Abstract
The method to analyze both eukaryotic and prokaryotic microorganisms without preliminary microbial information of sample seemed to be useful not only for research and investigation of microorganisms but also for industry using microorganisms. In the present manuscript, preparation of a new DNA primers, new reference database for 18S rDNA for our newly developed method [1]- [3], and analyses of eukaryotic and prokaryotic microorganisms in fermentation products were presented. In komekouji, Aspergillus spp., was enumerated to be 46.5 × 106 MPN g-1, and Penicillium spp., was enumerated to be 1.5 × 106 MPN g-1. In dry yeast, Saccharomyces group, were enumerated to be 8600 × 106 MPN g-1. In komekouji-miso, no eukaryotic microorganism was detected, while the other Bacillus spp., was numerically dominant (21.5 × 106 MPN g-1) as prokaryotic microorganisms, followed by B. subtilis group (4.65 × 106 MPN g-1), and the other Firmicutes (3.7 × 106 MPN g-1). The komekouji-miso included lower number of Actinobacteria (0.15 × 106 MPN g-1), Burkhokderia sp. (1.5 × 106 MPN g-1), and the other α,β,γ-proteobacteria (0.12 × 106 MPN g-1). In sake-kasu, both prokaryote and eukaryote were not detected by the method. Present results indicated that using both universal primers for eukaryotic and prokaryotic microorganisms, each groups of prokaryotic and eukaryotic microorganisms were enumerated without any preliminary information nor setting up standard curve, which were required for real time PCR.
Highlights
By PCR-based analysis methods such as real time quantitative PCR or multiplex PCR, eukaryotic and prokaryotic microorganisms [4]-[7] could be detected or quantified by the same procedure by changing used DNA primers
By quantitative PCR (qPCR) using universal primer for the both microorganisms, total numbers of eukaryote and prokaryote in sample could be enumerated, we could not know what kinds of microorganisms were mainly included without analyzing amplified DNA [4]
As numbers of each microbial groups were estimated by MPN not by an amount of the amplified DNA in this method as described in the former manuscripts [1] [2], the difference in the primers did not affect the numbers and composition of numerically dominant microorganisms but their effect was limited in numerically minor microorganisms
Summary
By PCR-based analysis methods such as real time quantitative PCR (qPCR) or multiplex PCR, eukaryotic and prokaryotic microorganisms [4]-[7] could be detected or quantified by the same procedure by changing used DNA primers. By qPCR using universal primer for the both microorganisms, total numbers of eukaryote and prokaryote in sample could be enumerated, we could not know what kinds of microorganisms were mainly included without analyzing amplified DNA [4]. By the multiplex PCR or qPCR using several selective primers, the exact numbers of each specific microorganisms could be detected or enumerated, we could not know what kinds of microorganisms were mainly included, because selective primer targeted the specific microorganisms and afford no-information as to microorganisms which were not covered by the used primers [4] [8]-[10]. We could not know relative abundance of each microbial group exactly, because of the PCR bias, which altered the ratio of each microbial groups in the amplified DNA from that of original DNA in sample [15]-[18]. We could not compare a number of eukaryote to that of prokaryote obtained by different PCR primers, respectively [11]-[14], because the amplification rate and PCR bias were varied with the used DNA primers
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