Comparison and transfer testing of multiplex ligation detection methods for GM plants

BMC Biotechnology

Research Article

Gabriella Ujhelyi¹  Henriek G Beenen²  Dany Morisset³  Kristina Gruden³  AM Angeline Van Hoef⁴  Esther J Kok⁴  Jeroen P van Dijk⁴  Theo W Prins⁴  Marleen M Voorhuijzen⁴ 

[1] CFRI - Central Food Research Institute, Herman Ottó út 15. H-1022, Budapest, Hungary;Laboratory of Phytopathology (WUR), 6708 PB, Droevendaalsesteeg 1, Wageningen, the Netherlands;NIB - Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia;RIKILT - Institute of Food Safety (WUR), Akkermaalsbos 2, 6708 WB, Wageningen, the Netherlands;

关键词: Ligation Reaction;    Padlock Probe;    Microarray Detection;    Transfer Laboratory;    Polymerase Chain Reaction Parameter;   

DOI  :  10.1186/1472-6750-12-4

 received in 2011-07-19, accepted in 2012-01-19,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundWith the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i) hybridisation and ligation of specific probes, ii) amplification of the ligated probes and iii) detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs) with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study.ResultsOf the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands).ConclusionsFrom the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the interlaboratory exchange study it was shown that the selected method meets the 0.1% sensitivity criterion. The present study thus shows that specific and sensitive multidetection of GMO targets is now feasible.

【 授权许可】

Unknown   
© Ujhelyi et al; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.