Antisense Primer Design for Ostreopsis cf. ovata Detection
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Solution Overview
Problem
Current methods for detecting microalgae, such as those of the genus Ostreopsis, in seawater samples are inadequate, particularly for species like Ostreopsis cf. ovata, leading to underestimation or false negatives due to incorrect primer sequences, which affects the accuracy of toxicity monitoring in coastal areas.
Innovation Solution
An improved detection method involving enhanced DNA extraction through filtration, mechanical lysis, and the use of a consensus plasmid for quantification, along with a specific mixture of antisense primers (e.g., CCA RGA R.Y. ATGCCTACATTCAA) for accurate amplification of Ostreopsis and Ostreopsis cf. ovata DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR methods with standard primer pairs are used for detection, then the detection process is simple and quick, but the detection accuracy is low leading to underestimation or false negatives for Ostreopsis cf. ovata
Solution Approach 1:
The patent modifies the primer sequences (changing specific nucleotide positions) to improve binding specificity for Ostreopsis cf. ovata. The forward primer was changed from 5'-AAAACGATATGAAGAGTGCAGC-3' to 5'-AAAACGATATGAAGAGTGCAGT-3' and the reverse primer from 5'-CCAGGAGTATGCCTACATTCAA-3' to 5'-CCAGGAGTATGCCTACATTCTA-3'. These parameter changes in the primer sequences resolve the technical contradiction by enhancing detection accuracy while maintaining the simplicity of the PCR methodology.
2Quantity of substance
If DNA extraction is performed without mechanical lysis, then the extraction process is faster and simpler, but the DNA extraction yield is insufficient for reliable detection
Solution Approach 1:
The patent incorporates mechanical lysis (bead beating) as a preliminary step before chemical extraction. The filter containing microalgae is mechanically disrupted using glass beads in a bead beater for 30-60 seconds, which breaks open cell walls and releases DNA. This preliminary mechanical action ensures sufficient DNA yield is obtained before proceeding to chemical purification steps.
3Quantity of substance
If filtration with small pore size is used to concentrate microorganisms, then the concentration of microorganisms in retentate is high, but the filtration time is longer
Solution Approach 1:
The patent uses different pore sizes for different filtration stages: a 3 μm pore size filter is used for initial concentration of microorganisms from large water volumes, then a 0.45 μm pore size filter is used for final DNA extraction. This local quality approach optimizes each filtration stage - the 3 μm filter quickly concentrates cells without excessive filtration time, while the 0.45 μm filter ensures complete retention for DNA extraction.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method provides reliable and accurate detection and quantification of microalgae, reducing errors and ensuring the presence of Ostreopsis and Ostreopsis cf. ovata are correctly identified, enhancing monitoring and safety in coastal environments.
Implementation Method 1
the lysis step advantageously comprises placing the filter in a container in the presence of a lysis solution, so that the filter and the retentate present on the filter are in contact with the lysis solution
Implementation Method 2
then preferably mechanically grinding the lysate thus obtained
Implementation Method 3
amplification of the DNA from the purified DNA solution with at least one primer pair specific to the microorganism to be detected
Data Source
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AI summary
The present invention relates to a method for detecting at least one microorganism, preferably a microalga, in a sample, preferably a seawater sample, comprising the following steps: filtering the sample, extracting, purifying, and amplifying the DNA, and determining the presence and/or quantifying the microorganism to be detected in the sample. The microorganism is preferably a microalga of the genus Ostreopsis, a microalga of the species Ostreopsis cf. siamensis, or a microalga of the species Ostreopsis cf. ovata. The present invention also relates to a mixture of antisense primers suitable for the detection of a microalga of the genus Ostreopsis and/or a microalga of the species Ostreopsis cf. ovata, a kit comprising said mixture of antisense primers, and their use for the detection of at least one microalga of the genus Ostreopsis and/or a microalga of the species Ostreopsis cf. ovata.