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PCR amplification primer for detecting and identifying northern pacific sea area ommatostrephes species by using environmental DNA as well as detection method and application thereof

A technology for amplification primers and detection methods, which is applied in the field of PCR amplification primers and detection methods, can solve the problems of ecological system damage, time-consuming and labor-intensive fishing operations, etc., and achieve the effect of less destructive and high accuracy

Pending Publication Date: 2020-07-24
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the detection of squid mainly relies on traditional fishing methods, but fishing operations are time-consuming and laborious, and at the same time cause serious damage to the entire ecosystem

Method used

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  • PCR amplification primer for detecting and identifying northern pacific sea area ommatostrephes species by using environmental DNA as well as detection method and application thereof
  • PCR amplification primer for detecting and identifying northern pacific sea area ommatostrephes species by using environmental DNA as well as detection method and application thereof
  • PCR amplification primer for detecting and identifying northern pacific sea area ommatostrephes species by using environmental DNA as well as detection method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0028] The detection of the validity of the PCR amplification primers of soft fish detection of the present embodiment comprises the following steps:

[0029] (1) Use DNeasy Blood and Tissue kits (Qiagen) kit to extract DNA from the muscle tissue of squid and its two closely related species, Pacific pleated squid and squid. After measuring the DNA concentration, dilute the obtained DNA to 100pg / μL for use. Amplified by PCR.

[0030] (2) The DNA diluted in step (1) is used for PCR amplification. The PCR reaction system is: 2 μL of DNA template, 15 μL of PCR mixture, 1 μL of 10 μM primers (OMBA-F, OMBA-R) each, and 20 μL of ultrapure water. 50°C, 2min→95°C, 10min→(95°C, 15s→60°C, 1min)×55 cycles.

[0031] (3) the PCR product of step (2) is detected by 2% agarose gel electrophoresis (results such as figure 1 Shown), a band of about 155 bp can be observed in the soft fish tissue samples, but no bands are observed in the samples of closely related species. The amplified fragmen...

Embodiment 2

[0033] The method for detecting squid in North Pacific waters using environmental DNA comprises the following steps:

[0034] (1) Use a water collector to collect 2L of surface water samples, vacuum filter them with a nitrocellulose filter membrane with a diameter of 47 mm and a pore size of 0.4 μm, fold the filtered filter paper in half, wrap it with tin foil, and record the information of the sample , -20°C cryopreservation.

[0035] Precautions for the above steps: a. Put on disposable gloves before collecting water samples, and then wash the sampled bottle 3 times (use the water from the sampling place). Gloves must be replaced at each sampling point; b. After collecting the water sample, add a reagent with a final concentration of 0.01% benzalkonium chloride to prevent DNA degradation; c. Mix the reagent and the collected water sample evenly, shake it up and down about 5 times, and at the same time Keep samples out of direct sunlight as much as possible.

[0036] (2) Bi...

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Abstract

The invention provides a PCR amplification primer for detecting and identifying northern pacific sea area ommatostrephes species by using environmental DNA as well as detection method and applicationthereof. In the PCR amplification primer, the sequence of an upstream primer OMBA-F is 5 '-CGAAGGTTAATCTGTCTCCATCT-3', and the sequence of a downstream primer OMBA-R is 5 '-CCCAATTAAAATTTATATACCACCT-3'. Wherein the DNA molecular marker for detecting the ommatostrephes species is located in the 16S gene of the ommatostrephes; through comparing the environmental DNA detection result with the DNA molecular marker site sequence, the ommatostrephes species can be determined if the comparison result is 100% matching; according to the detection method disclosed by the invention, the detection of thesquid species is realized by only collecting the water sample from the water body, and through DNA sequence comparison, the detection method causes little damage to an ecological system, and the obtained result has the characteristic of high accuracy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a PCR amplification primer for environmental DNA detection and identification of squid species in the North Pacific sea area, a detection method and an application thereof. Background technique [0002] Soft fish (Ommastrephes bartrami) belongs to cephalopods, and its life cycle is 1 year. Its population is widely distributed in the North Pacific Ocean and is one of the important oceanic economic fish species in the world. Squid is mainly divided into two breeding groups: autumn and winter and spring, and the western group of winter and spring is the main fishing target in my country. At present, the detection of squid mainly relies on traditional fishing methods. However, fishing operations are time-consuming and laborious, and at the same time cause serious damage to the entire ecosystem. In multicellular organisms, for example, metabolic waste, damaged tissue or exfo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6806C12N15/11
CPCC12Q1/6888C12Q1/6806C12Q2531/113C12Q2565/125C12Q1/6853C12Q1/686
Inventor 陈新军钨倩倩刘洋刘必林王丛丛
Owner SHANGHAI OCEAN UNIV
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