Wspólnotowy Serwis Informacyjny Badan i Rozwoju - CORDIS

Development of a quantitative real-time PCR assay for A. carbonarius

The highly conserved regions of calmodulin gene were considered as target sequences for the assay. In an alignment of sequences representing Aspergillus spp. occurring on grapes, primers and probes sequences were chosen. The primers and probe sequences were completely conserved among all sequences examined within the A. carbonarius strains. Primer/probe combination successfully amplified DNA extracted from pure cultures of A. carbonarius fungal species, whereas no fluorescence could be measured with any of the other species within the Aspergillus genera.

Occasionally, the fluorescence value of negative control (containing water instead template) started to increase after 36 - 38 cycles. This is not unusual when use SYBR Green and is caused by primer-dimers formation (especially at low level of target template). Even though this effect was almost eradicated by reducing the primer concentration, any increase in fluorescence after this point was deemed unreliable and not used to detect or quantify samples. It was avoided using a sequence specific assay, as TaqMan method.

The TaqMan real time PCR is based on the cleavage of an internal probe by the 5’-/3’ endonuclease activity of the Taq polymerase. During each cycle of the extension phase one molecule of the reporter dye is released for each molecule amplified resulting in generation of fluorescent emission of the reporter dye. The threshold cycle (Ct values) indicates the increase of reporter fluorescence and it is the number of cycles before the fluorescence emitted passes a fixed limit. The log10 of the number of targets initially present is proportional to the Ct value and can be measured using a standard curve.

To develop a standard curve for the quantitative PCR reactions were used serial dilutions of A. carbonarius strains. A pure culture of A. carbonarius was used to extract the DNA and make two fold dilutions for obtaining the standard curve useful for Real Time PCR. Using the TaqMan method, large numbers of samples can be processed allowing a much more intensive monitoring of the population dynamics. To evaluate intra-assay variability, each dilution was assayed in triplicate. Relative standard deviations of the Ct values within the same sample was lower than 1.5%.

Application of this method to a known conidial suspension (CFU/ml) provided a positive correlation between CFU and haploid genome weight. Genomic DNA weight of A. carbonarius (not known) was assumed to be similar to that of A. niger (4.13 x 10-5ng, for a haploid genome) reported in the website http://www.broad.mit.edu/annotation/fungi/fgi/history.h tml. To verify this assumption an absolute quantification of total DNA was made for DNA extracted from a known conidial suspension (3x102 CFU/ul) of A. carbonarius, by real-time quantitative PCR assay.

For a suspension of 3x105 conidia 10.95ng of total DNA was obtained, corresponding to 3.65x10-5 ng for a single CFU or conidium, which is very close to the genome weight of A. niger (35.9 Mb). In order to study the usefulness of the TaqMan PCR assay as a tool in food quality/safety control the quantification of A. carbonarius DNA was carried out in triplicate on 15 samples of grapes having a known content of OTA. The regression curve of A. carbonarius DNA content versus OTA content showed a very good correlation (R2=0.92), despite the finding of low levels of DNA in 4 ochratoxin-free samples.

Reported by

National Research council of Italy
Via Amendola 122/O
70126 Bari
Italy
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