Humic substances cause fluorescence inhibition in real-time PCR.
Forskningsoutput: Tidskriftsbidrag › Artikel i vetenskaplig tidskrift
Real-time PCR (qPCR) is the cornerstone of DNA analysis, enabling detection and quantification of minute nucleic acid amounts. However, PCR-based analysis is limited, in part, by the presence of inhibitors in the samples. PCR inhibition has been viewed solely as failure to efficiently generate amplicons, i.e. amplification inhibition. Humic substances (HS) are well known inhibitors of PCR amplification. Here we show that HS from environmental samples, specifically humic acid (HA), are very potent detection inhibitors, i.e. quench the fluorescence signal of dsDNA binding dyes. HA quenched the fluorescence of the commonly used qPCR dyes EvaGreen, ResoLight, SYBR Green I and SYTO 82, generating lowered amplification plots although amplicon production was unaffected. For EvaGreen, 500 ng HA quenched almost all fluorescence, whereas 1000 ng HA completely inhibited amplification when applying Immolase DNA polymerase with BSA. Fluorescence spectroscopy measurements showed that HA quenching was either static or collisional, and indicated that HA bound directly to the dye. Fulvic acid did not act as a qPCR detection inhibitor, but inhibited amplification similarly to HA. Hydrolysis probe fluorescence was not quenched by HA. Detection inhibition is an overlooked phenomenon that needs to be considered to allow for development of optimal qPCR assays.
|Enheter & grupper|
Ämnesklassifikation (UKÄ) – OBLIGATORISK
|Status||Published - 2015|
|Peer review utförd||Ja|
Maja Sidstedt, 2019 maj 24, Lund University, Division of Applied Microbiology: Department of Chemistry, Lund University. 172 s.
Forskningsoutput: Avhandling › Doktorsavhandling (sammanläggning)