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Multiple PCR detection method of G. spinigerum, G. nipponicum and G. doloresi and primer for detection

A technique for echinostomum and gnathostomum, applied to the multiplex PCR detection of gnathostomum japonica and gnathostomum donovani, primers for gnathostomum japonica, primers for gnathostomum dunovani, and simultaneous detection of gnathostomum, capable of Solve the problems of heavy workload, time-consuming and high-cost identification of gnathostomy nematodes, and achieve the effects of short time-consuming, reduced manpower consumption and low cost

Inactive Publication Date: 2013-05-01
SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF P R C
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It aims to solve the problems of time-consuming, high cost, and heavy workload in the identification of gnathostomum, and also lays the foundation for further simultaneous identification of all 13 gnathostomy species

Method used

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  • Multiple PCR detection method of G. spinigerum, G. nipponicum and G. doloresi and primer for detection
  • Multiple PCR detection method of G. spinigerum, G. nipponicum and G. doloresi and primer for detection
  • Multiple PCR detection method of G. spinigerum, G. nipponicum and G. doloresi and primer for detection

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Embodiment

[0045] 1. First extract DNA from Echinostomum, Nematode japonica, and Nematode duninii

[0046] The DNA extraction method describes the DNA extraction method with the DNA extraction kit (DNeasy Blood & Tissue kit) of QIAGEN Company: pick three kinds of single known worm bodies (the sources of these three strains of worms: G. spinigerum (Yizhou strain), G. doloresi (Fujian strain) was donated by Zhang Hongman, Chief Physician of Guangxi Epidemic Prevention and Control Center, G. nipponicum (Guangzhou strain) was isolated from loach by Shanghai Entry-Exit Inspection and Quarantine and identified by morphology and molecular biology, and stored in 70% ethanol) Put it into a sterilized centrifuge tube, add sterilized distilled water, repeat washing 3 times, discard the distilled water in the centrifuge tube after washing, crush the worms in the centrifuge tube with a grinding rod, add 180 μl Buffer ATL, 20 μL proteinase K, mix well Then incubate at 56°C for 1h to 3h until the diges...

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Abstract

The invention discloses a multiple PCR detection method of G. spinigerum, G. nipponicum and G. doloresi. The method comprises the following steps: 1, extracting DNA from Gnathostomas, and simultaneously designing specific primers of G. spinigerum, G. nipponicum and G. doloresi; 2, optimizing PCR reaction conditions of the designed primers to determine an optimal multiple PCR mode; and 3, experimenting according to the optimal multiple PCR mode, verifying the single PCR specificity, the multiple PCR specificity, the PCR sensitivity and the clinical sample operation feasibility of each primer. The invention also discloses a primer sequence for simultaneously detecting G. spinigerum, G. nipponicum and G. doloresi. Various verifications show that G. spinigerum, G. nipponicum and G. doloresi can be simultaneously identified through the method of the invention; and the method which has the characteristics of rapidness, sensitivity, specificity and synchronous identification of the three Gnathostomas can be applied to the identification and the quarantine of Gnathostomas of aquatic products.

Description

technical field [0001] The invention relates to the technical field of agriculture and animal quarantine, in particular to a detection method for Echinostoma japonicum and Gillostomum donovani, in particular to Echinostoma japonicum and Gillostomum donovani Multiplex PCR detection method. In addition, the present invention also relates to primers for simultaneous detection of Echinostomum, Nematode japonicum and Nematode duniani. Background technique [0002] Gnathostoma larvae are zoonotic parasitic diseases caused by human consumption of raw or undercooked freshwater fish containing larvae of the genus Gnathostoma. There are 13 effective species in the genus G. gnathostomum. The pathogenic species that have been reported so far include G. spinigerum, G. hispidum, G. doloresi, Japan G. nipponicum, G. malaysiae and G. binucleatum, therefore, the identification of G. nipponicum larvae species detected in food is critical to the disease. Diagnosis and prevention are of grea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 李树清李雯雯张鸿满陈韶红黄维义陈志飞张子群王巧全张强王艳李健
Owner SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF P R C