Kit for detecting cysticercus cellulosae, swine trichinella and swine toxoplasmosis and application

A technology for Trichinella spiralis and Toxoplasma gondii, which is applied to the kits for three food-borne parasites of Toxoplasma gondii, to detect cysticercosis and Trichinella pigs, and can solve the problems of backward slaughtering and quarantine technology, threats to consumers’ health, and problems affecting meat quality. Improve the accuracy of detection, save time and effort on reagents and consumables, and reduce the false positive rate

Active Publication Date: 2012-01-04
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the problem of food safety and sanitation is very serious, but the slaughtering and quarantine technology is very backward
Some items that must be inspected in slaughter and quarantine, such as trichinella and cysticerci, are only diagnosed by naked eye observation, which can easily cause missed inspections, seriously affect the quality of meat, and pose a huge threat to the health of consumers

Method used

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  • Kit for detecting cysticercus cellulosae, swine trichinella and swine toxoplasmosis and application
  • Kit for detecting cysticercus cellulosae, swine trichinella and swine toxoplasmosis and application
  • Kit for detecting cysticercus cellulosae, swine trichinella and swine toxoplasmosis and application

Examples

Experimental program
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Effect test

Embodiment 1

[0024] The kit of the present invention comprises: sterile deionized water, PCR reaction solution, Taq DNA polymerase, GoldView DNA dye, bromophenol blue loading buffer, standards, controls.

[0025] Wherein, the PCR reaction solution contains PCR buffer, MgCl 2 , dNTPs, primers for detection.

[0026] The detection primers were 6 upstream primers and downstream primers for the detection of 3 parasites (Table 1):

[0027]

[0028] The standard sequence is Cysticercus porcine ( T. solium ) has the nucleotide sequence of SEQ ID No: 7, Trichinella pigs ( T. spiralis ) has the nucleotide sequence of SEQ ID No: 8, Toxoplasma gondii ( T. gondii ) has a nucleotide sequence of SEQ ID No: 9, see the sequence listing for the specific sequence.

[0029] 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 Cysticercus suis, Trichinella pigs and Toxoplasma gondii, and t...

Embodiment 2

[0031] Example 2 Multiplex PCR Detection of Three Parasite Standards of Cysticercus porcine, Trichinella pigs, and Toxoplasma gondii

[0032] 1. Materials

[0033] 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.

[0034] 2. Primer design and synthesis (Table 2)

[0035]

[0036] 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.

[0...

Embodiment 3

[0041] Example 3 Multiplex PCR detection of three parasite-positive samples of cysticercus porcine, trichinella pigs, and toxoplasma gondii

[0042] 1. Sample DNA extraction

[0043] Collect 9 grams of pig diaphragm specimens from 4 cases of pig cysticercus, Trichinella pigs, and toxoplasmosis pigs diagnosed by traditional methods, and 2 healthy pig diaphragm samples without the three pathogens of cysticercus pigs, Trichinella pigs, and Toxoplasma gondii. 9 g, extract DNA according to the instructions of the Universal Genomic DNA Extraction Kit, and adjust the DNA concentration to 60 ng / μl with sterile deionized water.

[0044] 2. Specimen testing

[0045] Take out the PCR reaction solution and melt it in an ice bath, take 21.87 μl, add 0.13 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...

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Abstract

The invention provides a kit for detecting cysticercus cellulosae, swine trichinella and swine toxoplasmosis. The kit comprises sterile deionized water, polymerase chain reaction (PCR) liquid, thermus aquaticus deoxyribonucleic acid (Taq DNA) polymerase, a GoldView DNA dye, bromophenol blue sample loading buffer solution, a standard substance and a reference substance. In the detection kit provided by the invention, DNA of the cysticercus cellulosae, the swine trichinella and the swine toxoplasmosis in a specimen to be detected can be detected quickly and accurately only by one reaction, and the kit can also be used for the molecular epidemiological survey and curative effect monitoring of cysticercus cellulosae, swine trichinella and swine toxoplasmosis infection. The method has simple preparation steps and is low in cost, easy to operate, time-saving and labor-saving, a large number of reagents and consumables are saved, and the work efficiency is improved. The method is high in detection sensibility, specificity and accuracy, and the false positive rate is reduced.

Description

technical field [0001] The invention relates to biotechnology, in particular to a kit for detecting three kinds of food-borne parasites including Cysticercus suis, Trichinella pigs and Toxoplasma gondii. Specimens of Toxoplasma gondii were tested for differential diagnosis. Background technique [0002] At present, more and more people keep pets in our country, the mobility of the crowd 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 parasitism Insect diseases have constituted a huge threat to public health. Trichinosis is caused by Trichinella spiralis ( Trichinella spiralis ) is a disease caused by parasitic adult worms in the intestinal tract of mammals and parasitic larvae in muscle tissue, which is a zoonotic disease that seriously endangers public health safety. Trichinella can infect most mammals. Pigs have the highest prevalence among domestic animals except...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 杜爱芳张德福周前进王素华
Owner ZHEJIANG UNIV
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