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Use of an intron containing grapevine gene as internal control for validation of cDNA synthesis in virus detection by RT-PCR

Oláh, Róbert, Deák, Tamás, Turcsán, Mihály, Szénási, Márta, Bordé, Ádám, Szegedi, Ernő
European journal of plant pathology 2017 v.149 no.3 pp. 765-770
RNA, RNA viruses, Vitis vinifera, complementary DNA, essential genes, gene expression, introns, phosphoenolpyruvate carboxylase, reverse transcriptase polymerase chain reaction, viruses
Detection of viruses in grapevine plants is important to prevent their spreading with propagating stocks. For this purpose PCR based protocols are commonly and routinely used. Since most grapevine viruses are RNA viruses, the detection procedure starts with RNA purification followed by cDNA synthesis prior to PCR. The cDNAs should be validated by an internal control for which usually constitutively expressed housekeeping genes are used. Here we publish a new set of primers designed in the Vitis vinifera phosphoenolpyruvate carboxylase gene to encompass two or three introns. PCR products amplified from remaining genomic DNA in the RNA samples and from cDNA can be clearly distinguished since cDNA-derived products are smaller in size compared to those amplified from genomic DNA. Thus these primers may be useful reference markers in RT-PCR-based virus detection assays both for the validation of cDNA synthesis and the detection of trace amounts of genomic DNA.