PCR primer design method for identification of closely related species

Abstract

The invention discloses a PCR primer design method for identification of closely related species. The PCR primer design method comprises the following steps: obtaining multiple DNA sequences of a same gene of a target species and a non-target species, and performing multi-sequence alignment to obtain a multi-sequence alignment file; designing multiple upstream primers and multiple downstream primers according to a conventional primer design method, and excluding unidirectional primers with sensitivity and specificity lower than set thresholds; pairing the remained upstream primers and the remained downstream primers with each other to obtain primer pairs, and excluding unreasonable primer pairs and primer pairs with relatively low sensitivity and specificity; calculating P values of the remained primer pairs, performing cluster analysis, and taking the primer pair with the minimum P value in each class as a designed PCR primer. Through the adoption of the PCR primer design method for the identification of the closely related species, a PCR primer with high specificity and high sensitivity for the target species can be quickly designed, thus reducing the difficulty of species-specific PCR primer design.

Description

technical field [0001] The invention relates to a method for designing specific primers, in particular to a method for designing PCR primers for identifying closely related species. Background technique [0002] The identification of closely related species has always been an important task in the fields of environmental monitoring, aquatic product species identification, animal and plant species identification, disease diagnosis, and import and export inspection and quarantine. However, the traditional classification and identification methods based on morphology have many defects, such as: most closely related species have very little morphological difference, making it difficult to identify; some species have large morphological differences in different growth periods or different growth environments, which further increases the identification Difficulty; after being processed as a raw material, it will lose its original morphological characteristics, resulting in indisti...

AI Technical Summary

Problems solved by technology

Therefore, the designed primers will have poor specificity (primers are located in conserved regions, cannot effectively distinguish between two species, and false positives) or poor sensitivity (primers are located in hypervariable regions, and can only identify some species within the species, resulting in false negatives) The problem

Therefore, it is difficult to design PCR primers that meet the requirements

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