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Humic substances cause fluorescence inhibition in real-time PCR.

Sidstedt, Maja LU ; Jansson, Linda LU ; Nilsson, Elin; Noppa, Laila; Forsman, Mats; Rådström, Peter LU and Hedman, Johannes LU (2015) In Analytical Biochemistry 487. p.30-37
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Analytical Biochemistry
volume
487
pages
30 - 37
publisher
Elsevier
external identifiers
  • pmid:26170001
  • wos:000361254800005
  • scopus:84939457793
ISSN
1096-0309
DOI
10.1016/j.ab.2015.07.002
language
English
LU publication?
yes
id
d38837fd-6151-4d06-8392-f22358a58303 (old id 7744668)
date added to LUP
2015-09-13 20:26:24
date last changed
2017-11-19 03:18:55
@article{d38837fd-6151-4d06-8392-f22358a58303,
  abstract     = {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.},
  author       = {Sidstedt, Maja and Jansson, Linda and Nilsson, Elin and Noppa, Laila and Forsman, Mats and Rådström, Peter and Hedman, Johannes},
  issn         = {1096-0309},
  language     = {eng},
  pages        = {30--37},
  publisher    = {Elsevier},
  series       = {Analytical Biochemistry},
  title        = {Humic substances cause fluorescence inhibition in real-time PCR.},
  url          = {http://dx.doi.org/10.1016/j.ab.2015.07.002},
  volume       = {487},
  year         = {2015},
}