Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection |
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Authors: | Meti Buh Gašparič Torstein Tengs Jose Luis La Paz Arne Holst-Jensen Maria Pla Teresa Esteve Jana Žel Kristina Gruden |
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Institution: | (1) Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia;(2) Section for food bacteriology and GMO, National Veterinary Institute, P.O. Box 750, Sentrum, 0106 Oslo, Norway;(3) Consorci CSIC-IRTA and IBMB-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain |
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Abstract: | Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative
real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes
and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared
systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes
(SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes,
comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published
experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries
were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall
applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics.
None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology
as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but
may need more optimization prior to use. |
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