A detection kit for Echinococcus shiqu based on rpa technology and its application

A technology of Echinococcus shiqu and a kit, which can be applied in the biological field and can solve problems such as the application of RPA technology that has not been seen

Active Publication Date: 2022-06-21
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no application of RPA technology in the detection of Echinococcus

Method used

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  • A detection kit for Echinococcus shiqu based on rpa technology and its application
  • A detection kit for Echinococcus shiqu based on rpa technology and its application
  • A detection kit for Echinococcus shiqu based on rpa technology and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Embodiment 1, the RPA primer and probe that is used to detect Echinococcus shiquilla and its detection method

[0080] 1. RPA primers and probes for detection of Echinococcus shiqu

[0081] 1. Obtain the sequence information of the target gene

[0082] The partial sequence of the mitochondrial genome of Echinococcus shiquli was used as the target gene for detection. According to the complete mitochondrial genome sequences of Echinococcus shiquilla (NC_009460) and other common tapeworms with canines as the terminal host in the GenBank database [GenBank accession no.NC_014768 (Taenia taenia, Taenia taeniaeformis), GU569096 (Taenia bean), NC_021145 (Taenia canis), GQ228818 (Taenia polycephalus), GQ228819 (Taenia vesicularis), NC_009460 (Echinococcus Tapeworm), AB786664 (Echinococcus granulosus), KU601616 (Echinococcus granulosus), NC_000928 (E. Echinococcus canadensis G10) and AB235847 (Echinococcus canadensis G7)], using the online software Clustal Omega to perform sequ...

Embodiment 2

[0112] Embodiment 2, the specific fluorescence RPA detection method of Echinococcus shiqu

[0113] Using the genomic DNAs of Taenia polycephala, Taenia vesicularis, Taenia bean, Echinococcus multilocularis, Echinococcus granulosus and Echinococcus shiqu as templates, using SEQ ID No.2 and SEQ ID No.3 The primers, with Es-b-probe3 as the probe, were used for specific fluorescent RPA detection according to the instructions of the RPA kit Basic exo, and the signal detection was performed by a fluorescence quantitative PCR instrument. The detailed operation steps are as follows:

[0114] ①Add the following reagents: upstream primer (10 μmol / L) 2.1 μL, downstream primer (10 μmol / L) 2.1 μL, Es-b-probe (10 μmol / L) 0.6 μL, Rehydration buffer 29.5 μL, template (genome) 1 μL, ddH 2 Add 12.2μL of O to a 1.5mL centrifuge tube, vortex and mix;

[0115] ②Add 47.5μL of reaction system to the freeze-dried reaction reaction tube in the kit, and mix by pipetting up and down until all the part...

Embodiment 3

[0124] Embodiment 3. Sensitivity analysis of Echinococcus Shiqu specific fluorescence RPA detection

[0125] 1. Construction of Plasmid Standards

[0126] ① Using the genomic DNA of Echinococcus shiqu as template, using Es-T-F and Es-T-R primers to carry out ordinary PCR amplification to obtain the target fragment shown in SEQ ID No.1. The primer sequences are as follows:

[0127] Es-T-F: 5'-TAGTGTGATGATGGTTTTTGA-3' (SEQ ID No. 10);

[0128] Es-T-R: 5'-CACACACCCAAAATCAGTAC-3' (SEQ ID No. 11).

[0129] PCR amplification reaction system (total volume 50 μL) is as follows: upstream primer 1 μL, downstream primer 1 μL, Ex Taq premix enzyme 25 μL, template (genome) 2 μL, ddH 2 O 21 μL.

[0130] The PCR amplification reaction procedure was as follows: firstly, denaturation at 98°C for 5 min; then 35 cycles, respectively: denaturation at 95°C for 30s; annealing at 56°C for 30s; extension at 72°C for 50s, and finally extension at 72°C for 10 min.

[0131] ②Use the PCR product pur...

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Abstract

The invention discloses a detection kit for Echinococcus shiquegus based on RPA technology and its application. The present invention designs and screens a set of primers and probe combinations that can be used for RPA detection according to the mitochondrial gene sequence of Echinococcus shiqu, and establishes a specific real-time fluorescent RPA detection method for Echinococcus shiqu. The method adopts recombinase and single-strand binding protein to cooperate to realize the specific combination of primer and template at normal temperature, and the primer is extended under the action of DNA polymerase to generate a new DNA chain. The specific fluorescent RPA detection method for Echinococcus shiquense of the present invention is simple, fast, and specific, and is suitable for rapid on-site detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection kit for Echinococcus shiquilla based on RPA technology and application thereof. Background technique [0002] Echinococcus shiquicus was first discovered and named in 2005 in Shiqu County (32°19'-34°20'N, 97°20'-99°15'E) in Ganzi Tibetan Autonomous Prefecture, Sichuan Province, China and was identified as an independent species, belonging to the genus Echinococcus (Echinococcus) tapeworm members. Echinococcus tapeworms belong to the animal kingdom (Animalia), the Platyhelminthes (Platyhelminthes), the Cestoda (Cestoda), the Cyclophyllidea (Cyclophyllidea), and the Taeniidae in the taxonomy. The classification of Echinococcus tapeworms has been controversial. With the continuous development and deepening of molecular biology technology, different classification methods and standards have been proposed, and now they are becoming unified. Echinococcus tapeworms are now reco...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6844
CPCC12Q1/6888C12Q1/6844C12Q2521/507C12Q2522/101
Inventor 贾万忠吴燕涛闫鸿斌李立朱国强李双男姚刚田文俊
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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