Utilization of specific primers in legume allergens based polymorphism screening

Abstract

Different types of allergies became a part of life of many people around the world. The research activities connecting to allergens are actually not oriented only for protein and immunological interactions, but to the genomic and transcriptomic background of them, too. Analysis and description of genomic variability of allergens in plant food resources will help to manage the allergen based strategies in the future. Here, the bioinformatic approach was used to develop and validate the specific primers for genomic screening of polymorphism of profilins (Profilin Based Amplicon Polymorphism; PBAP) and vicilins (Vicilin Based Amplicon Polymorphism; VBAP) among the legumes. The alignment of existing public databases data for these allergens in the group of legumes was performed. Subsequently, specific primers were designed and their ability to generate polymorphic amplicons were tested for three legumes – bean, lentil and chickpeas. In all cases, amplicons were generated and polymorphism was detected in all three species for profilin as well as for vicilin.

Polymorphism of Bolivian accessions of Arachis hypogaea L. revealed by allergen coding DNA markers

“…Profilin was first documented as an allergen found in birch pollen, called Bet v 2 (Valenta et al 1991), and is recognised for causing an allergic reaction to pear, peach, apple, melon, tomato, celery, pumpkin seeds, and peanuts (Breiteneder and Ebner 2001). Completely different amplicon profiles of PBAP were obtained for some of the previously analysed legume species (Klongová et al 2021), where the amplicons generated were within a range of 46 bp to 669 bp, and this technique differentiated species analysed in the study. Here, PBAP fingerprints of peanuts were distributed among 30 different loci, with their length ranging from 78 bp to 1 642 bp.…”

“…PCR amplification and data analysis. Degenerate primers were used according to a previously reported study for BBAP (Žiarovská and Zeleňáková 2018) and PBAP (Klongová et al 2021), and VBAP analysis. The sequences of primers were checked in silico to match the peanut allergen sequences with the positive match for A. hypogaea Ara h 8 allergen mRNA, complete cds (AY328088.1) as well as for Arachis hypogaea profilin (Ara h 5) mRNA, complete cds (AF059616.1).…”

Variability of Allergen-Based Length Polymorphism of Glycine max L. Varieties

“…Profilin variability was examined by two set of primers-specific and degenerated. Specific primers were designed to produce profilins [11], degenerated primers were designed on the base of conservative parts of profilin found in sequences of Rosaceae (taxid:3745) in the NCBI database (Table 2).…”

Utilization of plant profilins as DNA markers

“…Two types of primers were used in PCR analysis: nondegenerate, with unique sequences designed for conserved regions of the profilin genes and degenerate which are specially designed to be able to simultaneously hybridize with multiple divergent regions of profilin homologues. The sequences of non-degenerate primers used were as follows: F: 5' ACC GGC GAT CTG GTT TT 3' and R: 5' AGG TAG TCT CCC AAC CTC TC 3' (Klongová et al, 2021). Forward and reverse degenerate primer with sequences F: 5'-AGA GAA TTC CAT ATG TCG TGG CAR RCG TAC GT 3' (degeneracy = 4), and R: 5' AGA AAG CTT YTA CAK GCC YTG TTC ABV AGG TA 3' (degeneracy = 72) were used.…”