PCR detection and identification of Alternaria species-groups in processed foods based on the genetic marker Alt a 1

Quantification of disease progression of Alternaria spp. on potato using real-time PCR

“…Further molecular techniques are needed to guarantee rapid and reliable pathogen quantification. Here, quantitative polymerase chain reaction (real-time PCR) can be helpful to quantify and differentiate pathogens because of its increased sensitivity (Schena et al 2004;Pavón et al 2010). Since its development in the 1990s, real-time PCR has emerged as a reliable and sensitive method for detecting and quantifying phytopathogenic and antagonistic fungi (Schena et al 2004), and represents a highly sensitive and specific technique for the detection and quantification of nucleic acids (Tylor et al 2001).…”

A real-time reverse-transcriptase PCR technique for detection and quantification of viable Alternaria spp. in foodstuffs

“…tested. Compared to previous DNA-based results using primers targeting the Alt a 1 gene (Pavón et al, 2010) or the same ITS-based primers in a PCR method (Pavón et al, 2011), the real-time RT-PCR assay described herein improved 100-fold the sensitivity. Such improvement can be explained because of the presence of a higher number of RNA copies and the use of a real-time format.…”

“…in foods. They include conventional or real-time PCR using specific primers (Andersen, Smedsgaard, Jorring, Skouboe, & Pedersen, 2006;Guillemette, Iacomi, & Simoneau, 2004;Konstantinova, Bonants, van GentPelzer, van der Zouwen, & van den Bulk, 2002;Pavón, González, Pegels, Martín, & García, 2010;Pavón et al, 2011;Zur, Shimoni, Hallerman, & Kashi, 2002), and also PCR amplification followed by complementary techniques such as sequencing, PCR-RFLP, PCR-RAPD or PCR-AFLP (Bensassi, Zid, Rhouma, Bacha, & Hajlaoui, 2009;Diguta, Vincent, Guilloux-Benatier, Alexandre, & Rousseaux, 2011;Pryor & Michailides, 2002;Somma et al, 2011). Because DNA is a stable molecule, DNA-based methods cannot distinguish between living and dead cells.…”

PCR-based assay for the detection of Alternaria species and correlation with HPLC determination of altenuene, alternariol and alternariol monomethyl ether production in tomato products

“…Total DNA extraction from raw tomato samples and tomato products was performed using the Wizard Ò DNA Clean-up System kit (Promega Corp., Madison, WI) as previously described (Pavón, González, Pegels, Martín, & García, 2010). DNA concentration was measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Montchanin, DE).…”

“…The resulting DNA fragments were visualized by UV transillumination and analysed using a Chemidoc XRS System. PCR products obtained from samples that tested negative for the presence of ALT, AOH or AME were gel-purified and sequenced as previously described (Pavón et al, 2010). The sequences obtained were searched for homology to those available at the GenBank-EMBL database using the BLAST program (NCBI software package).…”