RFQ-PCR detecting method for skeletonema costatum cytochrome b gene
An RFQ-PCR and cytochrome technology, applied in the detection field of planktonic diatoms, can solve the problem that the basic physiological and ecological parameters such as the growth rate of planktonic algae cannot be measured on site, and achieve high reliability of the standard curve, accurate and stable detection results. good effect
Inactive Publication Date: 2010-06-16
OCEAN UNIV OF CHINA
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Problems solved by technology
[0008] The technical problem to be solved by the present invention is to provide a RFQ-PCR detection method for Scleracea costa cytochrome b gene. Housekeeping genes to study the changes of phytoplankton populations, construct ecological dynamic models and even red tide prediction, etc., to overcome the defects in the prior art that basic physiological and ecological parameters such as the growth rate of a single species of phytoplankton cannot be measured
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[0039] In the embodiment of the present invention, the culture process of Skeletonemacostatum is: Skeletonemacostatum (Skeletonemacostatum) is cultivated to logarithmic growth phase with F / 2 medium in the laboratory, culture conditions: light / dark cycle is 12h / 12h , the light intensity is 4000Lux, and the culture temperature is 22-25°C.
[0040] According to the Cytochrome B gene sequence of Thalassiosira pseudonana obtained from the GenBank database, the primers for Scutella costa were designed as follows: TpCbfp5-ATA CGC TTG GAG TTT TGG GTC TTC T -3, TpCbrp5-ATGAGC CGG AGT TGA CAT AGG ATC T-3.
[0041] Collect 5 parts of Skeletalum costanum (cell density is 4.1×107 cells / mL) cultured for one week about 3.8×10 7 cells (the weight of the algae concentrate is about 150mg). Use the following RNA extraction methods to extract:
[0042] (1) Collect algae cells and grind them into fine powder with a mortar under the condition of continuously adding liquid nitrogen;
[0043] (2)...
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Abstract
The present invention discloses a RFQ-PCR detection method of skeletonema costatum cytochrome b gene. Said method includes the following steps: (a), extracting skeletonema costatum RNA as standard sample; (b), separating and cloning skeletonema costatum cytochrome b gene to obtain skeletonema costatum cytochrome b gene sequence, and obtaining cytochrome b sequences of several algal seeds which areapproaching to the skeletonema costatum in status from GenBank, and constructing system tree; (c), making RFQ-PCR; and (d) utilizing obtained skeletonema costatum cytochrome b gene sequence with 700-740bp to construct standard curve and utilizing said standard curve to detect the skeletonema costatum cytochrome b gene to be detected.
Description
technical field [0001] The invention relates to a method for detecting planktonic diatoms, in particular to an RFQ-PCR method for detecting cytochrome b gene of Sclerostetes costa. Background technique [0002] S. costatum is a long chain of multiple cells, the cells are lens-shaped cylindrical cells with a diameter of 6-7 microns, 8-30 spines on the shell margin, and the intercellular space is generally larger than the length of the cells. Chromosomes are usually 2, located on the shell surface. The nucleus is in the center. Scutellaria mesocostalis is a typical representative of wide temperature and wide salt. It is widely distributed, from the arctic to the equator, from high salt water in open seas to coastal low salt water masses, and even in brackish water, but it is most common in the coast. Individual ecology studies have indicated that S. costaris has a high growth rate and nitrate uptake rate, so it is better adapted to high temperature and low salt environments ...
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IPC IPC(8): C12Q1/68
Inventor 甄毓何闪英孙静赵丽媛于志刚米铁柱
Owner OCEAN UNIV OF CHINA