as low as 0.1 ng / μL, two target bands can still be observed. Therefore, the limit of detection for the multiplex PCR amplification method established in this study at the genomic DNA level is 0.1 ng / μL, which has high detection sensitivity.

[0031] 2.3 Specificity test The results are as follows Figure 4 It can be seen that the DNA of Taenia vesicularis, Trachomatis macrocarpa, Taenia multicephalus, Sarcocystis suis, Sarcocystis molluscina, and Ascaris suis cannot be amplified in the same system, indicating good specificity.

[0032] 2.4 Clinical Sample Test Results like Figure 5 As shown in Figures A, B, and C, during the artificial sample testing, Kunming rats were orally fed 20 and 50 Toxoplasma gondii parasites respectively in groups of two, with five replicates. Another group of two Kunming rats was orally fed 200 and 600 Trichinella spiralis parasites, with five replicates. At 14, 21, and 35 days after infection, one rat from each group was sacrificed, and its leg, brain, diaphragm, small intestine, and heart were collected. The collected tissues were sonicated to extract genomic DNA, which was then used as a template for multiplex PCR amplification. At 14 days, corresponding bands appeared on the nucleic acid gel electrophoresis images. Specifically, bands were observed in the heart, diaphragm, muscle, and small intestine at a feeding dose of 600 mg / L Trichinella spiralis, and in the brain at a feeding dose of 50 mg / L Toxoplasma gondii. At 21 days, corresponding bands appeared on the nucleic acid gel electrophoresis images. Again, bands were observed in the heart, diaphragm, muscle, and small intestine at a feeding dose of 600 mg / L Trichinella spiralis, with a brighter band in the diaphragm. A band was also observed in the brain at a feeding dose of 50 mg / L Toxoplasma gondii. At 35 days, corresponding bands appeared on the nucleic acid gel electrophoresis images. At a feeding dose of 600 mg / L Trichinella spiralis, bands were observed in the heart, diaphragm, and muscle, with a brighter band in the diaphragm. No bands were observed in the small intestine. A band was observed in the brain at the Toxoplasma gondii testing site, indicating a high detection rate.

[0033] To examine the impact of routine food handling practices on these two parasites, such as... Figure 5As shown in Figures D, E, and F, muscle tissue from the above samples was taken and divided into three equal parts. DNA was extracted immediately by sonication, extracted by sonication after 24 hours at room temperature, and extracted by sonication after thawing at -20°C for 24 hours. At days 14 and 21, when the feeding dose of *Toxoplasma gondii* was 50 and that of *Trichinella spiralis* was 600, bands were observed for both *Toxoplasma gondii* and *Trichinella spiralis* after immediate or 24-hour treatment following meat extraction, and after freezing and thawing. Bands were also observed in the brain region, the site of *Toxoplasma gondii* testing. At day 35, when the feeding dose of *Toxoplasma gondii* was 50 and that of *Trichinella spiralis* was 600, bands were observed for both *Toxoplasma gondii* and *Trichinella spiralis* after immediate or 24-hour treatment following meat extraction, and after freezing and thawing. This demonstrates that the kit of this invention has practical detection capabilities in real-world applications.

Claims

1. A double PCR detection kit for Trichinella and Toxoplasma, characterized by, The kit comprises an upstream primer pair for detecting Trichinella, an upstream primer pair for detecting Toxoplasma, 2x Taq Master Mix and enzyme-free water; the primer pair for detecting Trichinella is specific to the complete sequence of the small subunit of ribosome gene of Trichinella T1 isolate ISS 003, with the GenBank accession number JQ823166.1; the primer pair for detecting Toxoplasma is specific to the 529REP repeat sequence region of Toxoplasma gondii H66R isolate, with the GenBank accession number PP712056.1; The nucleotide sequence of the upstream primer for detecting Trichinella is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2; the nucleotide sequence of the upstream primer for detecting Toxoplasma is shown as SEQ ID NO. 3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.

4.

2. The Trichinella and Toxoplasma dual PCR detection kit according to claim 1, characterized by, The kit further comprises a DNA template; the total volume of the kit reaction is 20 μl, comprising 2x Taq Master Mix 9 μl, two kinds of upstream and downstream primers each 0.5 μL, two kinds of parasite DNA templates each 2 μl, and enzyme-free water 5 μl.

3. The Trichinella and Toxoplasma dual PCR detection kit according to claim 2, characterized by, The concentration of the two kinds of upstream and downstream primers is 10 μmol / L.

4. The dual PCR detection kit for Trichinella spiralis and Toxoplasma gondii as described in claim 2, characterized in that, The reaction conditions of the PCR detection kit are as follows: 95 ℃ pre-denaturation for 10 min; 95 ℃ denaturation for 30 s, followed by 60 ℃ for 30 s, for 20 cycles; 72 ℃ extension for 10 min.

5. The application of a Trichinella and Toxoplasma double-PCR detection kit according to any one of claims 1-4 in the prevention and control of parasite transmission.

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