Applicability of the real-time PCR assay in the ampliﬁcation of cyanobacterial DNA from preserved samples

Churro, Catarina; Valério, Elisabete; Pereira, Paulo; Vasconcelos, Vitor

http://hdl.handle.net/10400.18/3775

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Abstract(s)

The study and monitoring of cyanobacterial blooms often involves the use of preserved samples to avoid cellular degradation. However, preserved samples may not be suitable for molecular biology studies because preservation methods can interfere with DNA quality/quantity. Real-time quantitative PCR analysis (qPCR) has been widely applied in molecular analysis and is considered a promising method for monitoring purposes. This study intended to evaluate the applicability of the real-time qPCR technique in samples that were subjected to different methods of preservation: (1) 15% Lugol’s iodine solution (2) 4% formaldehyde and (3) 25% glutaraldehyde. The ability to amplify and quantify DNA extracted from Planktothrix agardhii was assessed using the rpoC1 gene as the target fragment in both raw water samples and in vitro cultures. No reliable DNA ampliﬁcation was obtained from glutaraldehyde-preserved samples. Successful ampliﬁcation was obtained from Lugol’s and formaldehyde-preserved samples. In this case, however, the quantiﬁcation that was achieved by real-time PCR cannot be used to infer cell numbers, because the Ct values that were obtained from the Lugol’s and formaldehydepreserved samples were signiﬁcantly higher than the Ct values that were obtained from the unpreserved samples. Therefore real-time PCR can be used for the detection and identiﬁcation of cyanobacteria in preserved samples but no reliable cell quantiﬁcation can be performed using this method.