Method for discriminating overwintering-recovery period boundary of overwintering cyanobacteria

A recovery period and boundary technology, which is applied in the field of distinguishing the boundary of overwintering cyanobacteria overwintering recovery period, can solve the problems of pseudovacuum easy to collapse, etc., and achieve good repeatability, repeatability and accuracy

Inactive Publication Date: 2015-03-04
NANJING UNIV OF TECH
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  • Method for discriminating overwintering-recovery period boundary of overwintering cyanobacteria
  • Method for discriminating overwintering-recovery period boundary of overwintering cyanobacteria
  • Method for discriminating overwintering-recovery period boundary of overwintering cyanobacteria

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Embodiment 1

[0038] 1. Sample Collection and RNA Extraction

[0039] From November 2012 to July 2013, water samples from Meiliang Bay (N2) in Taihu Lake were collected monthly. At the sampling point, 100ml of water samples were immediately filtered with GF / C filter membranes, quickly stored in liquid nitrogen, and brought back to the laboratory for storage. Store at -70°C until RNA extraction. Take 40 mL of Microcystis grown indoors in logarithmic growth phase (the medium for indoor cultured Microcystis is BG-11, the culture temperature is 30 °C, and the light intensity is 30 μE·(m 2 s) -1 , photoperiod of 12 h:12 h), the algal cells were collected by centrifugation, and then RNA was extracted. RNA was extracted from field samples and indoor samples using RNeasy Plant Mini Kit from QIAGEN. The field samples and indoor samples were transferred to the corresponding numbered lysis tube B (Lysing MatrixB, diameter 0.1 mm), and then 500 μL Buffer RLT was added to each lysis tube B, and the l...

Embodiment 2

[0046] 1. Sample Collection and RNA Extraction

[0047] From November 2012 to May 2013, water samples from the center of Taihu Lake (S2) were collected every month. At the sampling point, 100 mL of water samples were immediately filtered with GF / C membranes, quickly stored in liquid nitrogen, and brought back to the laboratory for storage. Store at -70°C until RNA extraction. Take 40 mL of Microcystis grown indoors in logarithmic growth phase (the medium for indoor cultured Microcystis is BG-11, the culture temperature is 30 °C, and the light intensity is 30 μE·(m 2 s) -1 , and the photoperiod is 12h-12h), the algal cells were collected by centrifugation, and then RNA was extracted. The extraction of RNA from field samples and indoor samples was performed using RNeasy Plant Mini Kit from QIAGEN. The field samples and indoor algae were transferred to the corresponding numbered lysis tube B (Lysing MatrixB, diameter 0.1 mm), and then 500 μL Buffer RLT was added to each lysis ...

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Abstract

The invention discloses a method for discriminating an overwintering-recovery period boundary of overwintering cyanobacteria. The method comprises the following steps of carrying out RNA extraction and purification on a water sample from a cyanobacteria bloom region and indoor cultured microcystis; carrying out reverse transcription to obtain cDNA; with the cDNA as a template, carrying out real-time fluorescence quantitative PCR amplification; amplifying genes of microcystis gas vesicles by virtue of a primer gvpC; specifically amplifying microcystis 16srRNA reference genes by virtue of a primer Micr; after the PCR is completed, exporting correspondingly threshold cycle values of which the difference values are taken as corrected cell copy numbers, wherein the difference between the cell copy number of the wild sample and the cell copy number of the indoor sample is taken as a relative cycle number of the wild target genes and marked as deltadelta Ct; calculating the relative expression level by virtue of 2-deltadelta Ct, plotting a curve with time as an abscissa and the values of 2-deltadelta Ct as an ordinate and determining the overwintering-recovery period boundary of overwintering cyanobacteria according to the curve. By the method, a plurality of samples can be simultaneously analyzed and the method has the advantages of good repeatability and accuracy and short detection time.

Description

technical field [0001] The invention relates to a method for judging the limit of overwintering recovery period of overwintering cyanobacteria, belonging to the technical field of water environment treatment. Background technique [0002] In recent decades, the eutrophication of lakes in my country has developed rapidly. With the aggravation of eutrophication in large shallow lakes (such as Taihu Lake), Microcystis aeruginosa ( Microcystis aeruginosa ) is the bloom of the dominant species. Algae blooms mostly occur in summer and have obvious seasonal characteristics. According to the basic theory of ecology and the in-situ detection of the bloom formation process in the field, some researchers divided the formation of cyanobacterial blooms into four distinct but continuous processes: sinking and overwintering, recovery, biomass increase, Floats gather to form water blooms. This hypothesis provides a new idea of ​​"staged" governance for the control of cyanobacterial b...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/89
CPCC12Q1/689C12Q1/6851C12Q2600/158C12Q2531/113C12Q2545/113C12Q2561/113C12Q2563/107
Inventor 虞龙张金丽李玉燕于洋杜明勇
Owner NANJING UNIV OF TECH
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