Real-Time Detection of Genetically Modified Soya Using Lightcycler and ABI 7700 Platforms with TaqMan, Scorpion, and SYBR Green I Chemistries

Abstract

A comparative cross platform evaluation of real-time polymerase chain reaction detection of DNA sequences present in Roundup Ready soya was undertaken using the ABI 7700 and Roche Lightcycler detection systems in combination with 3 different detection chemistries: TaqMan, Scorpion primers, and SYBR Green I fluorescent dye. Various copy numbers of a plasmid containing the soya lectin sequence were used to determine the sensitivity and reproducibility of the different technology combinations and to examine both inter and intra machine variability. To examine the relative accuracy of each technology, the genetically modified soya content of baked products containing known amounts of Roundup Ready soya was determined by detection of lectin and the EPSPS transgene. It was determined that the combination of TaqMan detection chemistry and the ABI 7700 platform represented the best method for quantitative detection of genetically modified organisms in terms of both precision and accuracy.

The Development and Standardization of Testing Methods for Genetically Modified Organisms and their Derived ProductsF

“…Because of its ease of use, high throughput ability, decreased post‐PCR manipulation, and lack of cross‐contamination of PCR amplicons, the RT‐PCR method is becoming the new gold standard method for nucleic acid quantification (Yang et al 2008b). Currently, a number of RT‐PCR fluorogenic signal reagents have been developed and applied for quantitative purposes (Buh Gasparic et al 2008, 2010), for instance sequence unspecific DNA‐binding dyes (e.g., SYBR Green I) (Hernandez et al 2003b), fluorescence resonance energy transfer (FRET) probes (Wittwer et al 1997), TaqMan/MGB probes (Terry et al 2002), LNA (locked nucleic acid) probe (Salvi et al 2008), Plexor technology (Buh Gasparic et al 2008), light upon extension (LUX) probe (Nazarenko et al 2002), molecular beacons (Andersen et al 2006) and their derivatives (Amplifluor, Sunrise, and scorpion primers) (Whitcombe et al 1999; Thelwell et al 2000; Li et al 2002), and universal template (UT) probe (Zhang et al 2003) etc. Among them, TaqMan/MGB probes and SYBR green I are the most commonly used RT‐PCR chemistries.…”

Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms

“…Of all TaqMan ® and/or SYBR ® Green chemistries are being the most widely used [ 2 ]. The reason could lie in the numerous data available on the performance of these two methods, while only a few comparisons of the performance of alternative chemistries have been published [ 3 - 5 ].…”

“…In the field of GMO detection MGB, Molecular Beacon [ 3 ], Amplifluor ® [ 4 ] and Scorpion ® chemistries [ 5 ] have already been compared to TaqMan ® and SYBR ® Green performance. To supplement the available knowledge we tested additional four new technologies: Locked Nucleic Acid (LNA ® ) Probes (Sigma Proligo, Paris, France), Cycling Probe Technology (CPT) (Takara, Shiga, Japan), Light Upon eXtension (Lux™) Fluorogenic Primers (Invitrogen Corporation, Carlsbad, United States) and Plexor™ Technology (Promega, Madison, United States).…”

Molecular Monitoring of Minimal Residual Disease in Patients with Multiple Myeloma

“…As the "ASO probe strategy" is most suited for MRD detection in patients with MM, we recommend the use of a Taqman probe for RQ PCR, because the small size of 10-60 bp of the patientspecific CDR3 favours the design of one Taqman probe over that of two HybProbes. The availability of one or the other detection equipment does not necessarily influence the choice of probes to use, as Taqman probes can be easily used in combination with the LightCycler system for IgH RQ PCR [103] as has been shown for other target sequences before [95,[104][105][106][107][108][109][110][111][112][113].…”