Kit for identifying five target tapeworms by using specific primers and application of kit

Abstract

The invention relates to the technical field of tapeworm identification, and provides a kit for identifying five target tapeworms by using specific primers and application of the kit. All mainstream genotypes of genes of Echinococcus granulosus, Echinococcus multilocularis, Taenia vesicula, Taenia multiceps and Taenia canis are compared, and a primer pair capable of universally detecting nucleic acids of five tapeworms is obtained by taking conserved regions of the genes as diagnostic targets. The rapid detection of the five pathogens is realized by combining with a digoxin-labeled nucleic acid lateral chromatography test paper. A product obtained by isothermal amplification is detected by a nucleic acid detection test strip and then is directly observed by eyes, and the result is interpreted within 15-20 minutes, so that the operation is simple and convenient. The method provided by the invention has wide application prospects in the aspects of large-scale screening and detection of whether dogs are infected with echinococcus granulosus, echinococcus multilocularis, taenia vesicula, taenia multiceps and tapestoma dogs or not in pasturing areas, especially in areas with epidemic echinococcosis, and evaluation of the insect expelling effect of the dogs fed with praziquantel on the tapeworms.

Description

Technical Field

[0001] This invention relates to the field of tapeworm identification technology, specifically to a kit and its application for identifying five target tapeworms using specific primers. Background Technology

[0002] Echinococcosis, also known as hydatid disease, was first discovered in Tuscany, Italy, in the late 18th to mid-19th centuries. It is a zoonotic parasitic disease caused by the larvae of *Echinococcus granulosus* and is listed as a globally important neglected tropical disease (NTD) by the World Health Organization. The disease is prevalent in livestock-based areas, with *Echinococcus granulosus* and *Echinococcus multilocularis* being the most common pathogens. Its life cycle involves canines (definitive hosts) such as dogs and wolves, and livestock such as sheep and cattle, or humans (intermediate hosts). Humans are infected through ingestion of food or water contaminated with the eggs, or through close contact with dogs carrying the eggs. The eggs hatch into larvae in the human body, primarily invading the liver and lungs, forming slowly growing hydatid cysts that compress surrounding tissues, leading to functional impairment. In severe cases, it can cause anaphylactic shock and even death.

[0003] Cysticercosis is a parasitic disease caused by the larvae of Taenia solium, specifically the cysticercus (commonly known as "water bell"). It primarily affects livestock such as sheep and pigs, and occasionally cattle. The larvae parasitize the liver, mesentery, and abdominal cavity, causing symptoms such as acute hepatitis and peritonitis, which can lead to death in young animals in severe cases. The disease is transmitted through a cycle of "dogs (definitive host) - environment - livestock (intermediate host)." Dogs become infected by ingesting the internal organs of infected animals containing larvae; the eggs in their feces contaminate pastures, feed, and water sources, infecting livestock that consume these products. This disease is widespread globally, particularly in free-range or pastoral areas, and is a significant cause of economic losses in livestock farming.

[0004] Cerebral echinococcosis, scientifically known as polycephalocystis, is a fatal parasitic disease caused by the larvae of the tapeworm *Taenia multicephalosporinus*—the polycephalosporin larvae—parasitizing the central nervous system of ruminants, especially sheep and goats. This disease is distributed globally and is particularly severe in pastoral and mountainous areas of my country. The larvae primarily parasitize the brain or spinal cord of sheep and cattle, forming vesicles the size of a pea to an egg, which compress nerve tissue. Affected animals exhibit typical neurological symptoms: circling movements, ataxia, visual impairment, and head and neck tilting, commonly known as "circling disease," and often die from exhaustion or paralysis.

[0005] Canine dipylidium caninum is a common parasitic disease caused by the tapeworm *Dipylidium caninum*, which parasitizes the small intestine of dogs and cats. This tapeworm requires fleas (such as *Ctenophora canis*) as an intermediate host for its life cycle. Dogs and cats become infected by licking fleas containing infective larvae (similar to cysticerci). Adult worm infestation can cause indigestion and weight loss in animals; typical symptoms include perianal itching and the presence of characteristic "rice-grain-sized" mobile gravid protuberances in the feces.

[0006] Currently, all of the above-mentioned worms are tapeworms, with dogs being the definitive host. Adult tapeworms parasitize the dog's intestines, while the larval stage harms intermediate hosts, including cattle, sheep, and humans. Domestically and internationally, the main methods for detecting these diseases are PCR, real-time quantitative PCR, and microscopic observation. Although these methods play an important role in detection and diagnosis, they require certain levels of instrument and operator experience and generally suffer from drawbacks such as complex operation, long processing times, high equipment requirements, or overly complex primer designs. These methods are insufficient to meet the practical needs of clinical practice for rapid and visual on-site detection of dogs infected with Echinococcus granulosus, Taenia lentigines, Taenia multicephala, and Diploporum canis (dog feces), making them unsuitable for grassroots clinical settings.

[0007] In recent years, recombinase-mediated isothermal nucleic acid amplification (RAA) technology has shown great potential in disease diagnosis and detection, and has been widely used worldwide. Pathogen nucleic acid detection methods based on RAA and lateral flow immunochromatography have been successfully applied to the rapid diagnosis of SARS-CoV-2 and African swine fever virus (ASFV).

[0008] Since the entire RAA amplification process does not require complex and expensive instruments and equipment, and can be completed with just a thermostat or even body temperature, RAA technology is very suitable for development and application in rapid on-site detection. Summary of the Invention

[0009] The purpose of this invention is to provide a kit and application for identifying five target tapeworms using specific primers, in order to solve the technical problem of rapid and accurate on-site detection of Echinococcus granulosus, Echinococcus multilocularis, Taenia lentigines, Taenia multicephalus, and Diploporium canis gene infection.

[0010] The technical solution of the present invention is as follows: A specific primer is used for the identification of five target tapeworms. The specific primer includes the upstream primer shown in SEQ ID NO:1 and the downstream primer shown in SEQ ID NO:2; the 5' end of the upstream primer is labeled with DIG, and the 5' end of the downstream primer is labeled with Biotin; its sequence is as follows: SEQ ID NO: 1: 5'-ATCTGTTAGGTTTGAGGCTTGTTTTATGTG-3'; SEQ ID NO: 2: 5'-TCTCCATAATCAAATGGCGTACGATTAGTTTC-3'.

[0011] Further optimization involves the identification of nucleic acids from Echinococcus granulosus, Echinococcus multilocularis, Taenia vesicularis, Taenia multicephalus, and Diploporium canis in canine fecal samples.

[0012] A kit for identifying five target tapeworms includes specific primers, colloidal gold immunochromatographic nucleic acid detection strips, and RAA isothermal amplification reaction reagents; the colloidal gold immunochromatographic nucleic acid detection strips include a T line coated with avidin and a C line coated with Staphylococcus aureus A protein, and the binding pad of the colloidal gold immunochromatographic nucleic acid detection strips is coated with colloidal gold-labeled anti-DIG monoclonal antibody.

[0013] Further optimization includes positive plasmid controls and negative plasmid controls; the positive plasmid controls include target sequences of the genes of Echinococcus granulosus, Echinococcus multilocularis, Taenia vesicularis, Taenia multicephalus, and Diplopylidium canis; the negative plasmid controls are empty vector plasmids that do not contain homologous fragments of the genome sequences of the five target tapeworms.

[0014] Further optimizations include the inclusion of magnesium acetate solution in the RAA isothermal amplification reaction reagent; the kit also includes DNA extraction reagent and RAA microspheres.

[0015] The beneficial effects of this technical solution are: The specific primers used in this technology are designed for conserved regions of the genes of *Echinococcus granulosus*, *Echinococcus multilocularis*, *Taenia vesicularis*, *Taenia multicephalus*, and *Dipylidium canis*. After three rounds of screening and optimization, they can only specifically bind to the nucleic acid sequences of the above five target tapeworms and do not cross-react with the nucleic acids of other parasites such as *Ascaris lumbricoides*. At the same time, the DIG marker at the 5' end of the upstream primer and the Biotin marker at the 5' end of the downstream primer form dual specific recognition. Combined with the anti-DIG monoclonal antibody-colloidal gold marker and avidin-T line sandwich method of colloidal gold immunochromatographic nucleic acid detection test strip, the identification specificity is further improved, false positive results are effectively avoided, and the identification accuracy is ensured. A single pair of specific primers can simultaneously identify five target tapeworms, eliminating the need to design multiple sets of primers for a single species. This significantly simplifies the detection process. Compared to traditional PCR and real-time quantitative PCR, which require separate detection of different species, this technology reduces repetitive operations such as primer design and reaction system setup, shortens the detection cycle, and meets the high-efficiency requirements of large-scale sample screening. Based on RAA isothermal amplification technology, the detection can be completed within 5-20 minutes under a constant temperature of 37℃. It does not require expensive PCR instruments or other sophisticated equipment. It can be carried out with just a simple constant temperature device or even with the help of body temperature. After dilution, the amplification products can be directly interpreted by the naked eye through colloidal gold immunochromatographic nucleic acid test strips. No professional personnel or complex data analysis are required. It is suitable for rapid on-site testing scenarios in pastoral areas, grassroots communities and other places, and solves the pain points of traditional testing methods that are complicated to operate and have high requirements for instruments and personnel. Through primer optimization and reaction condition adjustment, the detection limit of this technology can reach 100 copies / μL, which can accurately capture low concentrations of target tapeworm nucleic acid in samples, enabling early detection of infection and providing timely evidence for disease prevention and control and evaluation of anthelmintic efficacy. At the same time, the kit is equipped with positive plasmid control and negative plasmid control, which can effectively monitor the reliability of the entire detection process and further ensure the accuracy of the detection results. Attached Figure Description

[0016] Figure 1 is a schematic diagram of the detection process for five types of target tapeworms in an embodiment of the present invention; Figure 2 shows the RAA-specific primer screening results in an embodiment of the present invention, wherein... Figure 2 A represents 10 different primer combinations from Table 1. 3 Image of nucleic acid gel electrophoresis results after 10 min of amplification of positive plasmid copies / μL; Figure 3 shows the optimization results of RAA primers in the embodiments of the present invention, wherein... Figure 3 A is a schematic diagram showing the amplification results of positive plasmids with different copy numbers diluted using the unoptimized GPCR-RAA-3F / GPCR-RAA-3R. Figure 3 B is a schematic diagram showing the amplification results of positive plasmids diluted with different copy numbers compared using the optimized GPCR-RAA-6F / GPCR-RAA-3R. Figure 4 shows the optimization results of the reaction time based on the RAA-LFA detection method in this embodiment of the invention. Figure 4 A is a schematic diagram showing the effect of primer dimers on the color development of the side-flow chromatography test strip at different amplification times; Figure 4 B is a schematic diagram of the color development effect of amplification of standard positive and negative templates when the amplification time is 10 min; Figure 5 shows the sensitivity test results of the RAA-LFA detection method in this embodiment of the invention; Figure 6 shows the specificity experimental results of the RAA-LFA detection method in this embodiment of the invention. Figure 6Figure A shows the RAA amplification results of each parasite sample in the sample dish using the GPCR-RAA-6F / GPCR-RAA-3R primer combination, and Figure 6B shows the RAA-LFA detection results of each parasite sample in the sample dish using the labeled primers. Figure 7 A schematic diagram of the PCR sensitivity test results recommended by OIE. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments. The components of the embodiments of the invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0018] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0019] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0020] A universal detection kit for Echinococcus granulosus, Taenia vesicularis, Taenia multicephalus, and Diploporium canis based on colloidal gold immunochromatographic nucleic acid detection test strips includes RAA primer pairs, DNA extraction reagent, RAA microspheres, colloidal gold immunochromatographic nucleic acid detection test strips, and positive control. The RAA primers were designed and optimized to target specific conserved regions of the genes of *Echinococcus granulosus*, *Taenia vesicularis*, *Taenia multicephalus*, and *Diplophora canis*. DIG and Biotin were then labeled at the 5' ends of the upstream and downstream primers, respectively. The colloidal gold immunochromatographic nucleic acid detection strip was independently developed. The hapten digoxigenin-BSA conjugate DIG-BSA was used as an artificial complete antigen to immunize BALB / c mice to screen for hybridoma cell lines that stably secrete anti-DIG monoclonal antibodies, and ascites was prepared. The ascites was then labeled with colloidal gold for specific detection of DIG-labeled nucleic acid molecules. Avidin (SA) was immobilized on the T line to capture the amplification products, and Staphylococcus aureus protein A was immobilized on the C line. SPA) is used as a quality control, and the qualitative detection of target nucleic acid is achieved by using DIG gold-labeled antibody sandwich method with avidin; the positive control is a standard positive plasmid containing the full length of Echinococcus granulosus and Echinococcus multilocularis genes, and the negative control is an empty vector plasmid without the genome of Echinococcus granulosus and Echinococcus multilocularis.

[0021] The specific primers include the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO: 2, with the following sequences: SEQ ID NO: 1: 5'-ATCTGTTAGGTTTGAGGCTTGTTTTATGTG-3'; SEQ ID NO: 2: 5'-TCTCCATAATCAAATGGCGTACGATTAGTTTC-3'.

[0022] Methods for detecting *Echinococcus granulosus*, *Echinococcus multilocularis*, *Taenia vesicularis*, *Taenia multicephalus*, and *Dipylidium canis* using colloidal gold immunochromatographic nucleic acid test strips, such as... Figure 1 As shown, it includes the following steps: S1. Amplify the sample DNA using the primer pairs from the kit and the RAA isothermal amplification reaction; S2. Use colloidal gold immunochromatographic nucleic acid test strips to detect the amplification products, and interpret the results based on the color development of the T and C lines. The interpretation criteria are as follows: If the test line is red, the result is positive; If the test line does not show color, the result is negative.

[0023] The specific detection method is as follows: Dissolve one unit of RAA reaction ball in 20.75 μL of deionized water, add 1.00 μL each of upstream and downstream primers and 1.00 μL of template positive plasmid, and finally add 1.25 μL of 280 mM magnesium acetate solution. Mix the RAA amplification system thoroughly and react in a 37℃ water bath for 10 min. After the reaction, obtain the RAA amplification product. Dilute the obtained RAA amplification product with 175 μL of buffer and add it to the sample wells of colloidal gold-labeled colloidal gold immunochromatographic nucleic acid test strip. Incubate at room temperature for 5-10 min and observe the results. If the test line is red, it is positive; if the test line is not colored, it is negative.

[0024] 1. Experimental materials The tissue materials used in the experiment, including Echinococcus granulosus, Echinococcus multilocularis, Taenia vesicularis, Taenia multicephalus, and Diploporium canis, were collected from veterinary hospitals. Whole-genome DNA was extracted using a DNA extraction kit and stored at -20°C for later use. RAA beads and DNA extraction reagents were customized by Suzhou Keer Life Technology Co., Ltd.

[0025] 2. Primer design and condition optimization The key to RAA amplification lies in primer design. However, RAA differs from conventional PCR reactions. This invention selects conserved regions as RAA amplification targets. Taking into account various factors such as (1) GC content (40~60%), (2) length (30~35 nt), (3) amplification product size (100~300 bp), etc., cross-pairing was performed on the designed upstream and downstream RAA primers, and the best primer pairing was screened in three rounds to obtain the optimal primers. See Table 1 for details.

[0026] Table 1. RAA primers designed for conserved regions of genes from five target tapeworms.

[0027] After RAA amplification using the RAA primer pairs designed in Table 1, the GPCR-RAA-3F / GPCR-RAA-3R primer pairs were selected by agarose gel electrophoresis as having the best amplification effect. Figure 2 As shown in Figures 2A, 2B, and 2C, these are 10 different primer combinations from Table 1. 3 The nucleic acid gel electrophoresis results after amplification of positive plasmid copies / μL for 10 min show good band specificity with no non-specific amplification. Other primer pairs, however, exhibited non-specific amplification and low amplification efficiency. Therefore, primer pair GPCR-RAA3F / GPCR-RAA-3R was selected for further primer optimization.

[0028] 3. Optimization after RAA primer screening Based on the screened RAA primers, following the design principles of RAA primers, GPCR-RAA-3F was designed to be GPCR-RAA-6F by single base shifts and base additions / reductions. Primer combinations of GPCR-RAA-3F / GPCR-RAA-3R and GPCR-RAA-6F / GPCR-RAA-3R were then used to create 10 primer pairs. 2 Comparing the nucleic acid gel electrophoresis results of positive plasmid amplification with copies / μL, it was found that the optimized primer combination had better band specificity, a cleaner background, and no non-specific amplification. Therefore, the optimized primer pair was selected as the combination for subsequent work.

[0029] 4. Sensitivity analysis of RAA primers After optimization, the sensitivity of the RAA reaction was further improved, as shown in Figure 3. Figure 3 A shows the amplification results of positive plasmids with different copy numbers diluted using the unoptimized GPCR-RAA-3F / GPCR-RAA-3R primer combination. The efficiency of the unoptimized primer combination can reach 10. 0 copies / μL; Figure 3B shows the amplification results of positive plasmids with different copy numbers diluted using the optimized GPCR-RAA-6F / GPCR-RAA-3R. The efficiency of the optimized primer combination can reach 10. 0 The positive results showed a cleaner background than the negative results; the final optimized GPCR-RAA-6F / GPCR-RAA-3R were labeled with DIG and Biotin at the 5' end, respectively.

[0030] The optimization of the reaction time for the RAA-LFA detection method followed the basic isothermal reaction procedure described above, with negative controls used as templates. The reaction was carried out in a 37℃ water bath for 0 min, 5 min, 10 min, 15 min, 20 min, and 25 min. After adding diluent, the solution was added and dropped into the sample wells of the colloidal gold-labeled lateral chromatography strip. The results are shown in Figure 4. Figure 4 A shows the effect of primer dimers on the color development of the lateral flow chromatography strip under different amplification times. When the amplification time is 15 min, the negative template has a longer RAA primer, and the primer dimer formed after amplification causes the colloidal gold-labeled lateral flow chromatography strip to show a positive result. Therefore, the optimal amplification time is 10 min. Figure 4 B represents the colorimetric effect of amplifying standard positive and negative templates at an amplification time of 10 min.

[0031] Sensitivity and specificity experiments of the RAA-LFA detection method Using a standard Echinococcus granulosus gene-positive plasmid sample as a template, the sample was serially diluted 10-fold with double-distilled water to a concentration of 10. 8 -10 0 The standard plasmid template was measured in copies / μL, with double-distilled water as a blank control. The detection was performed under optimized reaction conditions according to the RAA-LFA detection method described above. The result showed a minimum detectable template amount of 10 copies / μL. 0 copies / μL (see Figure 5); it has high sensitivity, and the detection method of the present invention is simpler and faster, especially suitable for on-site detection, and has more relaxed requirements for experimental conditions.

[0032] Nucleic acid samples extracted from positive samples were used as templates, and RAA-LFA detection was performed under optimized reaction conditions according to the RAA-LFA detection method. Detection was performed using gel electrophoresis and lateral chromatography. The experimental results are shown in Figure 6. Figure 6 Figure A shows the RAA amplification results of each parasite sample in the sample dish using the GPCR-RAA-6F / GPCR-RAA-3R primer combination. Figure 6B shows the RAA-LFA detection results of each parasite sample in the sample dish using the labeled primers. Only Echinococcus granulosus, Taenia vesicularis, Taenia multicephalus, and Diploporium canis were positive, while Ascariasis in dogs was negative. This indicates that the primer combination for RAA amplification of the present invention has extremely high specificity and the RAA-LFA detection system is extremely specific.

[0033] Using standard Echinococcus granulosus gene-positive plasmid samples as templates, the sensitivity was detected using the OIE-recommended PCR method, such as... Figure 7 As shown, the detection results are: template concentration from 10 8 -10 0 Gradual dilution with copies / μL resulted in progressively weaker bands as the concentration decreased. 10 5 No obvious band was observed after 1 copy / μL, and no band was observed in the negative control (NC), indicating that the limit of detection of this PCR method is approximately 10. 5 The detection sensitivity is weaker compared to the RAA-LFA detection method in this embodiment, with a sample size of copies / μL.

[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the description and drawings of the present invention shall also be included within the scope of protection of the present invention.

Claims

1. The application of a specific primer in the identification of five target tapeworms, characterized in that, The specific primers include the upstream primer shown in SEQ ID NO:1 and the downstream primer shown in SEQ ID NO:2; the 5' end of the upstream primer is labeled with DIG, and the 5' end of the downstream primer is labeled with Biotin; their sequences are as follows: SEQ ID NO: 1: 5'-ATCTGTTAGGTTTGAGGCTTGTTTTATGTG-3'; SEQ ID NO: 2: 5'-TCTCCATAATCAAATGGCGTACGATTAGTTTC-3'.

2. The application of the specific primer according to claim 1 in the identification of five target tapeworms, characterized in that, The sources of the identification targets include nucleic acids from Echinococcus granulosus, Echinococcus multilocularis, Taenia vesicularis, Taenia multicephalus, and Diploporium canis corresponding to canine fecal samples.

3. A kit for identifying five target tapeworms, characterized in that, The test includes the specific primers, colloidal gold immunochromatographic nucleic acid test strip, and RAA isothermal amplification reaction reagent as described in claim 1; the colloidal gold immunochromatographic nucleic acid test strip includes a T line coated with avidin and a C line coated with Staphylococcus aureus protein A, and the binding pad of the colloidal gold immunochromatographic nucleic acid test strip is coated with colloidal gold-labeled anti-DIG monoclonal antibody.

4. The kit for identifying five target tapeworms according to claim 3, characterized in that, It also includes positive plasmid controls and negative plasmid controls; the positive plasmid controls include target sequences of the genes of Echinococcus granulosus, Echinococcus multilocularis, Taenia vesicularis, Taenia multicephalus, and Diplopylidium canis; the negative plasmid controls are empty vector plasmids that do not contain homologous fragments of the genome sequences of the five target tapeworms.

5. The kit for identifying five target tapeworms according to claim 3, characterized in that, The RAA isothermal amplification reaction reagent includes magnesium acetate solution; the kit also includes DNA extraction reagent and RAA beads.

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