Trial of PCR to Detect Microbial Contamination in Ground Strata Underneath Osaka City

Abstract

In the present study, we checked the subsurface layers of Osaka city for microorganisms using PCR and investigated whether genetic diagnosis could be used to estimate the microbial contamination of the ground strata. The 145 soil specimens from 2.05 to 29.35 meters underground were obtained by boring surveys at Minami Izuo park in the west of Osaka city. Microbial DNA was detected by PCR using the primer sets P3MOD/PC5B for eubacteria, NS1/NS2 for fungi, protists, and green algae, and ZL-1675/ZL2548 for E. coli ., which were able to recognize microbial DNA in the soil dose-dependently. Increase in the number of inoculated organisms led to increase in the number of amplicons in the preliminary experiment. The PCR amplicons of protists and bacteria were detected well in the top-level sand stratum, but few signals were produced by these bands, especially protists, in the clay-stratum specimens from increasing depths. However, the NS1/NS2 band reappeared in the deeper sand stratum located at around 22.5 meters underground. Protists seem to be found more abundantly in sand than clay strata because they can move easily in and with groundwater. Only one of the 145 samples showed the positive reaction to the ZL-1675/ZL2548 primer set that suggests the presence of E. coli in the ground stratum. The results indicate that microbial DNA is present in the ground strata of Osaka city; helpful would be an investigation to assess how microbes reach the ground strata and contaminate groundwater.