Development of Plasmid Calibrators for Absolute Quantification of the β-parvalbumin gene in Lophius piscatorius

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00027022%3A_____%2F23%3AN0000005" target="_blank" >RIV/00027022:_____/23:N0000005 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216208:11310/23:10469637 RIV/60461373:22330/23:43927681

Výsledek na webu

<a href="https://link.springer.com/article/10.1007/s00217-023-04357-z" target="_blank" >https://link.springer.com/article/10.1007/s00217-023-04357-z</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1007/s00217-023-04357-z" target="_blank" >10.1007/s00217-023-04357-z</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Development of Plasmid Calibrators for Absolute Quantification of the β-parvalbumin gene in Lophius piscatorius

Popis výsledku v původním jazyce

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 β-parvalbumin (β-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 β-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 short-term stability study of the pDNA calibrants indicated its stability for 60 days at − 20 °C and 30 days at 4 °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.

Název v anglickém jazyce

Development of Plasmid Calibrators for Absolute Quantification of the β-parvalbumin gene in Lophius piscatorius

Popis výsledku anglicky

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 β-parvalbumin (β-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 β-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 short-term stability study of the pDNA calibrants indicated its stability for 60 days at − 20 °C and 30 days at 4 °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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/QK1910231" target="_blank" >QK1910231: Nové přístupy k průkazu falšování rybího masa pomocí genomové DNA</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2023

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

European Food Research and Technology

ISSN

1438-2377

e-ISSN

1438-2385

Svazek periodika

249

Číslo periodika v rámci svazku

12

Stát vydavatele periodika

DE - Spolková republika Německo

Počet stran výsledku

10

Strana od-do

3165–3174

Kód UT WoS článku

001089263300003

EID výsledku v databázi Scopus

2-s2.0-85168858155