open access

https://hdl.handle.net/2115/47550

Development of an Effective Method for Recovery of Viral Genomic RNA from Environmental Silty Sediments for Quantitative Molecular Detection

英語

2011-06

American Society for Microbiology

Applied and Environmental Microbiology

77(12)

3975-3981

Nine approaches to recover viral RNA from environmental silty sediments were newly developed and compared to quantify RNA viruses in sediments using molecular methods. Four of the nine approaches employed direct procedures for extracting RNA from sediments (direct methods), and the remaining five approaches used indirect methods wherein viral particles were recovered before RNA extraction. A direct method using an SDS buffer with EDTA to lyse viral capsids in sediments, phenol:chloroform:isoamyl alcohol to extract RNA, isopropanol to concentrate RNA, and magnetic beads to purify RNA resulted in the highest recovery rate (geometric mean of 11%, with a geometric standard deviation of 0.02; N = 7) of poliovirus 1 (PV1) inoculated in an environmental sediment sample. The direct method exhibiting the highest PV1 recovery was applied to environmental sediment samples. One hundred eight sediment samples were collected from the Takagi River and its estuary from November 2007 to April 2009, and the genomic RNA of enterovirus and human norovirus in these samples were quantified by RT-qPCR. The human norovirus genome was detected in one sample collected at the bay, although its concentration was below the quantification limit. Meanwhile, the enterovirus genome was detected in two samples at the river mouth and river at concentrations of 8.6 x 10^[2] and 2.4 x 10^[2] copies/g (wet), respectively. This is the first report to obtain quantitative data of a human pathogenic virus in a river and estuarine sediments using RT-qPCR.

学術雑誌論文

Accepted Manuscript

0099-2240