Detection of Microcystis in Lake Sediment using Molecular Genetic Techniques |
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Authors: | Sasidhorn Innok Masatoshi Matsumura Nantakorn Boonkerd Neung Teaumroong |
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Institution: | (1) School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 30000 Nakhon-Ratchasima, Thailand;(2) Institute of Applied Biochemistry, University of Tsukuba, 305-8572 Tsukuba, Ibaraki, Japan |
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Abstract: | Summary Microcystis, which are toxic microcystin-producing cyanobacteria, normally bloom in summer and drop in numbers during the winter season
in Senba Lake, Japan. Recently, this lake has been treated by ultrasonic radiation and jet circulation which were integrated
with flushing with river water. This treatment was most likely sufficient for the destruction of cyanobacterial gas vacuoles.
In order to confirm whether Microcystis viridis was still present, a molecular genetic monitoring technique on the basis of DNA direct extraction from the sediment was applied.
Three primer sets were used for polymerase chain reaction (PCR) based on rRNA intergenic spacer analysis (RISA), the DNA dependent
RNA polymerase (rpoC1) and a Microcystis sp.-specific rpoC1 fragment. The results from each primer were demonstrated on the basis of single strand conformation polymorphisms (SSCP).
Using the RISA primer showed different results from the rpoC1 and Microcystis sp.-specific rpoC1 fragment; meanwhile, the rpoC1 Microcystis sp.-specific fragment was more specific than the RISA primer. Therefore, the Microcystis sp.-specific rpoC1 fragment was further analysed by denaturing gradient gel electrophoresis (DGGE). The DNA pattern representing M. viridis could not be detected in any of the sediment samples. However, the results were confirmed with another technique, terminal
restriction fragment length polymorphisms (T-RFLP). Although T-RFLP patterns of 16S rDNA in sediment at 91 bp and 477 bp lengths
were matched with the T-RFLP of M. viridis (HhaI and MspI endonuclease digestion, respectively), the T-RFLP pattern of 75 bp length was not matched with M. viridis (both of HhaI and MspI endonuclease digestion) which were the major T-RFLP pattern of M. viridis. Therefore, the results most likely indicated that M. viridis seems to have disappeared because of the addition of the ultrasonic radiation and jet circulation to the flushing treatment. |
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Keywords: | Denaturing gradient gel electrophoresis (DGGE) DNA dependent RNA polymerase (rpoC1) Microcystis viridis rRNA intergenic spacer analysis (RISA) Terminal restriction fragment length polymorphism (T-RFLP) |
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