Development of a Multiplex Ligation Dependent Probe Amplification (MLPA) Module for Simultaneous Detection of Five Genetically Modified Rapeseed Events

Abstract

For the official surveillance of genetically modified crops, efficient and simple detection methods need to be on hand to implement the strict requirements regarding approval, labelling and traceability determined by the European Union. Therefore, a multiplex ligation dependent probe amplification (MLPA) module was developed for simultaneous detection of five genetically modified rapeseed events (MS8, RF3, GT73, Falcon GS40/90 and T45). Probes were designed and concentrations were adapted in order to obtain high sensitivity and specificity. This MLPA module was validated using certified reference materials and its applicability was tested analyzing routine honey, mustard and rapeseed samples. The limit of detection was determined by analyzing a dilution series (n = 16 for each concentration) of respective transgenic DNA. After optimization, the MLPA revealed limits of detection between 10 to 50 copies of the transgene DNA/assay. The method proved to be sensitive and highly reproducible. When analyzing routine samples, results obtained applying the MLPA module were similar compared to real-time PCR.

Multiplex Ligation-Dependent Probe Amplification for Simultaneous Identification of Bungarus multicinctus and Its Common Adulterants in a Single Assay

“…First, we recognized that the MLPA method avoids complicated steps, such as cell culture in medical diagnosis and significantly shortens the diagnosis period, but the long hybridization time is a disadvantage in identification of traditional Chinese medicine. In this work, we referred previous study to shorten the hybridization time to 3 h when evaluating the specificity of MLPA probes (Guertler et al, 2014), and each assay was repeated three times with expected and stable results. When analyzing the relative quantification of the five species in the composite samples, the hybridization time was extended to 16 hours in order to ensure sufficient hybridization of the DNA target and the probemix.…”

“…The multiplex ligation-dependent probe amplification (MLPA) (Schouten et al, 2002) as an emerging molecular diagnostic technique can detect the difference in nucleic acid sequences with a small amount of DNA and possess capability of detecting up to 50 genomic DNA sequence difference in one reaction relied on DNA denaturation, hybridization, ligation, and PCR reaction (Os and Schouten, 2011;Samelak-Czajka et al, 2017). Recently, the technology has been applied to the clinical diagnosis of diseases, such as Duchenne muscular dystrophy (Guertler et al, 2014), Gaucher disease (Schouten et al, 2002), Acute leukemia (Reyes-Núñez et al, 2017), and it also has been received wide attentions in the field of allergen detection (López-Calleja et al, 2016), genetically modified species identification (Holck et al, 2009), and herb identification (Bo et al, 2018).…”