Microcystis aeruginosa의 정량을 위한 mcyB 특이 초고속 실시간 유전자 증폭법의 개발 (Development of mcyB-specific Ultra-Rapid Real-time PCR for Quantitative Detection of Microcystis aeruginosa)

서지정보

· 발행기관 : 한국물환경학회
· 수록지 정보 : 한국물환경학회지 / 34권 / 1호 / 46 ~ 56페이지
· 저자명 : 정현철, 임병철, 임수진, 김병희, 윤병수, 이옥민

초록

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 timesof 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 countingand calculation by mcyB-specific UR-qPCR. The water samples from the river water with the Microcystis outbreakwere also measured by using both methods. The 1.0 × 108 molecules of mcyB-specific DNA was recognized inner 4minutes after beginning of UR-qPCR, while 1.0 × 104 molecules of mcyB-specific templates was detected inner 7minutes with quantitative manner. From the range of 1.0 × 102 to 1.0 × 108 initial molecules, quantification was wellestablished based on CT using mcyB-specific UR-qPCR (Regression coefficiency, R2 = 0.9977). Between the numbersof M. aeruginosa cell counting under microscope and calculated numbers using mcyB-specific UR-qPCR, somedifferences were often found. The reasons for these differences were discussed; therefore, easy compensation methodwas proposed that was dependent on the numbers of the cell counting. Additionally, to easily extract the genomicDNA (gDNA) from the samples, a freeze-fracturing of water-sample using liquid nitrogen was tested, by excludingthe conventional gDNA extraction method. It was also verified that there were no significant differences using theUR-qPCR with both gDNAs. In conclusion, the mcyB-specific UR-qPCR that we proposed would be expected to bea useful tool for rapid quantification and easy monitoring of M. aeruginosa in environmental water.

영어초록

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 timesof 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 countingand calculation by mcyB-specific UR-qPCR. The water samples from the river water with the Microcystis outbreakwere also measured by using both methods. The 1.0 × 108 molecules of mcyB-specific DNA was recognized inner 4minutes after beginning of UR-qPCR, while 1.0 × 104 molecules of mcyB-specific templates was detected inner 7minutes with quantitative manner. From the range of 1.0 × 102 to 1.0 × 108 initial molecules, quantification was wellestablished based on CT using mcyB-specific UR-qPCR (Regression coefficiency, R2 = 0.9977). Between the numbersof M. aeruginosa cell counting under microscope and calculated numbers using mcyB-specific UR-qPCR, somedifferences were often found. The reasons for these differences were discussed; therefore, easy compensation methodwas proposed that was dependent on the numbers of the cell counting. Additionally, to easily extract the genomicDNA (gDNA) from the samples, a freeze-fracturing of water-sample using liquid nitrogen was tested, by excludingthe conventional gDNA extraction method. It was also verified that there were no significant differences using theUR-qPCR with both gDNAs. In conclusion, the mcyB-specific UR-qPCR that we proposed would be expected to bea useful tool for rapid quantification and easy monitoring of M. aeruginosa in environmental water.

참고자료

· 없음