The application of quantitative real-time PCR to studies of the abundance and toxicity of cyanobacteria in Portuguese potable and recreational water supplies

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Accessing Planktothrix species diversity and associated toxins using quantitative real-time PCR in natural waters

Publication . Churro, Catarina; Vasconcelos, Vitor Manuel de Oliveira

The most common cyanotoxins in Portuguese freshwaters are microcystins and their occurrence has been mainly attributed to cyanobacteria from the Microcystis genus. However, most recently, it has been described the production of these toxins by species of the Planktothrix, suggesting that this genus is also a major producer of microcystin in Portuguese surface waters. Nevertheless, and conversely to the Microcystis species, the knowledge on the occurrence, distribution and toxigenesis of Planktothrix is still limited. Planktothrix species exhibits some particularities that difficult their sampling, identification, quantification and toxigenic characterization in natural samples - Chapter 2. Particularly, the morphology of Planktothrix colonies does not allow distinguishing easily the individual cells. This makes their identification and quantification by optical microscopy a very difficult task, although this is the method generally used in cyanobacteria monitoring. Moreover, the most common methods for the detection of microcystins (ELISA, HPLC) do not identify the producer strains. These strains can be identified by conventional PCR, but this method doesnt enable to quantify them. In resume, there is not yet available a method that allow simultaneously identify and quantify microcystin-producing strains. This work aimed to develop a method based on Real-Time PCR applied to the monitoring of toxic species of Planktothrix in surface freshwater reservoirs used as drinking water supply and for recreational activities. The experimental work was developed according to several phases that are listed and explained below. In a first approach, field surveys were conducted to access the occurrence and distribution of Planktothrix – Chapter 3. It was observed that Planktothrix has a wide distribution in Portuguese lakes and that Planktothrix agardhii is the most commonly found specie. Furthermore microcystin production was detected in isolates from this species. In a second stage, it was developed a method based on real-time PCR to detect and quantify Planktothrix agardhii - Chapter 4. The real-time PCR is a promising technique for cyanobacteria research and monitoring. The main advantage of real-time PCR over conventional PCR is the ability to quantify the target gene copy numbers on a sample. Thus, in addition to identifying Planktothrix strains, the real-time PCR also enables to quantify those strains, which constitutes an advantage over the procedures used in the routine monitoring of cyanobacteria. It should be noted that the determination of the cell FCUP Accessing Planktothrix species diversity and associated toxins using quantitative real-time PCR in natural waters vii density is critical in the risk assessment of toxic cyanobacteria, as the guideline values for cyanotoxins are based on cyanobacterial concentrations as well as on toxin cell quota. Another important aspect in cyanobacteria monitoring is the use of preserved samples. Preservation is used to maintain the morphologic features of cyanobacterial cells to further be used in their identification/quantification and also to avoid sample degradation during transport. In this work, the applicability of the real-time technique in the amplification of DNA from preserved samples was evaluated, by using the method previously developed for cell quantification – Chapter 5. The results indicate that real-time PCR is a robust technique applicable to those types of samples but that the most common preservation methods (Lugol’s solution, formaldehyde, glutaraldehyde) reduce the DNA quantity and quality. Since DNA degrades fast in those samples, the applicability of real-time PCR on preserved samples was tested using other preservation procedure – Chapter 6. The preservation in methanol 100% at -20ºC allowed maintaining the integrity of the samples both for morphologic and molecular analysis up to two years after preservation. The last chapter of this thesis reports the result of two years of monitoring of a reservoir having a persistent bloom of toxic P. agardhii (Appendix A) - Chapter 7. In this reservoir, high cell densities did not always correspond to high amounts of toxin and vice versa. Using the real-time PCR it was demonstrated that both toxic and non-toxic strains are present within the reservoirs and that they can flourish at different times. It was also detected a specie from another genus that also contributes to the production of microcystins. During the monitoring it was observed a chytrid parasite that infected the filaments of Planktothrix (Appendix A). The density of this parasite was also quantified by real-time PCR and the results showed that its development coincides with the increase of toxic Planktotrhix strains and of microcystin levels in the reservoir.

2015-12-17Doctoral thesis

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Evaluation of methanol preservation for molecular and morphological studies in cyanobacteria using Planktothrix agardhii

Publication . Churro, Catarina; Valério, Elisabete; Vieira, Luís; Pereira, Paulo; Vasconcelos, Vitor

Molecular studies on cyanobacteria often involve filtering and freezing of samples leading to loss of cell morphological features. Methanol is often used in preservation of biological materials in association with other fixatives. This study evaluates the application of methanol in the preservation of DNA for molecular studies as well as for the preservation of cell morphology for morphometric analysis in filamentous cyanobacteria. In the present study, both culture and environmental bloom samples were preserved using a cold methanol dehydration series (50, 70, and 100 %) and stored at −20 °C for up to 2 years. The DNA quantity and quality, nucleotide sequence retrieval, and real-time PCR quantification were analyzed over time. Morphometric cell analysis was performed on preserved samples. Results show that the DNA extracted from samples preserved up to 6 months was successfully quantified by real-time PCR. After that period, the DNA quantity decreased with the preservation time. Nevertheless, we were able to detect/amplify the target fragment in samples preserved up to 2 years. The DNA sequence and cell morphology were also maintained during the preservation time. Thus, methanol preservation is an adequate method to preserve molecular information and morphological features after long storage periods.

2015-10-29Journal article

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

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

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.

2015Journal article

Open access