| Title |
Development of mcyB-specific Ultra-Rapid Real-time PCR for Quantitative Detection of Microcystis aeruginosa |
| Authors |
정현철 ( Hyunchul Jung ) ; 임병철 ( Byoungcheol Yim ) ; 임수진 ( Sujin Lim ) ; 김병희 ( Byounghee Kim ) ; 윤병수 ( Byoungsu Yoon ) ; 이옥민 ( Okmin Lee ) |
| DOI |
https://doi.org/10.15681/KSWE.2017.34.1.46 |
| Keywords |
Cyanobacteria; Microcystin; Microcystis; Quantitative PCR; Ultra-Rapid PCR |
| Abstract |
A mcyB-specific Ultra-Rapid quantitative PCR was developed for the quantitative detection of Microcystis aeruginosa, which is often a dominant species in green tide. McyB-specific UR-qPCR was optimized under extremely short times of each step in thermal cycles, based on the specific primers deduced from the mcyB in microcystin synthetase of M. aeruginosa. The M. aeruginosa strain KG07 was used as a standard for quantification, after the microscopic counting and calculation by mcyB-specific UR-qPCR. The water samples from the river water with the Microcystis outbreak were also measured by using both methods. The 1.0 × 108 molecules of mcyB-specific DNA was recognized inner 4 minutes after beginning of UR-qPCR, while 1.0 × 104 molecules of mcyB-specific templates was detected inner 7 minutes with quantitative manner. From the range of 1.0 × 102 to 1.0 × 108 initial molecules, quantification was well established based on CT using mcyB-specific UR-qPCR (Regression coefficiency, R2 = 0.9977). Between the numbers of M. aeruginosa cell counting under microscope and calculated numbers using mcyB-specific UR-qPCR, some differences were often found. The reasons for these differences were discussed; therefore, easy compensation method was proposed that was dependent on the numbers of the cell counting. Additionally, to easily extract the genomic DNA (gDNA) from the samples, a freeze-fracturing of water-sample using liquid nitrogen was tested, by excluding the conventional gDNA extraction method. It was also verified that there were no significant differences using the UR-qPCR with both gDNAs. In conclusion, the mcyB-specific UR-qPCR that we proposed would be expected to be a useful tool for rapid quantification and easy monitoring of M. aeruginosa in environmental water. |