INTRODUCTION: The occurrence of Harmful Algal Blooms (HABs) in the Mediterranean Sea is an important and increasing problem involving human health and fishing industry. In fact, algal toxins can accumulate in mussels and, after consumption, can cause human syndromes as Paralytic Shellfish Poisoning (PSP) or Diarrhoeic Shellfish Poisoning (DSP). The dinoflagellates belonging to the genus Alexandrium are the most widespread toxic microalgae responsible for PSP intoxication in the Mediterranean basin (1). The monitoring of toxic microalgal species in the seawater actually requires accurate morphological identification and the enumeration of target phytoplankton species by using standard microscopy procedures. These procedures are time-consuming and demand a remarkable taxonomic experience, since identification is based on the recognition of morphological characteristics (2). In recent years the development of molecular methods as diagnostic tools has revolutionised the methodological approach for the identification of HAB species, because these methods can rapidly detect limited numbers of specific organisms in mixed ecological scenarios. In particular, the ribosomal DNA has been selected as targets for developing PCR assay in microalgae (3). In this contest, an effective and selective DNA extraction method could be helpful to increase sensitivity and specificity of a standard PCR assay. We tested a DNA extraction method based on silica-coated super paramagnetic nanoparticles conjugated to a capture DNA sequence complementary to a specific region of the Alexandrium rDNA. A. catenella cultured cells were used as target for the DNA extractions and a PCR assay was performed with primers specific for Alexandrium spp. (4) after each purification, in order to assess specificity and sensitivity of the method. MATERIALS AND METHODS: The capture probe AP (5'-CCC CCC TYG GGA TAT KCT TGA AGG TDT G-3') was designed on the 5.8S rDNA consensus sequence for Alexandrium spp. and was modified with a C6 amino linker at the 5' end. Moreover, 6 extra cytidine residues were incorporated at the 5' end to minimize inhibitory effects including steric hindrance. The amino-modified silica-coated nanoparticles were coupled with the amino-modified probe via glutaraldehyde-based chemistry. The capture efficiency was tested in the hybrid capture using a complementary fluorescent probe. The DNA purification was performed by adding the modified paramagnetic nanoparticels to the raw cell lysates containing various amount of target A. catenella cells. The nanoparticles were collected using a magnet, washed three times and the volume equivalent to 5 g of nanoparticles were added directly in the PCR tube, avoiding the DNA elution step, and amplification was carried out using Alexandrium-specific primers (4). The PCR products were visualized on agarose gel. RESULTS: The hybrid capture test showed that 105 pmoles of fluorescein labelled probe could be selectively captured and recovered onto 1 mg of AP-modified nanoparticles. In order to test the possibility to use the nanoparticles directly in PCR without elution of bound DNA, we performed PCR reactions with different amount of nanoparticles to see potentially inhibitory effects of these materials. We found that 5 g of nanoparticles can be used per reaction tube without any significant PCR inhibition. We used paramagnetic nanoparticles modified with the capture probe to selectively purify Alexandrium DNA from samples containing different amount of A. catenella cells mixed with different amount of Navicula spp. cells (diatoms). The percentages of A. catenella cells in the samples were from 20% to 0.01% of the total cells, while the absolute number of A. catenella cells in the samples were from 100 to 10. After DNA purification and PCR reaction we found an amplification product in all samples. In a further single experiment with a sample containing one A. catenella cell and 100,000 Navicula spp. cells as background (0.001%), we also had a positive result after PCR reaction. These results indicate that the use of probe-conjugated paramagnetic nanoparticles could be effective for the specific purification of microalgal DNA in mixed samples, ensuring sensitivity and specificity of the following PCR reaction. REFERENCES 1. Vila M et al. (2001) J. Plankton Res. 23, 497-514 2. Steidinger KA et al. (1997) in Identifying marine phytoplankton, Academic Press, Florida, 387-584 3. Penna A et al. (2000) J. Phycol. 36, 1183-1186 4. Galluzzi L et al. Harmful Algae submitted paper

SPECIFIC PURIFICATION OF DNA FROM ALEXANDRIUM SPP (DINOFLAGELLATE) USING A CAPTURE PROBE CONJUGATED TO PARAMAGNETIC NANOPARTICLES

BERTOZZINI, ELENA;GALLUZZI, LUCA;PENNA, ANTONELLA;MAGNANI, MAURO
2004-01-01

Abstract

INTRODUCTION: The occurrence of Harmful Algal Blooms (HABs) in the Mediterranean Sea is an important and increasing problem involving human health and fishing industry. In fact, algal toxins can accumulate in mussels and, after consumption, can cause human syndromes as Paralytic Shellfish Poisoning (PSP) or Diarrhoeic Shellfish Poisoning (DSP). The dinoflagellates belonging to the genus Alexandrium are the most widespread toxic microalgae responsible for PSP intoxication in the Mediterranean basin (1). The monitoring of toxic microalgal species in the seawater actually requires accurate morphological identification and the enumeration of target phytoplankton species by using standard microscopy procedures. These procedures are time-consuming and demand a remarkable taxonomic experience, since identification is based on the recognition of morphological characteristics (2). In recent years the development of molecular methods as diagnostic tools has revolutionised the methodological approach for the identification of HAB species, because these methods can rapidly detect limited numbers of specific organisms in mixed ecological scenarios. In particular, the ribosomal DNA has been selected as targets for developing PCR assay in microalgae (3). In this contest, an effective and selective DNA extraction method could be helpful to increase sensitivity and specificity of a standard PCR assay. We tested a DNA extraction method based on silica-coated super paramagnetic nanoparticles conjugated to a capture DNA sequence complementary to a specific region of the Alexandrium rDNA. A. catenella cultured cells were used as target for the DNA extractions and a PCR assay was performed with primers specific for Alexandrium spp. (4) after each purification, in order to assess specificity and sensitivity of the method. MATERIALS AND METHODS: The capture probe AP (5'-CCC CCC TYG GGA TAT KCT TGA AGG TDT G-3') was designed on the 5.8S rDNA consensus sequence for Alexandrium spp. and was modified with a C6 amino linker at the 5' end. Moreover, 6 extra cytidine residues were incorporated at the 5' end to minimize inhibitory effects including steric hindrance. The amino-modified silica-coated nanoparticles were coupled with the amino-modified probe via glutaraldehyde-based chemistry. The capture efficiency was tested in the hybrid capture using a complementary fluorescent probe. The DNA purification was performed by adding the modified paramagnetic nanoparticels to the raw cell lysates containing various amount of target A. catenella cells. The nanoparticles were collected using a magnet, washed three times and the volume equivalent to 5 g of nanoparticles were added directly in the PCR tube, avoiding the DNA elution step, and amplification was carried out using Alexandrium-specific primers (4). The PCR products were visualized on agarose gel. RESULTS: The hybrid capture test showed that 105 pmoles of fluorescein labelled probe could be selectively captured and recovered onto 1 mg of AP-modified nanoparticles. In order to test the possibility to use the nanoparticles directly in PCR without elution of bound DNA, we performed PCR reactions with different amount of nanoparticles to see potentially inhibitory effects of these materials. We found that 5 g of nanoparticles can be used per reaction tube without any significant PCR inhibition. We used paramagnetic nanoparticles modified with the capture probe to selectively purify Alexandrium DNA from samples containing different amount of A. catenella cells mixed with different amount of Navicula spp. cells (diatoms). The percentages of A. catenella cells in the samples were from 20% to 0.01% of the total cells, while the absolute number of A. catenella cells in the samples were from 100 to 10. After DNA purification and PCR reaction we found an amplification product in all samples. In a further single experiment with a sample containing one A. catenella cell and 100,000 Navicula spp. cells as background (0.001%), we also had a positive result after PCR reaction. These results indicate that the use of probe-conjugated paramagnetic nanoparticles could be effective for the specific purification of microalgal DNA in mixed samples, ensuring sensitivity and specificity of the following PCR reaction. REFERENCES 1. Vila M et al. (2001) J. Plankton Res. 23, 497-514 2. Steidinger KA et al. (1997) in Identifying marine phytoplankton, Academic Press, Florida, 387-584 3. Penna A et al. (2000) J. Phycol. 36, 1183-1186 4. Galluzzi L et al. Harmful Algae submitted paper
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11576/2564974
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