Multiple PCR detecting reagent case for diagnosis of oesophagostomiasis in pig

A detection kit and technology for nematode disease, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., to achieve simple operation, high sensitivity, and objective result judgment

Inactive Publication Date: 2008-07-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no convenient and specific detection kits have been developed yet.

Method used

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  • Multiple PCR detecting reagent case for diagnosis of oesophagostomiasis in pig
  • Multiple PCR detecting reagent case for diagnosis of oesophagostomiasis in pig

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Composition of the kit

[0057] The kit contains 30ml DNA lysis solution, which contains 100mM NaCl, pH8.0 10mM Tris-Cl, pH8.0 25mM EDTA, 1% (W / V) SDS and 1.7μg / μL proteinase K ; PCR reaction solution 100 reactions (25μL / reaction), final concentration of each 200μM dATP, dTTP, dGTP, dCTP, final concentration of each 0.5pmol / μL primers OdspF, OdspR2, OqspF and OqspR, final concentration of 3.5mM MgCl 2 , Taq enzyme 25μL (5U / μL); one for each positive control of dentate esophageal nematode DNA and one positive control of four-spinned esophageal nematode.

Embodiment 2

[0058] Example 2 Kit specificity test

[0059] Using 5 control samples of Ascaris suum, Trichinella spiralis, Trichinella, Clonorchis sinensis, and Schistosoma japonicum that have been validated by DNA validity, 1μL each of the DNA was used as a template to perform specific PCR amplification according to the reaction conditions of the kit. Blank control and kit positive control.

[0060] PCR amplification conditions: 94℃ pre-denaturation 5min

[0061]

[0062] Extension 5min after 72℃

[0063] After the PCR products were electrophoresed in a 1.2% TBE agarose gel, the results were observed under a UV transilluminator and photographed by a gel imaging system.

[0064] Results The kit has a DNA positive control of esophageal esophagus nematode and amplifies a band of about 130 bp. The positive control of four spines esophageal nematode DNA amplifies a band of about 330 bp. The mixed DNA of the two positive controls amplifies a band of about 130 bp and The band of 330b...

Embodiment 3

[0065] Example 3 Sensitivity test of the kit

[0066] First, the dentate esophageal mouth nematode sample and the four-spinned esophageal mouth sample nematode extract DNA, then dilute, vortex and mix, and detect the total DNA content according to the operating procedures of the Eppendorf Biophotometer nucleic acid protein analyzer. Dilute DNA by 5×, 10×, 20×, 50×, 100×, 200×, 400×. The PCR amplification conditions are the same as above, and a blank control is set. The PCR product was detected by 1.2% agarose gel electrophoresis to determine its sensitivity. The test results show that the PCR detection method is highly sensitive, and can detect 0.1ng DNA dentate esophagus nematodes and 0.12ng DNA four spines esophagus nematodes at the lowest.

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Abstract

The invention discloses a multiple PCR detection kit which can be used in the diagnosis of the swine esophagostomiasis. The detection kit comprises DNA lysis solution, PCR reaction solution, as well as the DNA positive control of toothed Oesophagostomum and the positive control of four-ratched Oesophagostomum. By using the ITS repeat DNA fragments of swine toothed Oesophagostomum and four-ratched Oesophagostomum as genetic markers, the invention builds up a fast, specific and sensitive multiple PCR methods which can be used for diagnosing swine esophagostomiasis. With simple and programmed operation and objective result judgments, the invention can be used for diagnosing swine esophagostomiasis, for investigating epidemiology, and for identifying and diagnosing toothed Oesophagostomum and four-ratched Oesophagostomum.

Description

Technical field [0001] The invention relates to the technical field of diagnosis and detection of swine esophagus nematode disease, and more specifically, to a PCR detection kit and a detection method for pig dentate esophagus nematodes and four spines esophagus nematodes. Background technique [0002] Esophageal mouth nematode disease is caused by a variety of nematodes of the esophagostomum genus (Oesophagostomum) parasitic in the intestines of animals. Because some species of larvae form nodular lesions in the intestinal wall, it is called nodule insects. It can be severely infected. Causes colitis. At present, this disease is widespread in cattle, sheep and pigs all over our country, and it is extremely widespread, causing great economic losses to animal husbandry production. Although esophageal mouth nematodes are usually only found in ruminants, pigs and monkeys, there have been reports of human infection, especially in certain areas of Africa, where the infection rate is v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04C12Q1/68
Inventor 朱兴全林瑞庆艾琳宋慧群
Owner SOUTH CHINA AGRI UNIV
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