0576025 - ÚEM 2025 RIV DE eng J - Journal Article
Mukherjee, S. - Hanák, Petr - Zdeňková, K. - Musilová, Z. - Horká, P. - Jilková, D. - Čermáková, E.
Development of plasmid calibrators for absolute quantification of the beta-parvalbumin gene in Lophius piscatorius.
European Food Research and Technology. Roč. 249, č. 12 (2023), s. 3165-3174. ISSN 1438-2377. E-ISSN 1438-2385
Institutional support: RVO:68378041
Keywords : plasmid calibrator * genetic marker * Pvalb * lophius * DNA quantification
Impact factor: 3, year: 2023
Method of publishing: Open access
https://link.springer.com/article/10.1007/s00217-023-04357-z

The real-time quantitative PCR (qPCR) calibration curves are highly reproducible and allow the generation of specific, sensitive, and reproducible data that can be used for gene quantification. However, it is important to rigorously validate the external calibration curve model in qPCR since absolute quantification is dependent on the standards used. We present a method for standardising qPCR-based quantification of the ss-parvalbumin (ss-pvalb) gene of Lophius piscatorius, a major fish allergen, using a plasmid DNA (pDNA) calibrator. In parallel experiments, standard curves were generated and compared from the genomic DNA ( gDNA) isolated from L. piscatorius and pDNA carrying the target, pvalb. The commutability of pDNA and gDNA calibrators for the quantification of ss-pvalb was assessed by employing a TaqMan qPCR, targeting the second intron of the pvalb gene of L. piscatorius. Higher PCR efficiencies, good linearity, and lower standard deviation (S.D.) values were observed with pDNA instead of gDNA calibrants. pDNA calibrants exhibited a lower bias in terms of closeness to the expected value of unknown samples than their genomic counterparts. The assay was specific and sensitive, where the limit of detection (LOD) and limit of quantification (LOQ) were five copies and ten copies per reaction. The shortterm stability study of the pDNA calibrants indicated its stability for 60 days at 20 degrees C and 30 days at 4 degrees C. The efficient results indicated a plasmid calibrator as a potential tool for absolute quantification of the pvalb gene and an alternative to conventional gDNA standards.
Permanent Link: https://hdl.handle.net/11104/0354668