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Primer pair and primer probe set for identifying echinococcus multilocularis and application thereof

A technology of Echinococcus multilocularis and primer probes, applied in the biological field, can solve problems such as difficult treatment, and achieve the effects of high sensitivity, strong specificity, and easy operation

Active Publication Date: 2020-02-28
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

Pastoral and semi-agricultural and semi-pastoral areas in Qinghai, Ningxia, Gansu, Xinjiang, Sichuan, and Tibet are high-incidence areas of multilocular echinococcosis in my country. The treatment of this disease is more difficult than cystic echinococcosis. Liver transplantation is required outside the lesion, which brings a huge financial burden to the patient himself and his family

Method used

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  • Primer pair and primer probe set for identifying echinococcus multilocularis and application thereof
  • Primer pair and primer probe set for identifying echinococcus multilocularis and application thereof
  • Primer pair and primer probe set for identifying echinococcus multilocularis and application thereof

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Experimental program
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Effect test

Embodiment 1

[0052] The design and screening of embodiment 1, primer and the determination of primer-probe group

[0053] 1. Primer design and screening

[0054] The amplification primer in RPA technology can be said to be the key to the whole reaction. Primers that are too short will seriously affect the activity of the recombinase. Long primers do not necessarily improve amplification performance, but instead increase the possibility of secondary structure formation and increase noise from primers. For the amplification primers in RPA technology, there is no definite design rule. Therefore, it is necessary to test and screen candidate primers (primer screening is mainly divided into the following steps: select the target region, design candidate primers, screen candidate primers, improve performance and re-screen).

[0055] After the above steps, the optimal amplification primer pair was obtained, consisting of the upstream primer Em-c-Fnew2 and the downstream primer Em-c-Rnew2 (named...

Embodiment 2

[0070] Embodiment 2, the specificity of primer probe set

[0071] The samples to be tested were Taenia polycephala (Tm), Taenia alveolar (Th), Taenia lentilis (Tp), Echinococcus multilocularis (Em), Echinococcus granulosus (Eg), Echinococcus shiqu Taenia (Es) or healthy voles liver tissue (NC).

[0072] 1. Extract the genomic DNA of the sample to be tested.

[0073] 2. Using the genomic DNA obtained in step 1 as a template, use the primer probe set (upstream primer Em-c-Fnew2, downstream primer Em-c-Rnew2, specific probe Em-c-probe) determined in Example 1 to carry out specific Sexual fluorescent RPA detection (using TwistAmp exo kit).

[0074] ① Add the following reagents into a 1.5mL centrifuge tube and vortex to mix: Em-c-Fnew2 (10μmol / L) 2.1μL, Em-c-Rnew2 (10μmol / L) 2.1μL, Em-c-probe (10μmol / L) / L) 0.6μL, Rehydration buffer 29.5μL, template (genome) 1μL, ddH 2 O 12.2 μL;

[0075] ② Add 47.5 μL of the reaction system prepared in step ① into the freeze-dried reaction re...

Embodiment 3

[0079] Embodiment 3, the sensitivity of primer probe set

[0080] 1. Construction of plasmid standards

[0081]The double-stranded DNA molecule shown in Sequence 1 of the sequence listing was inserted into the pMD19-T simple vector (TaKaRaBiotech) to obtain the recombinant plasmid pMD19-T-EmC, which was the plasmid standard product.

[0082] Second, the sensitivity of the primer probe set

[0083] 1. Prepare plasmid standard solutions of various concentrations, and the concentration of the recombinant plasmid pMD19-T-EmC is 10 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL.

[0084] 2. Using the plasmid standard solution obtained in step 1 as a template, use the primer probe set determined in Example 1 (upstream primer Em-c-Fnew2, downstream primer Em-c-Rnew2, specific probe Em-c-probe) Perform specific fluorescent RPA detection (using TwistAmp exo kit).

[0085] ① Add the following reagents into a 1.5mL centrifuge tube and...

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Abstract

The invention discloses a primer pair and a primer probe set for identifying echinococcus multilocularis and application thereof. The specific primer pair is composed of single-stranded DNA moleculesshown as a sequence 2 and single-stranded DNA molecules shown as a sequence 3. The primer probe set is composed of the specific primer pair and a specific probe, the specific probe is composed of a first segment, tetrahydrofuran and a second segment, the first segment is shown as a sequence 4, and the second nucleotide of 3' terminal is modified by fluorophore; the second segment is shown as a sequence 5, the second nucleotide of 5' terminal is modified by quencher, and C3 Spacer modification is performed on the 3' terminal. The specific primer pair or the primer probe set can be used for identifying echinococcus multilocularis and identifying whether a sample contains echinococcus multilocularis or not. The specific primer pair or the primer probe set is used for identifying echinococcusmultilocularis and is simple, convenient and quick in operation and high in specificity and sensitivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a pair of primers for identifying Echinococcus multilocularis, a primer-probe set and their application. Background technique [0002] Alveolar echinococcosis (AE), also known as alveolar echinococcosis, alveolar echinococcosis, alveolar echinococcosis, etc., is one of the most common echinococcosis in my country. It is a type of harm caused by the middle-stage larva of Echinococcus multilocularis - Echinococcus multilocularis (the pathogen of the disease) parasitizing the lungs, liver, intestines and other organs of intermediate hosts such as humans and animals. Severe zoonotic parasitic disease. The liver is the primary organ of the disease, and almost all clinical cases are hepatic cysts. The vesicles formed by Echinococcus multilocularis are grape-shaped in appearance, and after 1 to 2 years of invasive development, they can occupy almost the entire liver, and then s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6888C12Q1/6844
CPCC12Q1/6888C12Q1/6844C12Q2521/507C12Q2563/107
Inventor 贾万忠吴燕涛闫鸿斌李立朱国强李秀荣李双男姚刚付宝权
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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