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Anisakis body allergen PCR detection technology

Anisakis and detection technology, applied in the biological field, can solve the problems of difficult to diagnose allergic symptoms, and achieve the effect of improving specificity and sensitivity

Inactive Publication Date: 2015-09-30
舟山出入境检验检疫局综合技术服务中心
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Problems solved by technology

[0007] The purpose of the present invention is to provide a high-quality method for detecting the components of anisakis in order to solve the existing defects that the allergic symptoms caused by the components of anisakis are difficult to diagnose, which can reduce the risk of anisakis passing to humans. Risk PCR detection technique for anisakis allergen

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  • Anisakis body allergen PCR detection technology
  • Anisakis body allergen PCR detection technology

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Embodiment

[0033] A PCR detection technique for allergens of anisakis worms, comprising the steps of:

[0034] a) DNA extraction of Anisakis; DNA extraction is to wash the Anisakis preserved in glycerol with distilled water and apply Magnetic DNA Purification System for Food kit for DNA extraction;

[0035] b) Primer design:

[0036] Upstream primer ANISAKIS COI F: 5'-GGKCYATTAAYTYTATRACWACTAC-3' (SEQ ID NO.1)

[0037] Downstream primer ANISAKIS COI R5'—AAAGAWGTATTMARRTTACGRTCVG—3' (SEQ ID NO.2)

[0038] The annealing temperature is 54.5°C, and the expected amplified fragment size is 178bp;

[0039] c) Perform PCR amplification on the primers in step b), electrophoresis on agarose gel, and observe the results; the amount of each reagent in the PCR reaction system is: 10×PCR buffer 2 μL, dNTP 1.6 μL, upstream primer 0.4 μL, downstream primer 0.4 μL μL, DNA template 1 μL, ddH 2 O 14 μL, Taq enzyme 0.1 μL, total volume 20 μL;

[0040] Amplification program: 94°C for 5min→94°C for 30s...

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Abstract

The invention relates to an anisakis body allergen PCR detection technology. Specific primers are designed according to anisakis mitochondrial COI sequence published on GenBank. PCR specific amplification is carried out on a nematode sample. Results show that the size of an amplified target fragment is 178bp. The PCR detection system has high specificity, and no band can be amplified from other non-anisakis and fish meat DNA in a fish body. Sensitivity is high, and the minimum detected DNA content is 0.36pg. With successful establishment of the detection system, powerful technical supports are provided for detection, identification and epidemiological investigation of anisakis simplex.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a PCR detection technology for allergens of Anisakis nematodes. Background technique [0002] Anisakis is the largest family of fish parasitic nematodes. Adults live in marine mammals, and larvae live in fish or molluscs. Humans eat food containing live larvae of some Anisakis. Causes anisakiasis. [0003] With the rise of eating fashion such as eating seafood and processed seafood and the development of fishery and tourism, Anisakis, as an important food-borne parasitic disease in seafood, poses a huge threat to people's life and health. Not only live anisakis worms can cause anisakiasis, lead to acute abdomen and allergy, trigger allergic reactions of urticaria, and severe cases can cause death. Because Anisakis worms contain many heat-resistant and pepsin-resistant allergens, dead worms treated with high temperature and gastric juice can also cause allergic reactions. Shimakura...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6888
Inventor 李孝军杨赛军周圆陈璐敏单长林沈飚
Owner 舟山出入境检验检疫局综合技术服务中心
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