Departmental Bulletin Paper 異なるDNA精製法が定量PCRによるEdwardsiella tardaの計数に与える影響

朝重, 紅音  ,  菅, 向志郎  ,  金井, 欣也

99pp.7 - 11 , 2018-03 , 長崎大学水産学部
Quantitative PCR (qPCR) quantifies the template DNA from the number of threshold cycles (Ct value), but the probability of quantitative errors occurs due to the differences in recovery rates during DNA purification. In order to investigate the effect of different DNA purification kits in counting Edwardsiella tarda cells using qPCR, we compared two methods; the bacterial culture dilution method, in which the DNA is purified after 5 steps of a ten-fold serial dilution of E. tarda culture, and the DNA dilution method, which the DNA is diluted after purification with the optimum cell number of E. tarda. In both methods, we used three types of DNA purification kit: Chelex resin, Non-Silica membrane and Silica membrane. Our results showed the lowest Ct value and high DNA recovery rate were obtained using Chelex resin. Non-Silica membrane and Silica membrane Ct values in DNA dilution method were higher than that of Chelex resin. With the use of Non-Silica membrane, the calculated DNA using bacterial culture dilution method decreased by 67-87% compared to DNA dilution method at 1.0×10 4cells/μL of E. tarda.

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