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Highly Precise Measurement of HIV DNA by Droplet Digital PCR  [PDF]
Matthew C. Strain, Steven M. Lada, Tiffany Luong, Steffney E. Rought, Sara Gianella, Valeri H. Terry, Celsa A. Spina, Christopher H. Woelk, Douglas D. Richman
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0055943
Abstract: Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR (long terminal repeat) circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it.
Quantification of Zoonotic Bacterial Pathogens within Commercial Poultry Processing Water Samples Using Droplet Digital PCR  [PDF]
Michael J. Rothrock, Kelli L. Hiett, Brian H. Kiepper, Kim Ingram, Arthur Hinton
Advances in Microbiology (AiM) , 2013, DOI: 10.4236/aim.2013.35055

Raw poultry and poultry products are a significant source of zoonotic bacterial pathogen transmission; thus the sensitive detection of major zoonotic pathogens (Salmonella spp., Campylobacter jejuni, and Listeria monocytogenes) is a vital food safety issue. Recently, third generation PCR technology, known as droplet digital PCR (ddPCR) has been developed to be more accurate and sensitive to detect genetic targets than current quantification methods, but this technology has not been tested within an industrial setting. There is an on-going study within our laboratory is investigating the effects of sampling times and sampling methods on the cultural and molecular (via qPCR) quantification of dominant zoonotic pathogens within a poultry processing facility. This presents a unique opportunity to compare the quantification resulted from this emerging, third generation technology to traditional quantification methods currently employed by the poultry industry. The results show that ddPCR detected pathogen-specific genes from more pathogen:sampling time combinations than either the qPCR or culturing methods from the final scalder and chiller tanks at three stages of processing (Start, Mid, and End). In fact, both ddPCR and qPCR substantially outperformed culture methods commonly used in poultry processing food safety-related studies, with Salmonella recovered only from the Mid and End sampling times from the scalder tank. While neither C. jejuni nor L. monocytogenes were recovered culturally, ddPCR was able to detect their respective genes commonly throughout the processing day in both the scalder and chiller water samples. Additionally, the use of unfiltered processing water provided significantly greater detection of bacterial and pathogen-specific gene abundances than did an analysis of larger volumes of filtered water. Considering the ddPCR-derived concentrations of the bacterial pathogens were consistent with what was previously found culturally in commercial poultry processing operations, ddPCR represented a significant advancement in poultry processing zoonotic pathogen quantification.

Quantification of residual stresses in the weld by the hole-drilling method  [PDF]
Trebuňa, F.,?im?ák, F.,Bocko, J.,?arga, P.
Metalurgija , 2008,
Abstract: Residual stresses arise in the structures without loading during the technological processes, e.g. casting, rolling, welding, and pressing. In the paper is described the process of quantification of residual stresses by the hole-drilling method. For determination of residual stresses were used the procedures in which are supposed constant or linear distributions of stresses along the hole.
Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA  [PDF]
Maja Kiselinova, Alexander O. Pasternak, Ward De Spiegelaere, Dirk Vogelaers, Ben Berkhout, Linos Vandekerckhove
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0085999
Abstract: Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2 = 0.97, msRNA: R2 = 0.92) and patient-derived samples (usRNA: R2 = 0.51, msRNA: R2 = 0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used in clinical studies aimed at HIV eradication, but should be cross-validated by multiple laboratories prior to wider use.
Quantification of Residual Stresses in Hot Rolled Steel Sheets by the Hole Drilling Method  [PDF]
Trebuňa, F.,?im?ák, F.,Bur?ák, M.,Bocko, J.
Metalurgija , 2007,
Abstract: The paper deals with the problems of quantification of residual stresses in hot rolled sheets produced under various regimes. On the base of stress distribution along width and thickness of the belt is pointed out on possibilities of belt deformation from the plane as a result of action of torsional moment as an integral internal quantity in cross-section. For the non-uniformly distributed stresses along the thickness of the sheet the new method for residual stresses determination was developed. Application of the method is described in the paper.
An investigation of excess residual cytoplasm in human spermatozoa and its distinction from the cytoplasmic droplet  [cached]
Rengan Anil K,Agarwal Ashok,van der Linde Michelle,du Plessis Stefan S
Reproductive Biology and Endocrinology , 2012, DOI: 10.1186/1477-7827-10-92
Abstract: Recent studies have shown cytoplasmic droplets to be normal morphological occurrences in human male spermatozoa. When the cytoplasm around the sperm midpiece is present in large amounts, however, pathological effects may transpire. The cytoplasmic droplet then becomes known as excess residual cytoplasm, which can impair overall sperm function and produce higher levels of reactive oxygen species, potentially leading to male infertility. Though the distinction between cytoplasmic droplets and excess residual cytoplasm has been made, some studies fail to recognize the difference and incorrectly label the latter as a cytoplasmic droplet. This review attempts to clarify excess residual cytoplasm’s effect on fertility, examine the enzymes responsible, and suggest tests and possible treatment options for those affected by this defect.
Escherichia coli, an Intestinal Microorganism, as a Biosensor for Quantification of Amino Acid Bioavailability  [PDF]
Vesela I. Chalova,Sujata A. Sirsat,Corliss A. O’Bryan,Philip G. Crandall,Steven C. Ricke
Sensors , 2009, DOI: 10.3390/s90907038
Abstract: In animal diets optimal amino acid quantities and balance among amino acids is of great nutritional importance. Essential amino acid deficiencies have negative impacts on animal physiology, most often expressed in sub-optimal body weight gains. Over supplementation of diets with amino acids is costly and can increase the nitrogen emissions from animals. Although in vivo animal assays for quantification of amino acid bioavailability are well established, Escherichia coli-based bioassays are viable potential alternatives in terms of accuracy, cost, and time input. E. coli inhabits the gastrointestinal tract and although more abundant in colon, a relatively high titer of E. coli can also be isolated from the small intestine, where primary absorption of amino acids and peptides occur. After feed proteins are digested, liberated amino acids and small peptides are assimilated by both the small intestine and E. coli. The similar pattern of uptake is a necessary prerequisite to establish E. coli cells as accurate amino acid biosensors. In fact, amino acid transporters in both intestinal and E. coli cells are stereospecific, delivering only the respective biological L-forms. The presence of free amino- and carboxyl groups is critical for amino acid and dipeptide transport in both biological subjects. Di-, tri- and tetrapeptides can enter enterocytes; likewise only di-, tri- and tetrapeptides support E. coli growth. These similarities in addition to the well known bacterial genetics make E. coli an optimal bioassay microorganism for the assessment of nutritionally available amino acids in feeds.
Cloud-Enabled Microscopy and Droplet Microfluidic Platform for Specific Detection of Escherichia coli in Water  [PDF]
Alexander Golberg, Gregory Linshiz, Ilia Kravets, Nina Stawski, Nathan J. Hillson, Martin L. Yarmush, Robert S. Marks, Tania Konry
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0086341
Abstract: We report an all-in-one platform – ScanDrop – for the rapid and specific capture, detection, and identification of bacteria in drinking water. The ScanDrop platform integrates droplet microfluidics, a portable imaging system, and cloud-based control software and data storage. The cloud-based control software and data storage enables robotic image acquisition, remote image processing, and rapid data sharing. These features form a “cloud” network for water quality monitoring. We have demonstrated the capability of ScanDrop to perform water quality monitoring via the detection of an indicator coliform bacterium, Escherichia coli, in drinking water contaminated with feces. Magnetic beads conjugated with antibodies to E. coli antigen were used to selectively capture and isolate specific bacteria from water samples. The bead-captured bacteria were co-encapsulated in pico-liter droplets with fluorescently-labeled anti-E. coli antibodies, and imaged with an automated custom designed fluorescence microscope. The entire water quality diagnostic process required 8 hours from sample collection to online-accessible results compared with 2–4 days for other currently available standard detection methods.
Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR  [PDF]
Dany Morisset, Dejan ?tebih, Mojca Milavec, Kristina Gruden, Jana ?el
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0062583
Abstract: In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.
Residual-based localization and quantification of peaks in x-ray diffractograms  [PDF]
P. L. Davies,U. Gather,M. Meise,D. Mergel,T. Mildenberger
Statistics , 2007, DOI: 10.1214/08-AOAS181
Abstract: We consider data consisting of photon counts of diffracted x-ray radiation as a function of the angle of diffraction. The problem is to determine the positions, powers and shapes of the relevant peaks. An additional difficulty is that the power of the peaks is to be measured from a baseline which itself must be identified. Most methods of de-noising data of this kind do not explicitly take into account the modality of the final estimate. The residual-based procedure we propose uses the so-called taut string method, which minimizes the number of peaks subject to a tube constraint on the integrated data. The baseline is identified by combining the result of the taut string with an estimate of the first derivative of the baseline obtained using a weighted smoothing spline. Finally, each individual peak is expressed as the finite sum of kernels chosen from a parametric family.
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