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Primer, method and kit for detecting animal clonorchiasis sinensis specificity

The technology of Clonorchis sinensis and detection kit is applied in the field of genetic identification of Clonorchis sinensis in animals, can solve cumbersome problems and the like, and achieve the effects of simple operation, good detection effect, and objective result judgment.

Inactive Publication Date: 2009-11-25
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current methods that can extract DNA from feces either require cumbersome steps, such as immunomagnetic separation, gradient centrifugation, fluorescence-activated cell sorting, etc., or require the use of expensive DNA extraction kits

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 The specificity test of animal clonorchiasis specific primer

[0042] In this example, 11 negative control parasite samples that have been verified by DNA validity are selected: Schistosoma japonicum, Fasciola large, Fasciola hepatica, Orientobilax, Toxocara canis, Ancylostoma canis, Toxocara felis, Trichocephala pig Nematodes, Oesophagostoma nematodes, Ascaris suum and Opisthorchis sinensis.

[0043] The positive control of this embodiment is the positive DNA of Clonorchis sinensis.

[0044] With each 1 μ L of the DNA of the above-mentioned 11 control parasite samples as a template, the animal clonorchiasis specific primer of the present invention (the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2), specific PCR amplification was carried out according to the following PCR reaction conditions, and a blank control (ultrapure water) and a positive control were s...

Embodiment 2

[0051] The preparation of embodiment 2 kit

[0052] The test kit of the present embodiment is made up of following components:

[0053] a. DNA lysate 30ml: proteinase K containing 100mM NaCl, 10mM Tris-Cl pH8.0, 25mM EDTA pH8.0, 1% (W / V) SDS and 1.7μg / μL;

[0054] b.PCR reaction solution (100 reactions, 25 μ L / reaction), 2.5ml: contain animal clonorchiasis specific primer (the nucleotide sequence of upstream primer is shown in SEQ ID NO: 1, the nucleoside of downstream primer acid sequence as shown in SEQ ID NO: 2), the final concentration is 0.5pmol / μl; Tris-HCl (pH8.3) with a final concentration of 10mM, KCl with a final concentration of 50mM, dATP, dTTP, and dGTP with a final concentration of 200μM , dCTP, MgCl at a final concentration of 2.5mM 2 ;

[0055] c. Positive control substance: Clonorchis sinensis positive DNA 100μl;

[0056] d. Negative control substance: 100 μl of DNA-free ultrapure water;

[0057] e. Taq DNA polymerase, 5 U / μl, 12.5 μl.

Embodiment 3

[0058] The sensitivity test of embodiment 3 kit

[0059] The DNA concentration of representative adult samples of Clonorchis sinensis was measured with a nucleic acid protein analyzer, and then the DNA stock solution was diluted to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 and 10 -8 Eight dilutions. Take 1 μl of template DNA at each dilution, and use the kit prepared in Example 2. The total PCR reaction system is 25 μl, and the specific components are shown in Table 1. Pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, 61°C Refolding for 30 seconds, extension at 72°C for 30 seconds, 30 cycles; extension at 72°C for 5 minutes. At the same time, set a blank control to determine the lowest DNA concentration that can be detected by specific primers.

[0060] Table 1 PCR amplification system

[0061] Component

volume

PCR reaction solution

23.875μl

template DNA

1μl

Taq DNA polymerase (5U / μl)

0.1...

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Abstract

The present invention discloses a primer, a method and a kit for detecting the animal clonorchiasis sinensis specificity, a nucleotide sequence of an upstream primer of the primer is represented by SEQ ID NO:1, a nucleotide sequence of a downstream primer is represented by SEQ ID NO:2. the present invention implements an PCR amplification to a detectingformwork DNA by the primer, an amplifying outcome yield is processed by an agarose gel electrophoresis and observed under an ultraviolet light, if it is a positive result, there will be a specificity amplifying band, otherwise there will not be a band. According to an ITS zone sequence database OF THE animal clonorchiasis sinensis, the invention designs the primer, builds a rapid, special and sensitive PCR method, and it is capable of authenticating the animal clonorchiasis sinensis accurately. The operation of the kit of the invention is simple and programmable, the method specificity is strong, the sensibility is high, the result judgement is objective, and the invention is capable of being used for diagnosing the animal clonorchiasis sinensis and inquiring epidemiology.

Description

technical field [0001] The invention relates to the genetic identification of animal clonorchiasis, in particular to specific detection primers, detection methods and detection kits for animal clonorchiasis. Background technique [0002] Clonorchis sinensis is an important zoonotic parasitic disease caused by Clonorchis sinensis (Clonorchis sinensis) of the genus Clonorchis in the family Opisthorchiae parasitizing in dogs, cats, pigs and other animals and human bile ducts. Commonly known as liver fluke. [0003] Clonorchis sinensis parasitizes freshwater snails, freshwater fish, freshwater shrimp, dogs, pigs, cattle, mice, rabbits, guinea pigs, foxes, poultry and other animals. Humans are the final host. The disease can cause liver enlargement and other liver diseases Clinically, symptoms such as depression, loss of appetite, and diarrhea can be seen. Severe cases can cause liver cirrhosis or even cancer. A small number of children and adolescents can cause dwarfism. There ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 朱兴全林瑞庆唐剑栋宋慧群邓艳袁子国
Owner SOUTH CHINA AGRI UNIV
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