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Echinococcus shiquicus detection kit based on RPA technology and application of echinococcus shiquicus detection kit

A technology of Echinococcus shiqu and reagents, which is applied in the field of Echinococcus shiqu detection kits, can solve problems such as the application of RPA technology that has not been seen, and achieve the effects of short reaction time, strong specificity and high sensitivity

Active Publication Date: 2019-12-03
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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  • Echinococcus shiquicus detection kit based on RPA technology and application of echinococcus shiquicus detection kit
  • Echinococcus shiquicus detection kit based on RPA technology and application of echinococcus shiquicus detection kit
  • Echinococcus shiquicus detection kit based on RPA technology and application of echinococcus shiquicus detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1. RPA primers and probes for detecting Echinococcus shiquense and its detection method

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

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

[0082] The partial sequence of the whole mitochondrial genome of Echinococcus shiquense was used as the detection target gene, according to the complete mitochondrial genome sequence of Echinococcus shiquense (NC_009460) and other common tapeworms with canines as their final hosts in the GenBank database [GenBank accession no.NC_014768 (Tenia zoster, Taenia taeniaeformis), GU569096 (Taenia lentilis), NC_021145 (Pipipyra canis), GQ228818 (Tenia polycephalum), GQ228819 (Tenia alveolar), NC_009460 (Echinococcus shiqu tapeworm), AB786664 (Echinococcus granulosus), KU601616 (Echinococcus granulosus), NC_000928 (Echinococcus multilocularis), AB208063 (Echinococcus canadensis G6), AB235848 (Echinococcus canadensis G8), AB745463 ( Echinococ...

Embodiment 2

[0112] Embodiment 2, the specific fluorescent RPA detection method of Echinococcus shiquense

[0113] Using the genomic DNA of Taenia polycephala, Taenia alveolar, Taenia lentiformis, Echinococcus multilocularis, Echinococcus granulosus and Echinococcus shiqueri as templates, using SEQ ID No.2 and SEQ ID No.3 The primer uses Es-b-probe3 as the probe, and performs specific fluorescent RPA detection according to the instructions of the RPA kit Basic exo, and uses a fluorescent quantitative PCR instrument for signal detection. The detailed operation steps are as follows:

[0114] ① Prepare 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 O 12.2 μL was added to a 1.5mL centrifuge tube, and vortexed to mix;

[0115] ② Add 47.5 μL of the reaction system to the freeze-dried reaction reaction tube in the kit, and mix well by blowing up and down wi...

Embodiment 3

[0124] Example 3, Sensitivity analysis of Echinococcus shiqu specific fluorescent RPA detection

[0125] 1. Construction of plasmid standards

[0126] ①Using the genomic DNA of Echinococcus shiquense as a template, the Es-T-F and Es-T-R primers were used for 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'-TAGTGTGATGATGGTTTTGA-3' (SEQ ID No.10);

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

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

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

[0131] ②Purify and r...

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Abstract

The invention discloses an echinococcus shiquicus detection kit based on a RPA technology and application of the echinococcus shiquicus detection kit. A set of primer and probe combination capable ofbeing used for RPA detection is designed and screened according to the gene sequence of mitochondria of echinococcus shiquicus, and an echinococcus shiquicus specificity real-time fluorescent RPA detection method is established. According to the method, recombinant enzymes and single-stranded binding proteins are adopted to cooperate at the normal temperature to achieve specific binding of a primer and a template, and under the effect of DNA polymerases, the primer is extended to generate new DNA strands. The echinococcus shiquicus specificity fluorescent RPA detection method is easy, convenient and quick to operate, high in specificity, and suitable for site quick detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection kit for Echinococcus shiquegus based on RPA technology and its application. Background technique [0002] Echinococcus shiquicus was first discovered and named in 2005 in Shiqu County, Garze Tibetan Autonomous Prefecture, Sichuan Province, China (32°19'-34°20' north latitude, 97°20'-99°15' east longitude) and was identified as an independent species, belonging to one of the members of the Echinococcus (Echinococcus) tapeworm. Echinococcus tapeworms belong to the kingdom Animalia, the phylum Platyhelminthes, the class Cestoda, the order Cyclophyllidea, and the family Taeniidae in terms of taxonomy. The classification of Echinococcus tapeworms has always been controversial. With the continuous development and deepening of molecular biology techniques, different classification methods and standards have been proposed, which are now tending to be unified. There are currently...

Claims

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Application Information

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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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