Novel bioluminescent quantitative detection of nucleic acid amplification in real-time.

BACKGROUND: The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. PRINCIPAL FINDINGS: Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. CONCLUSIONS: The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings

Additional file 1 of MomsTalkShots, tailored educational app, improves vaccine attitudes: a randomized controlled trial

Additional file 1: Appendix 1. Impact of MomsTalkShots on Vaccine KABs of Women One Month After Their Infant's Birth individual survey items, organized by constructs, stratified by baseline vaccine intentionsa, dichotomous analysisb. Appendix 2. Impact of MomsTalkShots on Vaccine KABs of Women One Year After Their Infant's Birth individual survey items, organized by constructs, stratified by baseline vaccine intentionsa, dichotomous analysisb. Appendix 3. Impact of MomsTalkShots on Vaccine KABs of Women One Month After Their Infant's Birth individual survey items, organized by constructs, stratified by baseline vaccine intentionsa, continuous analysisb. Appendix 4. Impact of MomsTalkShots on Vaccine KABs of Women One Year After Their Infant's Birth individual survey items, organized by constructs, stratified by baseline vaccine intentionsa, continuous analysisb. Appendix 5. Impact of MomsTalkShots on Women's Perceived Risk of Infant Pertussis One Month After Their Infant's Birth individual survey items, stratified by baseline vaccine intentionsa and Tdap vaccination, dichotomous analysisb. Appendix 6. Impact of MomsTalkShots on Women's Perceived Risk of Infant Pertussis One Year After Their Infant's Birth individual survey items, stratified by baseline vaccine intentionsa and Tdap vaccination, dichotomous analysisb. Appendix 7. Survey Initiation and Completion stratified by study arm. Appendix 8. Follow-Up Survey Completion stratified by maternal and infant vaccine intentions at baseline. Appendix 9. Response Rates for Vaccine KAB Constructs at Each Study Timepoint. Appendix 10. Maternal and Infant Vaccine Intentions at Baseline stratified by 4 study arms

Re-deposited cryptotephra layers in Holocene peats linked to anthropogenic activity

Tephra layers can form useful age-equivalent stratigraphic markers for correlating palaeoenvironmental sequences and they provide information about the spatio-temporal nature of past volcanic ash fall events. The use of microscopic ‘cryptotephra’ layers has both increased the stratigraphic resolution of tephra sequences in proximal areas and extended the distal application of tephrochronology to regions of the world situated far from volcanoes. Effective tephrochronology requires the discrimination between in situ tephra deposited directly from volcanic plumes and tephras that have been remobilised since their initial deposition. We present tephrostratigraphic and glass chemistry data from two proximal peat profiles (one lowland, one upland) from the Shetland Islands, UK. Both profiles contain the Hekla-Selsund tephra (deposited c. 1800–1750 cal. BC), whilst the Hekla 4 ash (c. 2395–2279 cal. BC) is present in the upland record. Overlying the Hekla-Selsund tephra are a number of distinct peaks in tephra shard abundance. The geochemistry of these layers shows that they represent re-working of the Hekla 4 and Hekla-Selsund layers rather than primary air-fall deposits. Pollen analysis of the peat sequences illustrates that these re-deposited tephra layers are coincident with a rise in heather-dominated vegetation communities (heath and/or moorland) and a subsequent intensification of burning in the landscape. We suggest that burning caused increased erosion of peats resulting in the remobilisation of tephra shards. The study demonstrates both the need for caution and the opportunities created when applying tephrochronologies in regions heavily affected by past human activity that contain both reworked tephra layers and in situ fallout