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PCR based assay for detection garden pea lec gene
Vráblík, Aleš ; Hodek, Jan ; Ovesná, Jaroslava
The method was developed and verified by staff of National reference laboratory for GMO identification and DNA fingerprinting suited for governmental control laboratories and private laboratories that analyze food and feed derived from various plant matrices. Method can be applied for quantification of GM pea in a sample, when reference gene is used as a standard. The method describe internal garden pea specific gene, lektin in this case, detection using PCR and real-time PCR. The general principle of the assay relies on lektin specific sequence presence that represents a species specific gene of garden pea. In many matrices DNA is damaged so amplification of short stretches of DNA is used. After amplification of target exploiting newly developer species specific gene primers PCR products are separated by electrophoresis in agarose gel. Resulting band is visualized by UV light and its position is compare with size standard. Alternatively, real-time PCR and ABI platform in combination with SYBR® Green can be used. Reaction parameters are described and specificity of reaction was verified. LOD as well as LOQ was defined.
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Methods of DNA extraction from the fresh papaya fruit and from the candied
Ovesná, Jaroslava ; Hodek, Jan
Aim the work is to provide a working procedure for extraction of amplifiable DNA form papaya fruits (Carica papaya), which are a poor source of DNA. Methods are optimised for DNA extraction from papaya fruits and stones and from candied papaya- these both were predicted as suitable commodities for an eventually screening of non-approved GM papaya in the market of Czech Republic. The methodology was prepared on basis of demand of reference laboratories of public service, which are involved in GMO analyses and also as a flow-work of Methodics for Detection of GM papaya 55-1 and 63-1 (Hodek, Ovesná, Pavlátová 2008) and scientific publication Detection of transgenic papaya lines: extraction protocol optimisation and verification of DNA quality by PCR assay (Ovesná, Hodek 2009).
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