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PCR kit for detecting clonorchis sinensis in pigs based on mitochondrial gene nad2 and application

A Clonorchis sinensis and mitochondrial gene technology, applied in the field of PCR kits, can solve the problems of time-consuming, labor-intensive, low-efficiency, and high false-negative rates of microscopic examination, and achieve time-saving, labor-saving reagents and consumables, saving reagents and consumables, and improving The effect of work efficiency

Inactive Publication Date: 2019-04-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Microscopic examination is time-consuming, labor-intensive, low-efficiency, and low-sensitivity
The operation process of the SPA method is cumbersome, and the ELISA is very sensitive, but the false negative rate is high
None of the above three methods can realize on-site detection, and it is not easy to popularize and apply

Method used

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  • PCR kit for detecting clonorchis sinensis in pigs based on mitochondrial gene nad2 and application
  • PCR kit for detecting clonorchis sinensis in pigs based on mitochondrial gene nad2 and application
  • PCR kit for detecting clonorchis sinensis in pigs based on mitochondrial gene nad2 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] The PCR detection of embodiment 1 Clonorchis sinensis standard substance

[0019] 1. Materials

[0020] Taq DNA polymerase reaction system, pMD18-T cloning system, Universal Genomic DNA Extraction Kit (Universal Genomic DNA Extraction Kit Ver.3.0), 250bp DNA molecular weight standard markers were all purchased from Treasure Bioengineering (Dalian) Co., Ltd., and a small amount of plasmid was extracted The kits and gel recovery kits were purchased from Hangzhou Aisijin Co., Ltd. Commonly used reagents such as ampicillin were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd. The PCR instrument was purchased from Germany T-gradient PCR instrument.

[0021] 2. Primer design and synthesis

[0022]

[0023] Using the above target gene sequence as a template, Primer Premier 5.0 software was used to analyze and design primers, which were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0024] 3. Preparation of detectio...

Embodiment 2

[0028] The PCR detection of embodiment two clonorchis sinensis positive sample

[0029] 1. Sample DNA extraction

[0030] Collect respectively 9 grams of pig manure samples of 1 case of clonorchiasis swine confirmed by traditional methods and 9 grams of pig manure samples of 5 healthy pigs without clonorchiasis suis, and extract DNA according to the instructions of the general genome DNA extraction kit. Adjust the DNA concentration to 27ng / μl with sterile deionized water.

[0031] 2. Sample detection

[0032] Take out the PCR reaction solution and melt it in an ice bath, take 21.75 μl, add 0.25 μl of DNA polymerase, and then add 3 μl of DNA from the sample to be tested. In addition, three more PCR reaction tubes were set up at the same time, and the above reagents were added in sequence, but the DNA was replaced by the standard substance, the positive control substance, and the negative control substance respectively. The prepared PCR reaction reagent tube was blown and mix...

Embodiment 3

[0035] Application of Example 3 Clonorchis Sinensis Detection Kit

[0036] The method is the same as in Example 2, randomly collect pig manure samples, extract DNA, and amplify by PCR. For the results, see image 3 : The rightmost two lanes "+, -" are positive control and negative control respectively; lane 15 is positive for Clonorchis sinensis; other lanes are negative.

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Abstract

The invention discloses a PCR kit for detecting clonorchis sinensis in pigs based on a mitochondrial gene nad2. The kit comprises sterile deionized water, PCR reation liquid, Taq, DNA, polymerase, GoldView DNA dye, 6*Loading Buffer, a standard product and a reference product. The kit can quickly and accurately detect DNA of the clonorchis sinensis in pigs of a detected sample and can be applied toinvestigation of molecular epidemiology infected by the clonorchis sinensis in pigs and curative effect monitoring. Template DNA preparation is simple in steps, low in cost and simple in operation especially for large-scale detection of a large number of samples, saves time, labor, a large number of reagents and consumable materials, and improves the work efficiency. The kit can be applied to detection for the clonorchis sinensis in pigs. The application of the kit is a detection method of high sensitivity and high specificity based on the molecular biology.

Description

technical field [0001] The invention relates to biotechnology, and relates to a PCR kit for detecting Clonorchis sinensis based on the mitochondrial gene nad2, which can carry out differential detection on samples infected with Clonorchis swine. Background technique [0002] At present, the population mobility in our country is increasing, and more and more people eat raw seafood and game. Various animal-derived diseases have begun to spread in non-endemic areas, and food-borne parasitic diseases have become a huge threat to public health. threaten. Clonorchis sinensis is a zoonotic parasitic disease caused by Clonorchis sinensis parasitizing in the liver bile duct and gallbladder of humans and animals. The terminal host of Clonorchis sinensis is human and various mammals, and there are two intermediate hosts, the first intermediate host is a freshwater snail, and the second intermediate host is a variety of freshwater fishes and freshwater shrimps. Clonorchiasis is very p...

Claims

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Application Information

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IPC IPC(8): C12Q1/6893C12Q1/686C12N15/11
CPCC12Q1/6893C12Q1/686
Inventor 杨怡杜爱芳郭筱璐潘辰孙洪超陈学秋阳毅敏
Owner ZHEJIANG UNIV
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