Propidium monoazide combined with real-time quantitative PCR to quantify viable Alternaria spp. contamination in tomato products
Crespo Sempere, Ana
MetadataShow full item record
Alternaria is a common contaminating genus of fungi in fruits, grains, and vegetables that causes severe economic losses to farmers and the food industry. Furthermore, it is claimed that Alternaria spp. are able to produce phytotoxic metabolites, and mycotoxins that are unsafe for human and animal health.
DNA amplification techniques are being increasingly applied to detect, identify, and quantify mycotoxigenic fungi in foodstuffs, but the inability of these methods to distinguish between viable and nonviable cells might lead to an overestimation of mycotoxin-producing living cells. A promising technique to overcome this problem is the pre-treatment of samples with nucleic acid intercalating dyes, such as propidium monoazide (PMA), prior to quantitative PCR (qPCR). PMA selectively penetrates cells with a damaged membrane inhibiting DNA amplification during qPCRs. In our study, a primer pair (Alt4-Alt5) to specifically amplify and quantify Alternaria spp. by qPCR was designed. Quantification data of qPCR achieved a detection limit of 102 conidia/g of tomato. Here, we have optimized for the first time a DNA amplification-based PMA sample pre-treatment protocol for detecting viable Alternaria spp. cells. Artificially inoculated tomato samples treated with 65 μM of PMA, showed a reduction in the signal by almost 7 cycles in qPCR between live and heat-killed Alternaria spp. conidia. The tomato matrix had a protective effect on the cells against PMA toxicity, reducing the efficiency to distinguish between viable and nonviable cells. The results reported here indicate that the PMA-qPCR method is a suitable tool for quantifying viable Alternaria cells, which could be useful for estimating potential risks of mycotoxin contamination.
Is part ofInternational Journal of Food Microbiology, 2013, vol. 165, p. 214-220
Showing items related by title, author, creator and subject.
Occurrence of Alternaria mycotoxins and quantification of viable Alternaria spp. during the food processing of tomato products in Spain Estiarte, Núria; Crespo Sempere, Ana; Marín Sillué, Sònia; Sanchís Almenar, Vicente; Ramos Girona, Antonio J. (Wageningen Academic Publishers, 2018-05-15)The occurrence of two Alternaria mycotoxins, alternariol (AOH) and alternariol monomethyl ether (AME) and the presence of conidia from Alternaria spp., were investigated throughout the food production chain of two businesses, ...
Effect of 1-methylcyclopropene on the development of black mold disease and its potential effect on alternariol and alternariol monomethyl ether biosynthesis on tomatoes infected with Alternaria alternata Estiarte, Núria; Crespo Sempere, Ana; Marín Sillué, Sònia; Sanchís Almenar, Vicente; Ramos Girona, Antonio J. (Elsevier, 2016)Ethylene is a naturally produced plant regulator involved in several plant functions, such as regulation of fruit ripening. Inhibition of ethylene perception by using 1-methylcyclopropene (1-MCP) slows down the ripening ...
Exploring polyamine metabolism of Alternaria alternata to target new substances to control the fungal infection. Estiarte, Núria; Crespo Sempere, Ana; Marín Sillué, Sònia; Sanchís Almenar, Vicente; Ramos Girona, Antonio J. (Elsevier Ltd., 2017-02-03)Polyamines are essential for all living organisms as they are involved in several vital cell functions. The biosynthetic pathway of polyamines and its regulation is well established and, in this sense, the ornithine ...