Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection.
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.
Buh Gasparic
Meti
M
Department of Biotechnology and Systems Biology, National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia. meti.buh.gasparic@nib.si
Tengs
Torstein
T
La Paz
Jose Luis
JL
Holst-Jensen
Arne
A
Pla
Maria
M
Esteve
Teresa
T
Zel
Jana
J
Gruden
Kristina
K
D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000737:chemistry* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*
D018744:DNA, Plant