A highly sensitive and rapid enzymatic method using a biochemical automated analyzer to detect inorganic pyrophosphate generated by nucleic acid sequence-based amplification.
Biochemical automated analyzer
Enzymatic method
Inorganic pyrophosphate
Nitroso-PSAP
Nucleic acid sequence-based amplification
Journal
Clinica chimica acta; international journal of clinical chemistry
ISSN: 1873-3492
Titre abrégé: Clin Chim Acta
Pays: Netherlands
ID NLM: 1302422
Informations de publication
Date de publication:
Dec 2020
Dec 2020
Historique:
received:
18
09
2020
revised:
16
10
2020
accepted:
18
10
2020
pubmed:
24
10
2020
medline:
22
6
2021
entrez:
23
10
2020
Statut:
ppublish
Résumé
Polymerase chain reaction-based techniques require expensive equipment for fluorescence detection of the products. However, the measurement of inorganic pyrophosphate (PPi) released during DNA synthesis can be used to quantify target genes without such equipment. Here, we devised a high-sensitivity enzymatic assay for detection of PPi. In our assay method, PPi was converted to hypoxanthine by hypoxanthine phosphoribosyl transferase. Xanthine dehydrogenase converted the hypoxanthine to uric acid and yielded two molecules of NADH, which in turn reduced Fe The assay was able to detect PPi within 10 min. It was linear between 0 and 10 µmol/L PPi, and intra-run and inter-run coefficients of variation were 1%-2%. Other validation tests with a biochemical automated analyzer were satisfactory. The assay could potentially be used to directly quantify samples after isothermal nucleic acid sequence-based amplification of a target gene. The method developed here for detection of PPi can be used to measure nucleic acid biomarkers in biological samples in clinical practice using a high-throughput biochemical automated analyzer.
Sections du résumé
BACKGROUND AND AIMS
OBJECTIVE
Polymerase chain reaction-based techniques require expensive equipment for fluorescence detection of the products. However, the measurement of inorganic pyrophosphate (PPi) released during DNA synthesis can be used to quantify target genes without such equipment. Here, we devised a high-sensitivity enzymatic assay for detection of PPi.
MATERIALS AND METHODS
METHODS
In our assay method, PPi was converted to hypoxanthine by hypoxanthine phosphoribosyl transferase. Xanthine dehydrogenase converted the hypoxanthine to uric acid and yielded two molecules of NADH, which in turn reduced Fe
RESULTS
RESULTS
The assay was able to detect PPi within 10 min. It was linear between 0 and 10 µmol/L PPi, and intra-run and inter-run coefficients of variation were 1%-2%. Other validation tests with a biochemical automated analyzer were satisfactory. The assay could potentially be used to directly quantify samples after isothermal nucleic acid sequence-based amplification of a target gene.
CONCLUSION
CONCLUSIONS
The method developed here for detection of PPi can be used to measure nucleic acid biomarkers in biological samples in clinical practice using a high-throughput biochemical automated analyzer.
Identifiants
pubmed: 33096031
pii: S0009-8981(20)30508-8
doi: 10.1016/j.cca.2020.10.026
pii:
doi:
Substances chimiques
Diphosphates
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
298-305Informations de copyright
Copyright © 2020 Elsevier B.V. All rights reserved.