An enclosed in-gel PCR amplification cassette with multi-target, multi-sample detection for platform molecular diagnostics

This work describes a self-contained, simple, disposable, and inexpensive gel capillary cassette for DNA amplification in near point of care settings. The cassette avoids the need for pumps or valves during raw sample delivery or polymerase chain reaction (PCR) amplification steps. The cassette contains capillary reaction units that can be stored at room temperature for up to 3 months. The current cassette configuration format can simultaneously tests up to 16 patients for two or more targets, accommodates different sample types on the same cassette, has integrated positive and negative controls and allows flexibility for multiple geometries. PCR reagents in the cassette are desiccated to allow storage at room temperature with rehydration by raw sample at the time of testing. The sample is introduced to the cassette via a transfer pipette simply by capillary force. DNA amplification was carried out in a portable prototype instrument for PCR thermal cycling with fluorescence detection of amplified products by melt curve analysis. To demonstrate performance, raw genital swabs and urine were introduced to the same cassette to simultaneously detect four sexually transmitted infections. Herpes Simplex Viruses (HSV-1 and HSV-2) were detected from raw genital swabs. Ureaplasma Urealyticum (UU) and Mycoplasma Homonis (MH) were detected from raw urine. Results for multiple patients were obtained in as little as 50'. This platform allows multiparameter clinical testing with a pre-assembled cassette that requires only the introduction of raw sample. Modification of the prototype device to accommodate larger cassettes will ultimately provide high throughput simultaneous testing of even larger numbers of samples for many different targets, as is required for most clinical applications. Combinations of wax and/or polymer cassettes holding capillary reaction units are feasible. The components of the cassette are suited to mass production and robotic assembly to produce a readily manufactured disposable reaction cassette that can be configured for disease-specific testing panels. Rapid testing with a disposable reaction cassette on an inexpensive instrument will permit on the spot evaluation of patients in the clinic for faster medical decision-making and more informed therapeutic choices

Local spatial structure of forest biomass and its consequences for remote sensing of carbon stocks

Advances in forest carbon mapping have the potential to greatly reduce uncertainties in the global carbon budget and to facilitate effective emissions mitigation strategies such as REDD+. Though broad scale mapping is based primarily on remote sensing data, the accuracy of resulting forest carbon stock estimates depends critically on the quality of field measurements and calibration procedures. The mismatch in spatial scales between field inventory plots and larger pixels of current and planned remote sensing products for forest biomass mapping is of particular concern, as it has the potential to introduce errors, especially if forest biomass shows strong local spatial variation. Here, we used 30 large (8–50 ha) globally distributed permanent forest plots to quantify the spatial variability in aboveground biomass (AGB) at spatial grains ranging from 5 to 250m (0.025–6.25 ha), and we evaluate the implications of this variability for calibrating remote sensing products using simulated remote sensing footprints. We found that the spatial sampling error in AGB is large for standard plot sizes, averaging 46.3% for 0.1 ha subplots and 16.6% for 1 ha subplots. Topographically heterogeneous sites showed positive spatial autocorrelation in AGB at scales of 100m and above; at smaller scales, most study sites showed negative or nonexistent spatial autocorrelation in AGB. We further show that when field calibration plots are smaller than the remote sensing pixels, the high local spatial variability in AGB leads to a substantial “dilution” bias in calibration parameters, a bias that cannot be removed with current statistical methods. Overall, our results suggest that topography should be explicitly accounted for in future sampling strategies and that much care must be taken in designing calibration schemes if remote sensing of forest carbon is to achieve its promise

Population Dynamics and Diversity of Viruses, Bacteria and Phytoplankton in a Shallow Eutrophic Lake

We have studied the temporal variation in viral abundances and community assemblage in the eutrophic Lake Loosdrecht through epifluorescence microscopy and pulsed field gel electrophoresis (PFGE). The virioplankton community was a dynamic component of the aquatic community, with abundances ranging between 5.5 × 107 and 1.3 × 108 virus-like particles ml−1 and viral genome sizes ranging between 30 and 200 kb. Both viral abundances and community composition followed a distinct seasonal cycle, with high viral abundances observed during spring and summer. Due to the selective and parasitic nature of viral infection, it was expected that viral and host community dynamics would covary both in abundances and community composition. The temporal dynamics of the bacterial and cyanobacterial communities, as potential viral hosts, were studied in addition to a range of environmental parameters to relate these to viral community dynamics. Cyanobacterial and bacterial communities were studied applying epifluorescence microscopy, flow cytometry, and denaturing gradient gel electrophoresis (DGGE). Both bacterial and cyanobacterial communities followed a clear seasonal cycle. Contrary to expectations, viral abundances were neither correlated to abundances of the most dominant plankton groups in Lake Loosdrecht, the bacteria and the filamentous cyanobacteria, nor could we detect a correlation between the assemblage of viral and bacterial or cyanobacterial communities during the overall period. Only during short periods of strong fluctuations in microbial communities could we detect viral community assemblages to covary with cyanobacterial and bacterial communities. Methods with a higher specificity and resolution are probably needed to detect the more subtle virus–host interactions. Viral abundances did however relate to cyanobacterial community assemblage and showed a significant positive correlation to Chl-a as well as prochlorophytes, suggesting that a significant proportion of the viruses in Lake Loosdrecht may be phytoplankton and more specific cyanobacterial viruses. Temporal changes in bacterial abundances were significantly related to viral community assemblage, and vice versa, suggesting an interaction between viral and bacterial communities in Lake Loosdrecht

Application of lab-on-a-chip multiplex cassette PCR for the detection of enterohemorrhagic Escherichia coli

Abstract Background Fast molecular detection methods benefit from ready-to-run lab-on-a-chip molecular assays with minimum preparation time. Detection efficiency of such methods can improve if multiple targets are detected simultaneously per given reaction. Detection of food pathogens, i.e. Escherichia coli (E. coli), is generally performed in two stages with the detection of multiple targets in each stage.With simultaneous testing, screening for pathogens is fast and efficient. Results In this study, we show the application of multiplex PCR performed on a ready-made cassette to detect 10 targets each for eight samples known to harbor E. coli. In cassette PCR, the aluminum cassette (38.6 mm × 31.4 mm) contains 10 trenches having a total of 50 capillaries with microliter volumes of desiccated acrylamide gels holding all reagents required for the PCR including internal positive and negative controls. The gel contains LCGreen dye to detect double stranded DNA. Fluorescence monitoring allows the detection of the amplified products by melt curve analysis. In this application, each of the five capillaries in a given trench contains two of the primer sets for the detection of 10 targets in pathogenic E. coli, namely, O157, Eae, Stx1, Stx2 and six O-antigen genes. Primer specificity was confirmed. Each trench tests one sample. Eight minimally processed enriched beef carcass swab samples were analyzed for parallel detection of 10 targets within 1 h and 15 min. Samples were delivered to the capillaries by capillary forces thereby hydrating the gels. Multiplex cassette PCR results were confirmed with conventional multiplex PCRs performed in a commercial real-time PCR system. Conclusions Cassette PCR technology is ideally suited to multi-target detection of pathogens in food products. The cassette performs multiple PCR reactions in parallel, with multiplex detection of targets within each reaction unit. Cassette PCR/ melt curve analysis results for the simultaneous detection of 10 targets of pathogenic E.coli in beef carcass swab samples were confirmed with a conventional real-time PCR/ melt curve analysis as well as with agarose gel electrophoresis. Although designed for the detection of E. coli, this multiplex cassette PCR technique can be applied to any other assay where the fast detection of multiple targets is required

Comparison of a Miniaturized Cassette PCR System with a Commercially Available Platform for Detecting Escherichia coli in Beef Carcass Swabs

Detection sensitivity of cassette PCR was compared with a commercial BAX® PCR system for detection of eae and stx genes in Escherichia coli from 806 beef carcass swabs. Cassette PCR detects multiple genetic markers on multiple samples using PCR and melt curve analysis. Conventional PCR served as a gold standard. Overall, for positive and negative concordance, cassette PCR was 98.6% concordant with conventional PCR, and BAX PCR was 65.4% concordant. Of 806 beef carcass swabs, 339 by cassette PCR and 84 by BAX PCR harbored eae + stx+E. coli. For BAX PCR reactions, 84% of eae+ swabs, 79% of stx+ swabs, and 86% of eae + stx+ swabs were also detected by cassette PCR. For cassette PCR reactions, 457 swabs were eae+ with only 117 scored as eae+ using BAX PCR for 26% positive concordance. For stx primers, cassette PCR scored 480 samples as stx+ but only 215 samples were stx+ by BAX PCR, giving 45% positive concordance. Importantly, cassette PCR scored 339 swabs as harboring eae + stx+ E. coli, but BAX PCR detected only 71 positives giving only 21% positive concordance, with many false negatives. Cassette PCR is a highly sensitive method for detection of STEC genes in E. coli found in carcass swabs