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Kit for detecting porcine clonorchis sinensis and application thereof

A technology of Clonorchis sinensis and a kit, which is applied in the field of kits for detecting food-borne parasites of Clonorchis sinensis, can solve problems such as inability to realize on-site detection, difficulty in popularization and application, time-consuming and labor-intensive microscopic examination, etc. Achieve time-saving and labor-saving reagents and consumables, saving reagents and consumables, and high sensitivity

Active Publication Date: 2014-09-10
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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  • Kit for detecting porcine clonorchis sinensis and application thereof
  • Kit for detecting porcine clonorchis sinensis and application thereof
  • Kit for detecting porcine clonorchis sinensis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The object of the present invention is to provide a test kit for detecting Clonorchis sinensis, which comprises: sterile deionized water, fluorescent quantitative PCR reaction solution, standard substance and reference substance.

[0024] Among them, the fluorescent quantitative PCR reaction solution contains SYBR Green Real-time PCR Master Mix, ddH 2 O, primers for detection; wherein the primers for detection are 2 upstream primers and downstream primers for detecting Clonorchis sinensis (Table 1):

[0025]

[0026] The standard product is Clonorchis sinensis ( C. sinensis ) recombinant plasmid, its nucleotide sequence is shown in SEQ ID No: 3.

[0027] The reference substance is divided into a positive control substance and a negative control substance, the positive control is a DNA sample with a DNA fragment of Clonorchis sinensis, and the negative control is a DNA sample without a Clonorchis sinensis DNA.

[0028] This kit is stored at -20 ℃, and repeated free...

Embodiment 2

[0030] Example two Fluorescent quantitative PCR detection of Clonorchis sinensis standard substance

[0031] 1. Materials

[0032] The Universal Genomic DNA Extraction Kit (Universal Genomic DNA Extraction Kit Ver. 3.0) was a product of Treasure Bioengineering (Dalian) Co., Ltd., SYBR Green Real-time PCR Master Mix was purchased from TOYOBO, and the fluorescent quantitative PCR instrument was Applied Biosystems? 7500 Real- Real-time quantitative PCR instrument from Time PCR Systems.

[0033] 2. Primer design and synthesis (Table 2):

[0034]

[0035] Using the target gene sequence of the above insect species 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.

[0036] 3. Preparation of Assay Standards

[0037] The worm body of Clonorchis sinensis was taken, and the genomic DNA was extracted as a template according to the instructions of the general genom...

Embodiment 3

[0042] Example 3 Fluorescence quantitative PCR detection of positive samples of Clonorchis sinensis in swine

[0043] 1. Sample DNA extraction

[0044] Collect 10 grams of pig manure samples from pigs with clonorchiasis sinensis diagnosed by traditional methods and 10 grams of pig manure samples from healthy pigs without clonorchiasis suis, and extract DNA according to the instructions of the general genome DNA extraction kit. Adjust the DNA concentration to 1.5ng / μl with sterile deionized water.

[0045] 2. Sample testing

[0046] Take out the fluorescent quantitative PCR reaction solution and melt it in an ice bath, take 24 μl, and then add 1 μl of the sample DNA to be tested. In addition, PCR reaction tubes were set up at the same time, and the above reagents were added in sequence, but the DNA was replaced by standard substances, positive control substances, and negative control substances respectively, and three parallel replicates were required for each reaction tube...

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Abstract

The invention provides a kit for detecting porcine clonorchis sinensis. The kit consists of sterile deionized water, a fluorescent quantitative PCR (polymerase chain reaction) solution, a standard and a reference, wherein the fluorescent quantitative PCR solution contains SYBRGreenReal-timePCRMasterMix, ddH2O and detection primers. The kit has the advantages of strong specificity, high sensitivity, no pollution and high flux, and does not need electrophoresis so as to save the detection time. The kit can be used for quickly and accurately detecting the DNA of the porcine clonorchis sinensis in a detected sample, and can be used for molecular epidemiological investigation and curative effect monitoring of porcine clonorchis sinensis infection. The template DNA preparation step of the method is simple and low in cost, and the operation is simple, so that time and labor are saved, a large amount of reagents and consumables are saved, and the working efficiency is improved. The kit is applied in detection of the porcine clonorchis sinensis serving as a food-borne parasite.

Description

technical field [0001] The invention belongs to biotechnology, and relates to a kit for detecting clonorchis sinensis food-borne parasites, which can be used to identify and detect samples infected with clonorchis sinensis. 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. Clonorchiasis is caused by Clonorchis sinensis ( Clonorchis sinensis ) is a zoonotic parasitic disease caused by 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...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2561/113C12Q2563/107C12Q2545/113
Inventor 杜爱芳潘辰杨怡张红丽
Owner ZHEJIANG UNIV
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