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Development of plasmid calibrators for absolute quantification of the beta-parvalbumin gene in Lophius piscatorius
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SYSNO ASEP 0576025 Document Type J - Journal Article R&D Document Type The record was not marked in the RIV Subsidiary J Článek ve WOS Title Development of plasmid calibrators for absolute quantification of the beta-parvalbumin gene in Lophius piscatorius Author(s) Mukherjee, S. (CZ)
Hanák, Petr (UEM-P) ORCID, RID
Zdeňková, K. (CZ)
Musilová, Z. (CZ)
Horká, P. (CZ)
Jilková, D. (CZ)
Čermáková, E. (CZ)Number of authors 7 Source Title European Food Research and Technology. - : Springer - ISSN 1438-2377
Roč. 249, č. 12 (2023), s. 3165-3174Number of pages 10 s. Language eng - English Country DE - Germany Keywords plasmid calibrator ; genetic marker ; Pvalb ; lophius ; DNA quantification OECD category Biochemistry and molecular biology Method of publishing Open access Institutional support UEM-P - RVO:68378041 UT WOS 001089263300003 EID SCOPUS 85168858155 DOI https://doi.org/10.1007/s00217-023-04357-z Annotation 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. Workplace Institute of Experimental Medicine Contact Arzuv Čaryjeva, arzuv.caryjeva@iem.cas.cz, Tel.: 241 062 218, 296 442 218 Year of Publishing 2025
Number of the records: 1