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Method for rapidly detecting chain alexandrum by adopting LAMP (Loop-Mediated Isothermal Amplification) technology

A ring-mediated constant temperature and technology technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as the detection and identification of Alexandrium chains that have not yet been seen, and achieve low instrument requirements, strong specificity, The effect of high amplification efficiency

Inactive Publication Date: 2012-11-14
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no relevant literature report on the application of this technology to the detection and identification of Alexandrium catenella

Method used

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  • Method for rapidly detecting chain alexandrum by adopting LAMP (Loop-Mediated Isothermal Amplification) technology
  • Method for rapidly detecting chain alexandrum by adopting LAMP (Loop-Mediated Isothermal Amplification) technology
  • Method for rapidly detecting chain alexandrum by adopting LAMP (Loop-Mediated Isothermal Amplification) technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Design of Primers for LAMP Amplification of Alexandrium catensis

[0028] (1) Genomic DNA Extraction of Alexandrium catenella

[0029] Divide the Alexandrium catenella cell liquid cultured to the logarithmic phase into 2mL centrifuge tubes, centrifuge, pour off the supernatant, add 600μL extract (100mmol / L Tris-Hcl, 100mmol / L EDTA, 1.4mol / L NaCl, 2% CTAB, 2% β-mercaptoethanol), 65°C water bath for 1h. Add an equal volume of PCI extract solution (volume ratio, phenol: chloroform: isoamyl alcohol = 25: 24: 1), mix well, and centrifuge at 12500 rpm for 10 min at 4°C. Transfer the supernatant to a new centrifuge tube, add RNase to a final concentration of 1 μg / mL, and place at 37°C for 30 minutes. The PCI was extracted once more, and centrifuged at 12,500 rpm for 10 min at 4°C. Transfer the aqueous phase to a new centrifuge tube, add 2 times the volume of absolute ethanol, and place it at room temperature for 8-12 minutes to precipitate DNA. Centrifuge at 12500 rpm for ...

Embodiment 2

[0043] Alexandrium catenella and negative control experiments

[0044] (1) Genomic DNA extraction of red tide algae

[0045] Including the DNA extraction of 8 species of algae: Alexandrium catenella, Heterocapsa circularisquama, Heterosigma akashiwo, Amphidinium carterae, Lima original Dinoflagellates (Prorocentrum lima), Chaetoceros sp., Alexandrium minutum, Karenia mikimotoi.

[0046] Eight algae cultured to the logarithmic phase (Alexandrium catenella, Heterophyta annulus, Heterophyllum akashiwa, Anterior dichotoma carinii, Prorocentrum lima, Chaetoceros, Alexandrium microbiota, Michelia Lun algae) algae cell solution was divided into 2ml centrifuge tubes, centrifuged, poured off the supernatant, added 600μL extract (100mmol / L Tris-Hcl, 100mmol / L EDTA, 1.4mol / LNaCl, 2%CTAB, 2%β -mercaptoethanol), in a 65°C water bath for 1h. Add an equal volume of PCI extract (phenol: chloroform: isoamyl alcohol = 25:24:1), mix well, and centrifuge at 12500 rpm for 10 min at 4°C. Transf...

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PUM

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Abstract

The invention relates to a method for rapidly detecting chain alexandrum by adopting an LAMP (Loop-Mediated Isothermal Amplification) technology. The method comprises the following steps of: (1) extracting harmful algae gene group DNA (Deoxyribonucleic Acid); (2) forming four LAMP amplification primers: lz-f31: 5'-GTTTAATGCAAAGTAGCTTTCA-3', LZ-B31: 5'-CTTCAGCTTGTCCCAGTT-3', LZ-FIP1: 5'-CATCCAGGTTCAATGCAAAACAT-ATTTAATGCTGATTAGCATTGTTG-3' and LZ-BIP1: 5'-GTTTACAACCTAAACATGGTTTCGTGTGCACATTTACAAATATCAACC-3'; (3) forming an LAMP amplification reaction system; and (4) detecting an LAMP amplification product. The LAMP detection method of the invention has the advantages of simple and rapid operation, low requirement for instruments, strong specificity, high amplification efficiency, simple result judgment, and the like and is suitable for rapidly identifying and detecting the chain alexandrum.

Description

technical field [0001] The invention belongs to the field of DNA molecular detection of Alexandrium catenella, and in particular relates to a method for rapidly detecting Alexandrium catenella by a ring-mediated constant temperature amplification technique. Background technique [0002] In recent years, red tide hazards have occurred many times along our coast, which has caused huge economic losses to the fishery, and also caused serious impacts on the environment and ecological balance. [0003] Alexandrium catenella is a toxic red tide algae, which can produce paralytic shellfish poison (Paralytic Shellfish Poison) and other toxins, which are harmful to the survival, reproduction and development of phytoplankton, crustaceans, shellfish, fish, etc. Very adverse effects, consumption of fish and shellfish contaminated by this red tide algae will directly threaten human health. Alexandrium catenella is distributed in the north and south seas of my country, therefore, the stri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 蒋科技张凤英马春艳马凌波
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI