CN-122012720-A - Primer probe set and kit for identifying echinococcus species

Abstract

The invention relates to the technical field of molecular biology and discloses a primer probe set for identifying echinococcus species, which comprises a first primer probe set for targeting echinococcus granulosus, a second primer probe set for targeting echinococcus multilocellus and/or a third primer probe set for targeting echinococcus stonickii. The primer probe group realizes synchronous, rapid and high-specificity identification and detection of three echinococcus granulosus (Eg), echinococcus multinomial (Em) and echinococcus stonecrop (Es). The detection sensitivity of the primer probe group for identifying and detecting three echinococcus can reach 200 copies/mL, and the positive coincidence rate can reach 100%. The primer probe group realizes synchronous and accurate identification of echinococcus species, and has wide application prospect in molecular epidemiological investigation and port quarantine.

Inventors

• CAI HUIXIA
• GUO SHUAI
• LIU YUFANG
• MA XIAO
• WANG WEI
• QI TONGSHENG
• ZHANG QING
• LEI WEN
• Shi Kemei
• ZHAN PEIZHEN
• DING YULIAN
• ZHANG QINGWEN
• SHAO GAOXIANG
• LIU NA
• ZHANG YUE
• LIU JIA
• MENG FANWEI
• MA WANLI
• DU YINGXIA

Assignees

• 青海省地方病预防控制所
• 苏州中科苏净生物技术有限公司

Dates

Publication Date

20260512

Application Date

20251229

Claims (10)

1. 1. The primer probe group for identifying the echinococcus species is characterized by comprising a first primer probe group for targeting echinococcus granulosus, a second primer probe group for targeting echinococcus multiloculatus and/or a third primer probe group for targeting echinococcus lycopersicum.
2. 2. The primer probe set of claim 1, wherein the first primer probe set comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.1, a reverse primer with a nucleotide sequence shown as SEQ ID NO.2, and a probe with a nucleotide sequence shown as SEQ ID NO. 3.
3. 3. The primer probe set according to claim 1, wherein the second primer probe set comprises a forward primer having a nucleotide sequence shown in SEQ ID NO.4, a reverse primer having a nucleotide sequence shown in SEQ ID NO.5, and a probe having a nucleotide sequence shown in SEQ ID NO. 6.
4. 4. The primer probe set according to claim 1, wherein the third primer probe set comprises a forward primer having a nucleotide sequence shown in SEQ ID NO.7, a reverse primer having a nucleotide sequence shown in SEQ ID NO.8, and a probe having a nucleotide sequence shown in SEQ ID NO. 9.
5. 5. The primer probe set according to any one of claims 1 to 4, wherein a fluorescent group and a quenching group are labeled on the probe.
6. 6. The primer probe set of claim 5, wherein the probe is labeled with a fluorescent group at the 5 'end and a quenching group at the 3' end; optionally, the fluorescent group comprises at least one of FAM, VIC and ROX; Optionally, the quenching group comprises MGB.
7. 7. A kit for identification of echinococcus species, characterized in that the kit comprises the primer probe set of any one of claims 1-5.
8. 8. The kit of claim 7, further comprising a DNA polymerase, dNTPs, and a buffer.
9. 9. A method for identifying the species of Echinococcus, which is not diagnostic or therapeutic for diseases, comprising detecting a sample to be detected using the kit according to claim 7 or 8.
10. 10. Use of the primer-probe set of any one of claims 1 to 6 or the kit of any one of claims 7 to 8 or the species identification method of claim 9 for identifying the species echinococcus or for the preparation of a product for diagnosing a parasitic disease.

Description

Primer probe set and kit for identifying echinococcus species Technical Field The invention relates to the field of molecular biology, in particular to a primer probe set and a kit for identifying echinococcus species. Background Echinococcosis is an infectious parasitic disease caused by an adult echinococcus of cestodes that is parasitic to carnivores such as canines. Echinococcosis is an important zoonosis caused by echinococcus larvae infecting humans or herbivores. The circulation mode of echinococcus is mainly three modes of domestic animal circulation, wild animal circulation and mixed (wild-domestic) circulation. The echinococcosis is an important infectious source for spreading echinococcosis, adult echinococcosis is born in the small intestine of the echinococcosis canine (final host), and eggs are discharged along with feces to pollute food, drinking water and surrounding environment after the development of the worm body is mature, so that the infection of people or herbivores (intermediate host) is caused, focus occupation is formed in viscera tissues such as livers, lungs and the like of people or livestock, and the echinococcosis becomes a patient or a sick livestock. Predation between canines and herbivores maintains the circulation of echinococcosis in nature. The data of World Health Organization (WHO) statistics show that echinococcosis and echinococcosis are highly endemic in the pasture areas of the Mediterranean coast, eastern Europe, central Asia, southwest and China, wherein the prevalence rate of pasture areas of Western provinces such as Xinjiang, qinghai, tibet and Sichuan in China is as high as 3-10%. The presently recognized echinococcus species include echinococcus granulosus, echinococcus multinomi, echinococcus lycopodium, echinococcus fumago and echinococcus minor. The types of echinococcus which are mainly popular in China are three types of echinococcus granulosus, echinococcus multinomus and echinococcus stonecrop, wherein the echinococcus granulosus caused by the echinococcus granulosus (Echinococcus granulosus, eg) is most widely distributed and is popular in more than 100 countries worldwide, the echinococcus vesiculosus caused by the echinococcus multinomus (Echinococcus multilocularis, eg) is mainly distributed in cold temperate regions of the northern hemisphere, the mortality rate is over 90%, and the echinococcus stonecrop (Echinococcus shiquicus, es) is a special species of the Qinghai-Tibet plateau, and can cause the echinococcus stonecrop, and the biological evolutionary inheritance and public health significance of the echinococcus are increasingly prominent. Besides distribution, the parasitic host preference and pathogenic mechanism of three species of insects at different development stages are also obviously different, and taking larval stage as an example, the echinococcosis mainly infects cattle, sheep and human beings, forms cystic focus occupation in organ tissues, the echinococcosis prefers rodent animals and human beings, presents invasive focus occupation, and the echinococcosis mainly infects high-altitude mice and rabbits, presents cystic focus occupation in lung. Because the adult stages of the three species of insects can be parasitic in the small intestines of dogs and other dogs, whether the dogs are infected or not and the specific type of the infection are required to be determined by a traditional morphological inspection mode of collecting the adults by the section inspection dogs, and quick and accurate identification of the species of domestic dogs or wild dogs cannot be realized, a quick and accurate species identification method is required to be established for the three species of insects so as to accurately monitor, prevent and control the infectious agents and restrict the spread of the infectious agents in the crowd. At present, the diagnosis of echinococcosis of an intermediate host mainly depends on three types of imaging examination, serological examination and traditional PCR technology, wherein the imaging examination is only suitable for the situation of human examination, but other intermediate host animal epidemiology screening is difficult to implement, the disease type differential diagnosis is high in complexity and easy to cause misdiagnosis, the serological examination products sold at present cannot distinguish insect species and are affected by the specificity of antigen components, the diagnosis sensitivity and the specificity are poor, the problem of cross reaction with other tapeworms exists, the detection is not easy to apply in other intermediate host animal epidemiology screening, and the traditional PCR method can improve the specificity, but generally needs 3-4 hours to report, and is long in time. In addition, most of the existing echinococcus species identification commercial kits developed based on the conventional PCR method are based on a single detection target, and are difficult to meet the clinical r