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Evaluation of nucleic acid stabilization products for ambient temperature shipping and storage of viral RNA and antibody in a dried whole blood format.

ABSTRACT: Loss of sample integrity during specimen transport can lead to false-negative diagnostic results. In an effort to improve upon the status quo, we used dengue as a model RNA virus to evaluate the stabilization of RNA and antibodies in three commercially available sample stabilization products: Whatman FTA Micro Cards (GE Healthcare Life Sciences, Pittsburgh, PA), DNAst?ble Blood tubes (Biom?trica, San Diego, CA), and ViveST tubes (ViveBio, Alpharetta, GA). Both contrived and clinical dengue-positive specimens were stored on these products at ambient temperature or 37°C for up to 1 month. Antibody and viral RNA levels were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays, respectively, and compared with frozen unloaded controls. We observed reduced RNA and antibody levels between stabilized contrived samples and frozen controls at our earliest time point, and this was particularly pronounced for the FTA cards. However, despite some time and temperature dependent loss, a 94.6-97.3% agreement was observed between stabilized clinical specimens and their frozen controls for all products. Additional considerations such as cost, sample volume, matrix, and ease of use should inform any decision to incorporate sample stabilization products into a diagnostic testing workflow. We conclude that DNAst?ble Blood and ViveST tubes are useful alternatives to traditional filter paper for ambient temperature shipment of clinical specimens for downstream molecular and serological testing.

SUBMITTER: Dauner AL

PROVIDER: S-EPMC4497903 | biostudies-other | 2015 Jul

REPOSITORIES: biostudies-other

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Evaluation of nucleic acid stabilization products for ambient temperature shipping and storage of viral RNA and antibody in a dried whole blood format.

Dauner Allison L AL Gilliland Theron C TC Mitra Indrani I Pal Subhamoy S Morrison Amy C AC Hontz Robert D RD Wu Shuenn-Jue L SL

The American journal of tropical medicine and hygiene 20150504 1

Loss of sample integrity during specimen transport can lead to false-negative diagnostic results. In an effort to improve upon the status quo, we used dengue as a model RNA virus to evaluate the stabilization of RNA and antibodies in three commercially available sample stabilization products: Whatman FTA Micro Cards (GE Healthcare Life Sciences, Pittsburgh, PA), DNAstāble Blood tubes (Biomātrica, San Diego, CA), and ViveST tubes (ViveBio, Alpharetta, GA). Both contrived and clinical dengue-posit ...[more]

PMID: 25940193

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Project description:BackgroundStool metabolites provide essential insights into the function of the gut microbiome. The current gold standard for storage of stool samples for metabolomics is flash-freezing at - 80 °C which can be inconvenient and expensive. Ambient temperature storage of stool is more practical, however no available methodologies adequately preserve the metabolomic profile of stool. A novel sampling kit (OMNImet.GUT; DNA Genotek, Inc.) was introduced for ambient temperature storage and stabilization of feces for metabolomics; we aimed to test the performance of this kit vs. flash-freezing. To do this stool was collected from an infant's diaper was divided into two aliquots: 1) flash-frozen and 2) stored in an OMNImet.GUT tube at ambient temperature for 3-4 days. Samples from the same infant were collected at 2 different time points to assess metabolite changes over time. Subsequently, all samples underwent metabolomic analysis by liquid chromatography - tandem mass spectrometry (LC-MS/MS).ResultsPaired fecal samples (flash-frozen and ambient temperature) from 16 infants were collected at 2 time points (32 individual samples, 64 aliquots). Similar numbers of metabolites were detected in both the frozen and ambient temperature samples (1126 in frozen, 1107 in ambient temperature, 1064 shared between sample types). Metabolite abundances were strongly correlated between storage methods (median Spearman correlation Rs = 0.785 across metabolites). Hierarchical clustering analysis and principal component analysis showed that samples from the same individuals at a given time point clustered closely, regardless of the storage method. Repeat samples from the same individual were compared by paired t-test, separately for the frozen and OMNImet.GUT. The number of metabolites in each biochemical class that significantly changed (p < 0.05) at timepoint 2 relative to timepoint 1 was similar in flash-frozen versus ambient temperature storage. Changes in microbiota modified metabolites over time were also consistent across both methodologies.ConclusionAmbient temperature storage and stabilization of stool in the OMNImet.GUT device yielded comparable metabolomic results to flash freezing in terms of 1) the identity and abundance of detected biochemicals 2) the distinct metabolomic profiles of subjects and 3) changes in metabolites over time that are plausibly microbiota-induced. This method potentially provides a more convenient, less expensive home collection and storage option for stool metabolomic analysis.

Project description:In this study, 28 "historical" clinical freeze-dried nontuberculous mycobacterial isolates collected from 1948 to 1957, were analyzed by investigating their viability and performing whole genome sequencing (WGS) on DNA extracted (i) directly from freeze-dried cells versus (ii) after culturing, to determine cell properties and DNA quality after centuries of freeze-dried storage. The isolated DNA was sequenced on the Illumina MiSeq platform and data quality evaluated analyzing the per-base quality scores of paired-end sequencing reads as well as the overall contiguity of resulting de novo assemblies. After 72 years in storage, all freeze-dried isolates were viable, and showed no signs of cell damage and limited signs of contamination when reculturing. They were recultured without problems and identified through WGS with only four of 13 parameters showing statistical significance based on sequence data obtained directly from the freeze-dried cells versus after reculturing, indicating no DNA degradation. Thus, mycobacteria can be whole genome sequenced successfully directly from freeze-dried material without prior recultivation, saving laboratory time and resources, and emphasizing the value of freeze-drying for long-term storage. Our study lays the groundwork for further genomic investigations of freeze-dried bacterial isolates, and the approximately 4,000 historical isolates in our collection will provide a unique opportunity to investigate mycobacterial DNA from a variety of NTM species unexposed to antimicrobials, some maybe still undescribed species. IMPORTANCE The genus Mycobacterium was described more than a century ago and new species are continuously identified and described. There is an ongoing discussion about an increase in the incidence of disease caused by nontuberculous mycobacteria (NTM). How the different bacteria looked before exposure to antibiotics can only be investigated by looking at strains from before the antibiotic era. Strains from that era will be stored in different ways, for example by freeze-drying. The question is how to investigate these strains, and if they are still viable, whether they need to be cultured, and if that changes the DNA. Here, we test all these parameters on freeze-dried strains and show that NGS can be applied directly without culturing.

Project description:BACKGROUND: Stool metabolites provide essential insights into the function of the gut microbiome. The current gold standard for storage of stool samples for metabolomics is flash-freezing at - 80 °C which can be inconvenient and expensive. Ambient temperature storage of stool is more practical, however no available methodologies adequately preserve the metabolomic profile of stool. A novel sampling kit (OMNImet.GUT; DNA Genotek, Inc.) was introduced for ambient temperature storage and stabilization of feces for metabolomics; we aimed to test the performance of this kit vs flash-freezing. To do this stool was collected from an infant's diaper was divided into 2 aliquots: 1) flash-frozen and 2) stored in an OMNImet.GUT tube at ambient temperature for 3-4 days. Samples from the same infant were collected at 2 different timepoints to assess metabolite changes over time. Subsequently, all samples underwent metabolomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).RESULTS: Paired fecal samples (flash-frozen and ambient temperature) from 16 infants were collected at 2 timepoints (32 individual samples, 64 aliquots). Similar numbers of metabolites were detected in both the frozen and ambient temperature samples (1126 in frozen, 1107 in ambient temperature, 1064 shared between sample types). Metabolite abundances were strongly correlated between storage methods (median Spearman correlation Rs = 0.785 across metabolites). Hierarchical clustering analysis and principal component analysis showed that samples from the same individuals at a given timepoint clustered closely, regardless of the storage method. Repeat samples from the same individual were compared by paired t-test, separately for the frozen and OMNImet.GUT. The number of metabolites in each biochemical class that significantly changed (p < 0.05) at timepoint 2 relative to timepoint 1 was similar in flash-frozen vs ambient temperature storage. Changes in microbiota modified metabolites over time were also consistent across both methodologies.CONCLUSION: Ambient temperature storage and stabilization of stool in the OMNImet.GUT device yielded comparable metabolomic results to flash freezing in terms of 1) the identity and abundance of detected biochemicals 2) the distinct metabolomic profiles of subjects and 3) changes in metabolites over time that are plausibly microbiota-induced. This method potentially provides a more convenient, less expensive home collection and storage option for stool metabolomic analysis.

Dataset Information

Evaluation of nucleic acid stabilization products for ambient temperature shipping and storage of viral RNA and antibody in a dried whole blood format.

Publications

Evaluation of nucleic acid stabilization products for ambient temperature shipping and storage of viral RNA and antibody in a dried whole blood format.

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