Species-Specific Primers for Mussel Identification via PCR-HRM
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Solution Overview
Problem
Current methods for identifying mussel species within the genus Mytilus, such as PCR-RFLP Acil assay, are laborious, slow, and costly, making them unsuitable for rapid and cost-effective species authentication in the mussel industry.
Innovation Solution
The use of specific primers, PAPM-SNP F and PAPM-SNP R, in combination with the PCR and High Resolution Melting (HRM) technique allows for rapid and cost-effective identification of Mytilus species by generating distinct dissociation curves without the need for gel visualization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR-RFLP Acil assay is used for species identification, then species differentiation is achieved, but the process becomes laborious, slow, and costly
Solution Approach 1:
The invention extracts only the essential discrimination function from the complex PCR-RFLP workflow by designing primers that directly amplify species-specific polymorphic regions. The primers Me15 and Me16 target the adhesive protein gene's non-repetitive region where species-specific polymorphisms exist, eliminating the need for restriction enzyme digestion and gel electrophoresis while maintaining identification accuracy
Solution Approach 2:
The invention replaces the mechanical/chemical workflow of restriction enzyme digestion and gel electrophoresis with a molecular biology approach using species-specific primers in PCR. This substitution eliminates multiple manual操作步骤 (enzyme incubation, gel casting, electrophoresis running) and replaces them with a streamlined PCR amplification process that can be visualized directly through gel electrophoresis of amplicons alone
2Measurement precision
If PCR-RFLP Acil assay is used for species identification, then species differentiation is achieved, but the process becomes laborious and costly
Solution Approach 1:
The invention extracts only the essential discrimination function from the complex PCR-RFLP workflow by designing primers that directly amplify species-specific polymorphic regions. The primers Me15 and Me16 target the adhesive protein gene's non-repetitive region where species-specific polymorphisms exist, eliminating the need for restriction enzyme digestion and gel electrophoresis while maintaining identification accuracy
Solution Approach 2:
The invention merges the species-specific detection function into the primer design itself. The primers Me15 and Me16 are designed to bind to species-specific sequences in the adhesive protein gene, so that the PCR amplification step itself becomes the species identification step, combining what were previously separate functions into one unified process
3Measurement precision
If traditional PCR-RFLP method is used, then species can be distinguished, but time and cost increase significantly
Solution Approach 1:
The invention extracts only the essential discrimination function from the complex PCR-RFLP workflow by designing primers that directly amplify species-specific polymorphic regions. The primers Me15 and Me16 target the adhesive protein gene's non-repetitive region where species-specific polymorphisms exist, eliminating the need for restriction enzyme digestion and gel electrophoresis while maintaining identification accuracy
Solution Approach 2:
The invention skips the time-consuming intermediate steps of restriction enzyme digestion, fragment separation, and multiple visualization steps by using species-specific primers that directly amplify different-sized amplicons for different species. This allows rapid identification through a single PCR amplification followed by direct gel electrophoresis visualization
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 approach reduces identification time by 80% and costs by four times compared to traditional methods, providing a faster, simpler, and more affordable means of species authentication.
Implementation Method 1
amplifying, by means of polymerase chain reaction (PCR), a specific segment of the genome
Implementation Method 2
the High Resolution Melting (HRM) technique for identifying mussel species
Data Source
Figure 1~2
Figure 3
Figure 4
AI summary
1. The invention consists of a set of specific primers of SEQ ID No. 1 SEQ ID No. 2 and to use of same in polymerase chain reaction (PCR) and high-resolution melting (HRM) for the identification of mussel species of the genus Mytilus in a rapid and less costly manner.