CN-122012725-A - Kit for identifying five target tapeworms by using specific primers and application

Abstract

The invention relates to the technical field of tapeworm identification, and provides a kit for identifying five target tapeworms by using a specific primer and application thereof. The main flow genotypes of the echinococcus granulosus, echinococcus multilocus, echinococcus bubble-shaped, echinococcus multilocus and echinococcus canis genes are compared, and aiming at the conserved region of the main flow genotypes as diagnostic targets, the primer pair capable of detecting five nucleic acids of the echinococcus is obtained, and the rapid detection of the five pathogens is realized by combining with the nucleic acid lateral chromatography test paper for detecting digoxin marks. The product obtained by isothermal amplification is directly visualized after being detected by a nucleic acid detection test strip, and the result is interpreted within 15-20min, so that the operation is simple and convenient. The invention has wide application prospect in large-scale screening and detecting whether dogs are infected with echinococcus granulosus, echinococcus multilocularis, echinococcus bubbly, echinococcus multilocularis, echinococcus canis and evaluating the insect repellent effect of praziquantel on the above-mentioned cestodes after feeding dogs in pastoral areas, especially in areas with epidemic echinococcosis.

Inventors

• WAN BO
• CHEN ZHAOJIANG
• HUANG HUIHUI
• LI XUECHEN
• ZHENG HAOTIAN
• ZHANG JIAHAO
• ZHANG YUHANG
• Liu Guanshang
• HAO LILI
• HU MAN
• ZHANG BIAO
• LING XIANGHUI
• Cong Jiahao
• YUAN WENJU

Assignees

• 河南农业大学

Dates

Publication Date

20260512

Application Date

20260127

Claims (5)

1. 1. The application of the specific primer in five target cestode identification is characterized in that the specific primer comprises an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2, wherein the 5 'end of the upstream primer is marked with DIG, the 5' end of the downstream primer is marked with Biotin, and the sequence is as follows: SEQ ID NO: 1 :5'-ATCTGTTAGGTTTGAGGCTTGTTTTATGTG-3'; SEQ ID NO: 2 :5'-TCTCCATAATCAAATGGCGTACGATTAGTTTC-3'。
2. 2. The use of a specific primer according to claim 1 for the identification of five target taenides, wherein the source of the identified subject comprises corresponding echinococcus granulosus, echinococcus multilocularis, taenia blendensis, taenia polycephala and taenia canina nucleic acids in a canine fecal sample.
3. 3. A kit for identifying five target tapeworms is characterized by comprising a specific primer, a colloidal gold immunochromatographic nucleic acid detection test paper and an RAA isothermal amplification reaction reagent, wherein the colloidal gold immunochromatographic nucleic acid detection test paper comprises a T line coated with avidin and a C line coated with staphylococcus aureus A protein, and a binding pad of the colloidal gold immunochromatographic nucleic acid detection test paper is coated with a colloidal gold-labeled anti-DIG monoclonal antibody.
4. 4. The kit for identifying five target tapeworms according to claim 3, further comprising a positive plasmid control and a negative plasmid control, wherein the positive plasmid control comprises target sequences of echinococcus granulosus, echinococcus multilocularis, echinococcus bleacher, echinococcus multilocularis and echinococcus canis genes, and the negative plasmid control is an empty vector plasmid not comprising genomic sequence homologous fragments of the five target tapeworms.
5. 5. The kit for identifying five target tapeworms according to claim 3, characterized in that the RAA isothermal amplification reaction reagent comprises a magnesium acetate solution, and the kit further comprises a DNA extraction reagent and RAA pellets.

Description

Kit for identifying five target tapeworms by using specific primers and application Technical Field The invention relates to the technical field of tapeworm identification, in particular to a kit for identifying five target tapeworms by using a specific primer and application thereof. Background Echinococcosis, also known as echinococcosis, was first found in the toskan region of italy in the last 18 th to the middle of the 19 th century, a parasitic disease common to humans and animals caused by the larval parasitic of echinococcus granulosus/echinococcus, and is listed as global focus NEGLECTED TROPICAL DISEASE (NTD) by the world health organization. The disease is mainly prevalent in areas of animal husbandry, where pathogens are most common among echinococcus granulosus and echinococcus multilocularis. The life cycle of the strain relates to canine animals (final hosts) such as dogs and wolves, livestock animals such as sheep and cattle or humans (intermediate hosts). People are infected by eating food or water polluted by eggs by mistake or by closely contacting dogs carrying eggs, the eggs hatch into larvae in the human body, mainly attack the liver and the lung to form slow-growing cyst of the artemia, press surrounding tissues, cause dysfunction, and can cause anaphylactic shock and even death when serious. The cercaria spinosa is a parasitic disease caused by larvae of taenia tenacissima, namely cercaria spinosa (commonly called as 'water bell'). Mainly invade domestic animals such as sheep and pigs, and occasionally infects cattle. Larvae are parasitic to liver, mesentery and abdominal cavity, which causes symptoms such as acute hepatitis, peritonitis, etc., and can cause death of young animals when serious. The disease is transmitted through the "canine (end host) -environmental-livestock (intermediate host)" cycle. Dogs are infected with tapeworms by eating viscera of the sick livestock containing larvae, eggs in the feces pollute pastures, feeds and water sources, and the livestock is infected after eating. The disease is widely distributed worldwide, is especially common in free-range or pasture areas, and is one of important diseases causing economic losses of animal husbandry. Echinococcosis, a subject of the school name cercaria, is a fatal parasitic disease caused by the parasitic of larvae of taenia multiceps, the cercaria, in the central nervous system of ruminants (especially sheep, goats). The disease is distributed globally, and the hazard in pasture and mountain areas in China is serious. Larvae are mainly parasitic in brain or spinal cord of sheep and cattle, form vesicles with the size of peas to eggs, and press nerve tissues. The sick livestock show typical neurological symptoms such as rotary motion, ataxia, vision disorder, head and neck skew and the like, commonly called as 'rotary disease', and finally die due to failure or paralysis. The taeniasis canis is a common parasitic disease caused by the small intestine of dogs and cats by the taeniasis canis (Dipylidium caninum). The taeniasis history requires fleas (e.g., chlamydia canis) as an intermediate host. Dogs and cats are infected by licking fleas containing infectious larvae (cysticercosis-like). Adult parasitic may cause dyspepsia and wasting, typical symptoms are perianal itching and the appearance of characteristic "rice grain" active gestation nodes in faeces. At present, the insect species are tapeworms, dogs are terminal hosts, adults are born in the intestinal tracts of dogs, and the larval stage endangers intermediate hosts, including cattle, sheep and humans. The detection of the diseases at home and abroad mainly uses PCR, real-time fluorescence quantitative PCR and other etiology detection methods such as microscopic observation. Although the detection method plays an important role in detection and diagnosis, certain requirements on the experience of instruments and operators are met, the defects of complex operation, long time consumption, high requirements on instruments and equipment, excessively complex primer design and the like are generally overcome, and the actual requirements of on-site rapid and visual detection on dogs (feces of dogs) infected by the echinococcus granulosus/echinococcus, the taenia pisiformis, the taenia multiceps and the taenia canina are difficult to meet clinically, and the method is not suitable for the basic field. In recent years, recombinase-mediated isothermal nucleic acid amplification (RAA) technology has shown good application potential in disease diagnosis and detection, and has led to widespread use worldwide. Pathogen nucleic acid detection methods based on RAA and lateral flow immunochromatography have been successfully applied to the rapid diagnosis of novel coronaviruses (SARS-CoV-2) and African Swine Fever Viruses (ASFV). Because the whole RAA amplification process does not need complicated and expensive instruments and equipment, the detection can be completed by