PCR-RFLP detection kit for authenticating and differentiating infections of echinococcus multilocularis and echinococcosis shiquicus

A PCR-RFLP and multilocular echinococcosis technology is applied in the field of PCR-RFLP detection kits to achieve the effect of saving time.

Inactive Publication Date: 2014-11-26
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • PCR-RFLP detection kit for authenticating and differentiating infections of echinococcus multilocularis and echinococcosis shiquicus
  • PCR-RFLP detection kit for authenticating and differentiating infections of echinococcus multilocularis and echinococcosis shiquicus
  • PCR-RFLP detection kit for authenticating and differentiating infections of echinococcus multilocularis and echinococcosis shiquicus

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Embodiment 1

[0038] A PCR-RFLP detection kit for identifying and distinguishing Echinococcus multilocularis and Echinococcus shiqu infection, including: TransGen (Beijing) company 2× PCR SuperMix, TaKaRa (Dalian) CoR Ⅰ and Ssp Ⅰ endonucleases, universal primers, and DNA template standards for Echinococcus multilocularis and Echinococcus shiqu.

[0039] The sequence of the universal primer is:

[0040] F: 5'-TAA GWT RAG TGT GTG TGT TGG T-3',

[0041] R: 5'-TAA RCA AAC CTC TCA ACG AGA C-3',

[0042] Wherein, W=A / T, R=A / G.

Embodiment 2

[0043] Example 2 Identification of DNA Template Standards

[0044] (1) PCR amplification of DNA template standard

[0045] Use the PCR reagents, universal primers, and DNA template standards of two Echinococcus species provided in the kit in Example 1 to perform PCR amplification respectively. In order to verify that there may be mixed infection of Echinococcus multilocularis and Echinococcus shiquero in field conditions, a PCR reaction with half and half of the standard template was added. The PCR reaction system is as follows:

[0046]

[0047] The PCR reaction was carried out according to the following conditions: 94°C for 4min; 94°C for 30s, 54°C for 30s, 72°C for 2min, 35cycles; 72°C for 10min.

[0048] Take 5 μ L of PCR reaction product in 1.0% (w / v) agarose gel, carry out electrophoresis analysis under 120 volts voltage, the result is as follows: image 3 As shown, the Em DNA fragment of 1417 bp and the Es DNA fragment of 1426 bp were amplified respectively with t...

Embodiment 3

[0057] Example 3 Application to Clinical Sample Detection

[0058] The clinical samples were Echinococcus multilocularis Dari County strain (Echinococcus multilocularis isolated from Dari County, other strains are described here), Yushu strain and Jiuzhi County strain, Echinococcus Shiqu Jiuzhi County strain, Dari county strain (Dari county strain has 5 genotypes: N6 / C7, N3 / C3, N2 / C6, N4 / C4 and N5 / C5; N represents nad1 gene, C represents cox1 gene).

[0059] (1) Preparation of clinical sample DNA

[0060] The clinically collected cysts were isolated separately, and then the DNA of a single cyst was extracted according to the instructions of the tissue DNA extraction kit.

[0061] (2) Perform PCR amplification on clinical samples

[0062] Reaction system and condition are with embodiment 2. Take 5 μ L of the PCR product and put it in 1.0% (w / v) agarose gel, and carry out electrophoresis analysis under 120 volts, the result is as follows: Figure 5 .

[0063] (3) Purificati...

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Abstract

The invention discloses a PCR-RFLP detection kit for authenticating and differentiating the infections of echinococcus multilocularis (Em) and echinococcosis shiquicus (Es). The kit comprises an Em and Es universal primer shown in SEQ ID NO. 1 and 2, restriction endonuclease EcoR I and/or Ssp I, a PCR reagent, and echinococcus multilocularis and echinococcosis shiquicus DNA template reference substances. The purpose of authenticating and differentiating the infections of echinococcus multilocularis and echinococcosis shiquicus can be achieved by extracting the DNA of the single cyst of the sample to be detected as a template, carrying out PCR amplification by using the universal primer, purifying a PCR product, carrying out enzyme digestion by using EcoR I or Ssp I incision enzyme, and then carrying out electrophoresis on an enzyme digestion product. According to the kit disclosed by the invention, the time consumption and money consumption due to sequencing, and the cumbersome process of data processing are omitted, a complex operation system is not required, and infection of echinococcus multilocularis or echinococcosis shiquicus to wild rodents can be rapidly, conveniently and economically differentiated and authenticated.

Description

technical field [0001] The invention relates to the detection of parasites, in particular to a PCR-RFLP detection kit for identifying and distinguishing the infection of Echinococcus multilocularis and Echinococcus stonecanalis. Background technique [0002] Echinococcus multilocularis (Em) larvae and Echinococcus shiquicus (Es) larvae can both parasitize rodents (such as plateau pikas, Qinghai voles, etc.), and both have cystic shapes They are very similar, and they were considered to be the larvae of Echinococcus multilocularis for a long time. It was not until the adult Echinococcus schizophrenia was found in the intestinal tract of the Tibetan fox (Vuipes ferrilata) that Echinococcus schizophrenia and Echinococcus multilocularis were clearly identified. Coccidioides were distinguished, and Echinococcus shiqu was identified as a new species of Echinococcus. The adult eggs of Echinococcus multilocularis infect humans and various rodents, causing multilocular echinococcosi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2521/301C12Q2565/125
Inventor 贾万忠范彦雷娄忠子李立闫鸿斌蔡进忠史万贵李建秋刘聪暖杨玉荣唐纳德·麦克麦纳斯
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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