Molecular detection method for rapid quantification of diatom cell density

A molecular detection and diatom technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of relying on professional technicians and high-precision instruments, lack of specific quantitative detection methods, and heavy analysis tasks. , to achieve the effect of improving efficiency, simple and convenient operation method, accurate and objective data

Active Publication Date: 2019-03-12
INST OF AQUATIC LIFE ACAD SINICA
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The purpose of the present invention is to provide a molecular detection method for rapid quantitative diatom cell density. Heavy tasks, heavy reliance on professional technicians and sophisticated instruments, lack of specific quantitative detection methods, etc., based on the biological principles and working characteristics of fluorescent quantitative PCR, combined with the design of dia

Method used

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  • Molecular detection method for rapid quantification of diatom cell density
  • Molecular detection method for rapid quantification of diatom cell density
  • Molecular detection method for rapid quantification of diatom cell density

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Primer design and specificity verification:

[0028] A pair of specific primers rbc280-F / rbc280-R was designed for diatom rbcL gene:

[0029] rbc280-F: 5'-ACGTTTAGAAGATATGCGTATTC-3'

[0030] rbc280-R: 5'-GGTTAATACCTTCCATACAG-3'.

[0031] The primers rbc280-F / rbc280-R were used to verify the existing algae strains, and the results are shown in Table 1.

[0032] Table 1 Verification results of some algae strains

[0033]

[0034]

[0035] Note: "+" means positive detection, "-" means negative and not detected.

[0036] Table 1 uses specific primers to detect the existing algae strains cultivated in the laboratory, including 7 species of cyanobacteria, 14 species of green algae and 9 species of diatoms. From the test results, the detection rate of diatoms in this research method is 100%. , but not detected for cyanobacteria and green algae, showing the specificity of this primer.

[0037] figure 1 The method is used to detect field samples, and the detection ra...

Embodiment 2

[0039] A molecular detection method for rapidly quantifying diatom cell density, the steps of which are:

[0040] 1. Sample collection and DNA extraction:

[0041] In April 2018, water samples from the main channel of the South-to-North Water Transfer Project were collected for the quantification of diatoms, and there were 2 sampling points. In this example, 1 L of the water sample to be tested was collected according to the specific conditions of the algae biomass in the water body, and the water sample was filtered through a 0.22 μm cellulose acetate membrane, and the filtered sample was subjected to extraction and purification of total genomic DNA. The extracted DNA was dissolved in sterile double distilled water or deionized water, and stored at -20°C for later use.

[0042] 2. Fluorescence quantitative PCR

[0043] Specific diatom rbcL gene primers were used to amplify and quantify by fluorescent quantitative PCR. The target product size was 280 bp. After the reaction w...

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Abstract

The invention discloses a molecular detection method for rapid quantification of diatom cell density. The method comprises the following steps: 1, filtering a water sample to be tested by using a membrane, then extracting genomic DNA of the sample, and purifying; 2, carrying out fluorescence quantitative polymerase chain reaction (PCR) amplification on a rbcL gene of diatom by using a specific primer, and recording a Ct value; 3, taking the single cultured or pure cultured diatom with known cell density as a standard algae strain, extracting genomic DNA, carrying out gradient dilution and thencarrying out quantitative PCR amplification by using the diluted product as a standard template, and drawing a fluorescence quantitative PCR standard curve according to the Ct value; 4, calculating the copy number of the sample rbcL gene according to the Ct value and the standard curve, wherein the cell density of the diatom in the sample is equal to the copy number. The method is simple, convenient and easy to operate, has very high sensitivity and accuracy, can be used for rapidly and accurately quantifying the cell density of the diatom in the water sample, and is suitable for the specificquantitative detection of diatoms.

Description

technical field [0001] The invention belongs to the field of algae detection, and specifically relates to a method for rapid quantitative detection of diatom groups in water bodies by using a fluorescent quantitative PCR (polymerase chain reaction) method, which is applicable to oceans, rivers and lakes, open water channels, Rapid quantitative detection of diatom groups (forensic identification) contained in groundwater, glaciers and even living organisms can provide reliable data for related ecological environment investigations and scientific research. Background technique [0002] Diatoms are a type of unicellular eukaryotic microalgal plants with pigment bodies and highly silicified cell walls, often consisting of several or many individual cells connected into various groups; diatoms have existed on the earth for about 200 million years It is one of the most species-rich organisms in aquatic ecosystems. There are more than 16,000 species in the world, and the number of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/06
CPCC12Q1/6851C12Q2545/114C12Q2563/107C12Q2537/16
Inventor 毕永红刘洋米武娟宋高飞
Owner INST OF AQUATIC LIFE ACAD SINICA
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