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Research Containing: Specimen Handling

Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

by on 9 June 2015in Biology & Biotechnology No comment

The development of fast and easy on-site molecular detection and quantification methods for hazardous microbes on solid surfaces is desirable for several applications where specialised laboratory facilities are absent. The quantification of bacterial contamination necessitates the assessment of the efficiency of the used methodology as a whole, including the preceding steps of sampling and sample processing. We used quantitative real-time polymerase chain reaction (qrtPCR) for Escherichia coli and Staphylococcus aureus to measure the recovery of DNA from defined numbers of bacterial cells that were subjected to three different DNA extraction methods: the QIAamp DNA Mini Kit, Reischl et al.'s method and FTA Elute. FTA Elute significantly showed the highest median DNA extraction efficiency of 76.9% for E. coli and 108.9% for S. aureus. The Reischl et al. method and QIAamp DNA Mini Kit inhibited the E. coli qrtPCR assay with a 10-fold decrease of detectable DNA. None of the methods inhibited the S. aureus qrtPCR assay. The FTA Elute applicability was demonstrated with swab samples taken from the International Space Station (ISS) interior. Overall, the FTA Elute method was found to be the most suitable to selected criteria in terms of rapidity, easiness of use, DNA extraction efficiency, toxicity, and transport and storage conditions.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/21311936

Plant molecular biology in the space station era: utilization of KSC fixation tubes with RNAlater

by on 9 June 2015in Biology & Biotechnology No comment

Spaceflight experiments involving biological specimens face unique challenges with regard to the on orbit harvest and preservation of material for later ground-based analyses. Preserving plant material for gene expression analyses requires that the tissue be prepared and stored in a manner that maintains the integrity of RNA. The liquid preservative RNAlater (Ambion) provides an effective alternative to conventional freezing strategies, which are limited or unavailable in current spaceflight experiment scenarios. The spaceflight use of RNAlater is enabled by the Kennedy space center fixation tube (KFT), hardware designed to provide the necessary containment of fixatives during the harvest and stowage of biological samples in space. Pairing RNAlater with the KFT system provides a safe and effective strategy for preserving plant material for subsequent molecular analyses, a strategy that has proven effective in several spaceflight experiments. Possible spaceflight scenarios for the use of RNAlater and KFTs are explored and discussed.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/15736319

Bacterial monitoring with adhesive sheet in the international space station-"Kibo", the Japanese experiment module

by on 9 June 2015in Biology & Biotechnology No comment

Microbiological monitoring is important to assure microbiological safety, especially in long-duration space habitation. We have been continuously monitoring the abundance and diversity of bacteria in the International Space Station (ISS)-"Kibo" module to accumulate knowledge on microbes in the ISS. In this study, we used a new sampling device, a microbe-collecting adhesive sheet developed in our laboratory. This adhesive sheet has high operability, needs no water for sampling, and is easy to transport and store. We first validated the adhesive sheet as a sampling device to be used in a space habitat with regard to the stability of the bacterial number on the sheet during prolonged storage of up to 12 months. Bacterial abundance on the surfaces in Kibo was then determined and was lower than on the surfaces in our laboratory (10(5) cells [cm(2)](-1)), except for the return air grill, and the bacteria detected in Kibo were human skin microflora. From these aspects of microbial abundance and their phylogenetic affiliation, we concluded that Kibo has been microbiologically well maintained; however, microbial abundance may increase with the prolonged stay of astronauts. To ensure crew safety and understand bacterial dynamics in space habitation environments, continuous bacterial monitoring in Kibo is required.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/23603802