Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms.
BACKGROUND
The real-time polymerase chain reaction is currently the method of choice for quantifying nucleic acids in different DNA based quantification applications. It is widely used also for detecting and quantifying genetically modified components in food and feed, predominantly employing TaqMan and SYBR Green real-time PCR chemistries. In our study four alternative chemistries: Lux, Plexor, Cycling Probe Technology and LNA were extensively evaluated and compared using TaqMan chemistry as a reference system.


RESULTS
Amplicons were designed on the maize invertase gene and the 5'-junction of inserted transgene and plant genomic DNA in MON 810 event. Real-time assays were subsequently compared for their efficiency in PCR amplification, limits of detection and quantification, repeatability and accuracy to test the performance of the assays. Additionally, the specificity of established assays was checked on various transgenic and non-transgenic plant species. The overall applicability of the designed assays was evaluated, adding practicability and costs issues to the performance characteristics.


CONCLUSIONS
Although none of the chemistries significantly outperformed the others, there are certain characteristics that suggest that LNA technology is an alternative to TaqMan when designing assays for quantitative analysis. Because LNA probes are much shorter they might be especially appropriate when high specificity is required and where the design of a common TaqMan probe is difficult or even impossible due to sequence characteristics. Plexor on the other hand might be a method of choice for qualitative analysis when sensitivity, low cost and simplicity of use prevail.
Buh Gasparic
Meti
M
Department of Biotechnology and Systems Biology, National Institute of Biology, Vecna pot 111, SI-1000 Ljubljana, Slovenia. meti.buh.gasparic@nib.si
Cankar
Katarina
K
Zel
Jana
J
Gruden
Kristina
K
D018744:DNA, Plant / Q000235:genetics*; D005504:Food Analysis / Q000379:methods*; D030841:Food, Genetically Modified / Q000145:classification*; D030821:Plants, Genetically Modified / Q000145:classification* / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D018744:DNA, Plant