Development of Two Quantitative Real-Time PCR Methods Based on SYBR Green and TaqMan to Quantify Sterigmatocystin-Producing Molds in Foods

Rodríguez, Alicia; Córdoba, Juan J.; Gordillo, Rubén; Córdoba, Maria de Guı́a; Rodrı́guez, Mar

doi:10.1007/s12161-012-9411-9

Cited by 8 publications

(6 citation statements)

References 53 publications

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“…2) or DNA extracted from yoghurt prepared with L. paracasei FNU (Fig. 3), standard curves presented mean efficiency values of 91 % (pure strain CTT 7501), 95 % (pure strain FNU), and 103 % (yoghurt with strain FNU); suitable efficiency values shall be between 90 % and 110 % (Rodríguez et al 2012). The qPCR efficiency values obtained by Achilleos and Berthier (2013) ranged from 81.1 % to 99.5 % for L. paracasei Tuf qPCR assay.…”

Section: Discussionmentioning

confidence: 99%

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Comparison of real-time PCR assay and plate count for Lactobacillus paracasei enumeration in yoghurt

et al. 2015

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Lactobacillus paracasei is a mesophilic lactic acid bacterium technologically active in food fermentation. Culture-independent methods have rapidly been recognized as a valuable alternative to culture-dependent methods for lactic acid bacteria enumeration. In the present work, the efficacy of different protocols to extract DNA from yoghurt were compared, real-time PCR (qPCR) targeting tuf gene for L. paracasei enumeration was evaluated, and qPCR and plate counts of L. paracasei in yoghurt samples were compared. Total DNA concentrations from commercial yoghurts were higher using DNAzol method 2 than using the other tested methods. Standard curves presented suitable mean efficiency values of 91 % (pure L. paracasei strain CTT 7501), 95 % (pure L. paracasei strain FNU), and 103 % (yoghurt with L. paracasei strain FNU). Limit of detection is 3 log DNA copy number, corresponding to 2.78 log CFU, a suitable range of CFU enumeration for probiotic bacteria in yoghurt samples, considering that they should be present in large amounts. The L. paracasei (CFU) enumerated by qPCR were compared to culturable L. paracasei enumerated by plate counts at 7, 14, 21, and 28 days of yoghurt manufacture. Differences between qPCR and plate counts were observed only 28 days after yoghurt preparation, counts were similar at 7, 14, and 21 days. In conclusion, this qPCR assay is a useful and rapid tool to enumerate L. paracasei in yoghurt, although it does not distinguish dead and viable cells.

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Section: Discussionmentioning

confidence: 99%

“…These methods can be based on the direct analysis of DNA extracted from the food matrix (Achilleos and Berthier 2013;Rodríguez et al 2012). DNA extraction is a crucial step for reliable DNA quantification by real-time PCR, called qPCR (Cankar et al 2006;Garcia et al 2013;Oliveira et al 2013;Tian et al 2013).…”

Section: Introductionmentioning

confidence: 99%

Comparison of real-time PCR assay and plate count for Lactobacillus paracasei enumeration in yoghurt

et al. 2015

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“…In literature, the DCt model was used not only for the analysis of gene expression (Livak & Schmittgen, 2001) but also for the comparison of real-time methods at quantification of molds in foods (Rodríguez, C ordoba, Gordillo, C ordoba, & Rodríguez, 2012) and evaluation of DNA extraction methods (Branquinho, Ferreira, & Cardarelli-Leite, 2012). In this work, we used the DCt values to monitorthe changes in DNA amplicability in the course of pickling process.…”

Section: Quantitative Pcr Analysis Of Dnas Isolated From Processed mentioning

confidence: 99%

Application of magnetic polymethacrylate-based microspheres for the isolation of DNA from raw vegetables and processed foods of plant origin

Trojánek

Kovařı́k

Španová

et al. 2017

J Food Process Preserv

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A method for the microextraction of DNA from raw vegetables and highly processed foods of plant origin suitable for PCR analysis was developed. It is based on non-selective binding of DNA in the presence of PEG 6,000/NaCl to hydrophilic magnetic nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) -P(HEMA-co-GMA) microspheres covered by carboxyl groups.The quantitative polymerase chain reaction (qPCR) by in-house designed primers targeting a highly repetitive rDNA locus allowed for detection of plant DNAs in a wide range of concentrations (0.1 pg/lL to 10 ng/lL). The described procedure is fast and simple. We further demonstrate that relatively mild acidic treatment of vegetable at elevated temperatures resulted in a dramatic reduction of PCR efficiency indicating extensive degradation of DNA during pickling. The described method is suitable for the analysis of highly degraded DNA from pickled food products. Practical applicationsDNA-based methods, such as the polymerase chain reaction (PCR), represent an important genetic approach for identification of plant species composition of foods. DNA from processed foods varies in both quality and quantity between the protocols used. Plant samples are generally characterised by a complex composition containing various inhibitors of PCR. Current methods have been tailored for specific samples while no universal protocol is available. Development of a simple universal procedure leading to PCR-ready DNA would be beneficial. Isolation strategies based on solid phase systems have become popular for PCR-ready DNA isolations from complex matrices.Here we applied magnetic hydrophilic P(HEMA-co-GMA) microspheres to extract DNA from raw vegetables and highly processed foods of plant origin. Proposed small scale PCR-ready DNA extraction protocol was comparable with a silica-based DNeasy Plant Mini Kit (Qiagen) and classical CTAB extraction methods in quality and amplificability of DNA while it is less costly and time consuming.

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“…This may be satisfactory if DNA was stable and there would be little cause to doubt this if mutagens in growth media did not occur. DNA is susceptible to damage (Table 1; Paterson & Lima, 2013) and high concentrations of various secondary metabolites are produced in agars: high concentrations of mycotoxins in agars are reported in , , Rodríguez, Córdoba, Gordillo, et al (2012), and . These high concentrations are indicated when undertaking the analysis of fungal cultures using the "agar plug" technique developed by Frisvad (e.g.…”

Section: Self Mutagens In Fungimentioning

confidence: 99%

“…Equivalent methods to these were used in a subsequent paper concerning ochratoxin A (Luque, Córdoba, Rodríguez, Núñez, & Andrade, 2013) although considerations of mutated fungi or IAC were not provided. was cited in Rodríguez, Córdoba, Gordillo, et al (2012) for sterigmatocystin determination although an IAC was not employed, and the fungal mutant issue was not considered in this paper.…”

Section: Self Mutagens In Fungimentioning

confidence: 99%

Self mutagens affect detrimentally PCR analysis of food fungi by creating potential mutants

Paterson

Lima

2014

Food Control

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a b s t r a c tAnalysing fungi from food by PCR is increasing rapidly. However, food fungi produce mutagens which may mutate the fungi in culture so that fungi which produce mycotoxins may have a negative PCR result for genes in the mycotoxin metabolic pathway and vice versa: It is impossible to state unequivocally that the current PCR results obtained are accurate. For example, food containing a mycotoxin fungus may be considered safe if the isolates from the food were mutated into being negative for the mycotoxin (or other) gene and vice versa. Growth conditions affect which mutagens are produced and the conditions used by authors are assessed for the first time in the current report. Previous research assumed that NA was unaffected by how fungi were grown despite no supporting evidence. Individual research groups used similar growth conditions for disparate fungi for PCR analysis which were different from methods used by alternative workers. Rationales for using particular growth methods are unexplained. The fungi will be in almost continuous contact for long periods with various biochemical mutagens at high concentrations. Only partial solutions can be provided by suggesting alternative methods. Future methods need to state why particular conditions are employed when growing fungi and what was done to avoid mutagens.

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Development of Two Quantitative Real-Time PCR Methods Based on SYBR Green and TaqMan to Quantify Sterigmatocystin-Producing Molds in Foods

Cited by 8 publications

References 53 publications

Comparison of real-time PCR assay and plate count for Lactobacillus paracasei enumeration in yoghurt

Comparison of real-time PCR assay and plate count for Lactobacillus paracasei enumeration in yoghurt

Application of magnetic polymethacrylate-based microspheres for the isolation of DNA from raw vegetables and processed foods of plant origin

Self mutagens affect detrimentally PCR analysis of food fungi by creating potential mutants

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