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Echinococcus granulosus detection kit and application thereof

A technology of Echinococcus granulosus and detection kit, which is applied in the biological field, can solve the problems of backward slaughter and quarantine technology, threat to consumers' health, and influence on the quality of meat products, so as to improve detection accuracy, save time and labor, reagents and consumables. , the effect of reducing the false positive rate

Active Publication Date: 2014-09-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current food safety and hygiene problems are very serious, but the slaughter and quarantine technology is quite backward, which can easily lead to missed inspections
This has seriously affected the quality of the meat, but also poses a huge threat to the health of consumers

Method used

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  • Echinococcus granulosus detection kit and application thereof
  • Echinococcus granulosus detection kit and application thereof
  • Echinococcus granulosus detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The invention provides a test kit for Echinococcus granulosus, which consists of sterile deionized water, PCR reaction solution, Taq DNA polymerase, GoldView DNA dye, bromophenol blue loading buffer, standard, and reference.

[0026] The PCR reaction solution includes PCR buffer, MgCl 2 , dNTPs, and detection primers.

[0027] The detection primers were 2 upstream primers and downstream primers for the detection of Echinococcus granulosus (Table 1):

[0028]

[0029] The standard substance is Echinococcus granulosus ( E. granulosus ) recombinant plasmid, its nucleotide sequence is shown in SEQ ID No: 3.

[0030] The reference substance is divided into a positive control substance and a negative control substance, the positive control is a DNA sample with DNA fragments of Echinococcus granulosus, and the negative control is a DNA sample without Echinococcus granulosus DNA.

[0031] This kit is stored at -20 ℃, and repeated freezing and thawing should be minimiz...

Embodiment 2

[0032] Example 2 PCR detection of Echinococcus granulosus standard

[0033] 1. Materials

[0034] Taq DNA polymerase reaction system, pMD18-T cloning system, Universal Genomic DNA Extraction Kit Ver. 3.0, and DNA molecular weight standard markers were all purchased from Treasure Bioengineering (Dalian) Co., Ltd. Plasmid mini-extraction reagents The kit and gel recovery kit were products of Hangzhou Aisijin Co., Ltd. Ampicillin and other commonly used reagents were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd. The PCR instrument was Biometra® T-gradient PCR instrument from Germany.

[0035] 2. Primer design and synthesis (Table 2):

[0036]

[0037] 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.

[0038] 3. Preparation of Assay Standards

[0039] The worms of Ech...

Embodiment 3

[0042] Example 3 PCR detection of Echinococcus granulosus positive samples

[0043] 1. Sample DNA extraction

[0044]9 grams of pig liver specimens positive for Echinococcus granulosus confirmed by traditional methods were collected, and DNA was extracted according to the instructions of the Universal Genomic DNA Extraction Kit, and the DNA concentration was adjusted to 60 ng / μl with sterile deionized water.

[0045] 2. Specimen testing

[0046] Take out the PCR reaction solution and melt it in an ice bath, take 22.8 μl, add 0.2 μl of DNA polymerase, and then add 2 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 pipetted and mixed with a micropipette, then centrifuged at a high speed for 2 s,...

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Abstract

The invention provides an Echinococcus granulosus detection kit which comprises sterile deionized water, a PCR reaction solution, Taq DNA polymerase, a GoldView DNA dye, a bromphenol blue loading buffer solution, a standard substance and a control substance, wherein the PCR reaction solution comprises a PCR buffer solution, MgCl2, dNTPs and primers for detection. The kit provided by the invention can quickly and accurately detect DNA of Echinococcus granulosus in a detected specimen, and can also be used for molecular epidemic research and curative effect monitoring of Echinococcus granulosus infection. The template DNA preparation step of the kit is simple and low in cost; and especially during large-scale detection, the kit is time-saving and labor-saving, saves a large number of reagents and consumable materials, and increases the work efficiency. The kit provides a detection method having high sensitivity and high specificity on the basis of molecular biology, improves the detection accuracy, reduces the false positive rate, and can used in detection of the foodborne parasite Echinococcus granulosus.

Description

technical field [0001] The invention belongs to biotechnology, and relates to a kit for detecting food-borne parasites such as Echinococcus granulosus, which can be used for identification and detection of specimens infected with Echinococcus granulosus. Background technique [0002] Echinococcosis, commonly known as hydatid disease, is caused by Echinococcus granulosus (Echinococcus granulosus) It is a disease caused by parasitic larvae in the liver, lung, brain, spleen and other organs of humans, cattle, sheep, pigs and other animals. [0003] Echinococcosis is distributed worldwide, causing global public health and economic problems, and has attracted widespread attention. Echinococcus granulosus can infect cattle, sheep, pigs, horses, camels and other livestock as well as a variety of wild animals and humans. The harm of Echinococcus granulosus to the animal body is mainly the mechanical oppression and toxin effect on the organs, resulting in organ atrophy and dysfunct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2531/113
Inventor 杜爱芳俞颖超卓洵辉阳毅敏
Owner ZHEJIANG UNIV
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