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Method for detecting microcystos toxigenicity

A technology of microcystis and algae liquid, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., which can solve the problem of affecting the accuracy and reproducibility of PCR detection, and the inability to directly detect and decontaminate with on-site water bloom samples It is difficult to completely remove the agent and other problems, and achieve the effect of simple and easy operation, low requirements and short time required

Inactive Publication Date: 2003-07-09
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
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AI Technical Summary

Problems solved by technology

[0006] The disadvantage of this method is that it takes a long time to extract the total DNA of cells, the efficiency is low, and the sample needs a large amount, and the detergent used for DNA extraction is difficult to completely remove, which affects the accuracy and reproducibility of PCR detection. On the other hand, the reagents and equipment required for extracting total DNA are complex and cannot be used for direct detection of on-site water bloom samples, which limits its application range

Method used

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Experimental program
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Effect test

specific Embodiment approach 2

[0036] 1. Take the algae liquid containing 5 mg of Microcystis aeruginosa 937 (Microcystis aeruginosa 937), centrifuge at 4000 rpm for 5 minutes, and remove the supernatant;

[0037] 2. Add 5 ml of sterile deionized water, mix thoroughly, centrifuge at 6000 rpm for 5 minutes, and remove the supernatant;

[0038] 3. Repeat step 2 5 times, then add 5 ml of sterile deionized water, and mix well to obtain algae cell suspension;

[0039] 4. Synthesize two pairs of primers TOX1P / 1M and TOX2P / 2M according to the sequences of TOX1P / 1M and TOX2P / 2M. The sequences are as follows:

[0040] TOX1P: 5'-CGATTGTTACTGATACTCGCC-3'

[0041] TOX1M: 5'-TAAGCGGGCAGTTGCTGC-3'

[0042] TOX2P: 5'-GGAACAAGTTGCACAGAATCCGC-3'

[0043] TOX2M: 5'-CCAATCCCTATCTAACACAGTAACTCGG-3'

[0044] 5. Add the following reagents to the test tube: bovine serum albumin (BSA) 20 pmol, 2 microliters containing 1.5 mmol MgCl 2 10 times thermostable DNA polymerase reaction buffer, four kinds of single nucleotides (dNTPs) ...

specific Embodiment approach 3

[0048] 1. Take the algae liquid containing 3 mg of Microcystis aeruginosa 912 (Microcystis aeruginosa 912), centrifuge at 6000 rpm for 5 minutes, and remove the supernatant;

[0049] 2. Add 3 ml of sterile deionized water, mix thoroughly, centrifuge at 5000 rpm for 5 minutes, and remove the supernatant;

[0050] 3. Repeat step 2 4 times, then add 3 ml of sterile deionized water, and mix well to obtain algae cell suspension;

[0051] 4. Synthesize two pairs of primers TOX1P / 1M and TOX2P / 2M according to the sequences of TOX1P / 1M and TOX2P / 2M. The sequences are as follows:

[0052] TOX1P: 5'-CGATTGTTACTGATACTCGCC-3'

[0053] TOX1M: 5'-TAAGCGGGCAGTTGCTGC-3'

[0054] TOX2P: 5'-GGAACAAGTTGCACAGAATCCGC-3'

[0055] TOX2M: 5'-CCAATCCCTATCTAACACAGTAACTCGG-3'

[0056] 5. Add the following reagents to the test tube: bovine serum albumin (BSA) 20 pmol, 2 microliters containing 1.5 mmol MgCl 2 10 times thermostable DNA polymerase reaction buffer, four kinds of single nucleotides (dNTPs...

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Abstract

The present invention discloses a method for detecting toxinogeny of microcyst alga. According to the principle of that the microcyst alga toxic synthetase gene mcy B only is existed in toxigenic microcyst alga said invention uses mcyB sequence to design out specific primer TOX1P / 1M and TOX2P / 2M, and utilizes complete cell PCR, preparation of sample, synthesis of primer, PCR reaction and electrophoretic detection to define that the microcyst alga is toxigenic or not. Said invention is short in detection time, good in reproducibility and high in sensibility and accuracy, can be used for massively and quickly identifying microcyst algae in the culture or natural water sample which are toxigenic or not.

Description

Technical field: [0001] The invention relates to Microcystis in cyanobacteria, and more specifically relates to a method for detecting whether Microcystis produces toxin. Background technique: [0002] Due to the increasing eutrophication of water bodies in rivers and lakes, frequent algae blooms in water bodies have become a serious problem of water environmental pollution. Cyanobacteria bloom not only affects the ecological function of the water body, but also affects the tourism value of the landscape water body. What's more serious is that the toxins produced by some cyanobacteria blooms will directly or indirectly affect human health, and long-term drinking of water sources containing cyanotoxins may induce the risk of liver cancer. [0003] The vast majority of cyanobacterial blooms that occur in lakes and other water bodies in China are Microcystis blooms. Among Microcystis species, some species produce microcystins and others do not. Even within the same species, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04
Inventor 宋立荣潘卉翟超甘南琴刘永定
Owner INST OF AQUATIC LIFE ACAD SINICA
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