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Modeling of 5' nuclease real-time responses for optimization of a high-throughput enrichment PCR procedure for Salmonella enterica.

Knutsson, Rickard LU ; Löfström, Charlotta LU ; Grage, Halfdan LU ; Hoorfar, Jeffrey and Rådström, Peter LU (2002) In Journal of Clinical Microbiology 40(1). p.52-60
Abstract
The performance of a 5' nuclease real-time PCR assay was studied to optimize an automated method of detection of preenriched Salmonella enterica cells in buffered peptone water (BPW). The concentrations and interactions of the PCR reagents were evaluated on the basis of two detection responses, the threshold cycle (C(T)) and the fluorescence intensity by a normalized reporter value (DeltaR(n)). The C(T) response was identified as the most suitable for detection modeling to describe the PCR performances of different samples. DNA extracted from S. enterica serovar Enteritidis was studied in double-distilled H2O (ddH2O) and in two different enrichment media (brain heart infusion and BPW) with two PCR mixtures based on AmpliTaq Gold or rTTH: A... (More)
The performance of a 5' nuclease real-time PCR assay was studied to optimize an automated method of detection of preenriched Salmonella enterica cells in buffered peptone water (BPW). The concentrations and interactions of the PCR reagents were evaluated on the basis of two detection responses, the threshold cycle (C(T)) and the fluorescence intensity by a normalized reporter value (DeltaR(n)). The C(T) response was identified as the most suitable for detection modeling to describe the PCR performances of different samples. DNA extracted from S. enterica serovar Enteritidis was studied in double-distilled H2O (ddH2O) and in two different enrichment media (brain heart infusion and BPW) with two PCR mixtures based on AmpliTaq Gold or rTTH: A descriptive model was proposed and fitted to the available experimental data. Equivalent PCR performances for the two PCR mixtures were obtained when DNA was diluted in ddH2O. However, the level of detection of DNA was affected when BPW was present during amplification. Use of the rTth mixture generated a 1-log-unit wider linear range of amplification, and the DNA detection levels were 2 x 10(-13) g/microwell for the rTth mixture and 2 x 10(-12) g/microwell for the AmpliTaq Gold mixture. To verify the improved amplification capacity of the rTth mixture, BPW was inoculated with 1 CFU of S. enterica serovar Enteritidis per ml and the mixture was incubated at 30 degrees C. Samples for PCR were withdrawn every 4 h during a 36-h enrichment. Use of the rTth mixture resulted in an earlier PCR detection during enrichment than use of the AmpliTaq Gold mixture. For accurate detection (C(T) < or = 30) of S. enterica serovar Enteritidis inoculated in BPW, the rTth mixture required 8.4 h of enrichment, while the AmpliTaq Gold mixture needed 11.6 h. In conclusion, the principle applied can improve the methodology of 5' nuclease real-time PCR for numerical optimization of sample pretreatment strategies to provide automated diagnostic PCR procedures. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Models Biological, Peptones, Polymerase Chain Reaction : methods : standards, Salmonella enterica : genetics : isolation & purification : physiology, Taq Polymerase, Support Non-U.S. Gov't, DNA Bacterial : analysis : isolation & purification, Animal, Culture Media
in
Journal of Clinical Microbiology
volume
40
issue
1
pages
52 - 60
publisher
American Society for Microbiology
external identifiers
  • wos:000173121800010
  • pmid:11773092
  • scopus:0036006906
ISSN
1098-660X
DOI
10.1128/JCM.40.1.52-60.2002
language
English
LU publication?
yes
id
54c01e33-615c-4845-8d19-59d6c3309a2c (old id 106879)
date added to LUP
2007-06-28 14:16:13
date last changed
2017-08-20 04:28:45
@article{54c01e33-615c-4845-8d19-59d6c3309a2c,
  abstract     = {The performance of a 5' nuclease real-time PCR assay was studied to optimize an automated method of detection of preenriched Salmonella enterica cells in buffered peptone water (BPW). The concentrations and interactions of the PCR reagents were evaluated on the basis of two detection responses, the threshold cycle (C(T)) and the fluorescence intensity by a normalized reporter value (DeltaR(n)). The C(T) response was identified as the most suitable for detection modeling to describe the PCR performances of different samples. DNA extracted from S. enterica serovar Enteritidis was studied in double-distilled H2O (ddH2O) and in two different enrichment media (brain heart infusion and BPW) with two PCR mixtures based on AmpliTaq Gold or rTTH: A descriptive model was proposed and fitted to the available experimental data. Equivalent PCR performances for the two PCR mixtures were obtained when DNA was diluted in ddH2O. However, the level of detection of DNA was affected when BPW was present during amplification. Use of the rTth mixture generated a 1-log-unit wider linear range of amplification, and the DNA detection levels were 2 x 10(-13) g/microwell for the rTth mixture and 2 x 10(-12) g/microwell for the AmpliTaq Gold mixture. To verify the improved amplification capacity of the rTth mixture, BPW was inoculated with 1 CFU of S. enterica serovar Enteritidis per ml and the mixture was incubated at 30 degrees C. Samples for PCR were withdrawn every 4 h during a 36-h enrichment. Use of the rTth mixture resulted in an earlier PCR detection during enrichment than use of the AmpliTaq Gold mixture. For accurate detection (C(T) &lt; or = 30) of S. enterica serovar Enteritidis inoculated in BPW, the rTth mixture required 8.4 h of enrichment, while the AmpliTaq Gold mixture needed 11.6 h. In conclusion, the principle applied can improve the methodology of 5' nuclease real-time PCR for numerical optimization of sample pretreatment strategies to provide automated diagnostic PCR procedures.},
  author       = {Knutsson, Rickard and Löfström, Charlotta and Grage, Halfdan and Hoorfar, Jeffrey and Rådström, Peter},
  issn         = {1098-660X},
  keyword      = {Models Biological,Peptones,Polymerase Chain Reaction : methods : standards,Salmonella enterica : genetics : isolation & purification : physiology,Taq Polymerase,Support Non-U.S. Gov't,DNA Bacterial : analysis : isolation & purification,Animal,Culture Media},
  language     = {eng},
  number       = {1},
  pages        = {52--60},
  publisher    = {American Society for Microbiology},
  series       = {Journal of Clinical Microbiology},
  title        = {Modeling of 5' nuclease real-time responses for optimization of a high-throughput enrichment PCR procedure for Salmonella enterica.},
  url          = {http://dx.doi.org/10.1128/JCM.40.1.52-60.2002},
  volume       = {40},
  year         = {2002},
}