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This study addressed whether digital droplet PCR (ddPCR) could improve sensitivity and specificity of human-associated Bacteroidales genetic markers, BacHum and B. theta, and their quantification in environmental and fecal composite samples. Human markers were quantified by qPCR and ddPCR platforms obtained from the same manufacturer. A total of 180 samples were evaluated by each platform including human and animal feces, sewage, and environmental water. The sensitivity of ddPCR and qPCR marker assays in sewage and human stool was 0.85-1.00 with marginal reduction in human stool by ddPCR relative to qPCR (<10%). The prevalence and distribution of markers across complex sample types was similar (74-100% agreement) by both platforms with qPCR showing higher sensitivity for markers in environmental and composite samples and ddPCR showing greater reproducibility for marker detection in fecal composites. Determination of BacHum prevalence in fecal samples by ddPCR increased specificity relative to qPCR (from 0.58 to 0.88) and accuracy (from 0.77 to 0.94), while the B. theta assay performed similarly on both platforms (specificity = 0.98). In silico analysis indicated higher specificity of ddPCR for BacHum was not solely attributed to reduced sensitivity relative to qPCR. Marker concentrations measured by ddPCR for all sample types were consistently lower than those measured by qPCR, by a factor of 2.6 ± 2.8 for B. theta and 18.7 ± 10.0 for BacHum. We suggest that differences in assay performance on ddPCR and qPCR platforms may be linked to the characteristics of the assay targets (that is, genes with multiple versus single copies and encoding proteins versus ribosomal RNA) however further work is needed to validate these ideas. We conclude that ddPCR is a suitable tool for microbial source tracking, however, other factors such as cost-effectiveness and assay-specific performance should be considered.
This article was published in the following journal.
Name: Water research
Droplet microfluidics is a technique that has the ability to compartmentalize reactions in sub nano- (or pico-) liter volumes that can potentially enable millions of distinct biological assays to be p...
Digital polymerase chain reaction (dPCR) is increasingly being adopted by reference material producers and metrology insti-tutes for value assignment, and for homogeneity and stability studies of nucl...
An analog-digital hybrid optical chaos source and a corresponding secure key distribution (SKD) scheme are proposed. An analog-digital hybrid electro-optic feedback loop is introduced to enhance the r...
The recent development of novel Polymerase Chain Reaction (PCR) technologies that confer theoretical advantages over quantitative PCR has considerable potential in the diagnosis of low load infections...
Global change is shifting the seasonality of vegetation in ecosystems around the globe. High-frequency digital camera imagery, and vegetation indices derived from that imagery, is facilitating better ...
This study will use droplet digital PCR (ddPCR) method to quantify and track peripheral blood plasma mutant allele frequency (MAF) in MDS and AML patients before, during and after chemothe...
The purpose of this study is to carry out a field trial to evaluate the effectiveness and added value of the b-SLIM digital Super Coach in a convenient sample of overweight and obese adult...
This pilot study examines concurrent and predictive relationships between eye tracking and clinical outcomes during a 16-week behavioral intervention (PRT) for children with ASD. Eye track...
This study will assess the effects of exercise and non-concussive bodily contact on eye-tracking scores collected by the EYE-SYNC eye-tracking device.
The purpose of this study is to evaluate whether bacterial DNA clearance measured with droplet digital Polymerase Chain Reaction (ddPCR) can be used as a measure of bacterial load in septi...
The process of converting analog data such as continually measured voltage to discrete, digital form.
Enumeration by direct count of viable, isolated bacterial, archaeal, or fungal CELLS or SPORES capable of growth on solid CULTURE MEDIA. Each colony (i.e., microbial colony-forming unit) represents the progeny of a single cell in the original inoculum. The method is used routinely by environmental microbiologists for quantifying organisms in AIR; FOOD; and WATER; by clinicians for measuring patients' microbial load; and in antimicrobial drug testing.
A frequent complication of drug therapy for microbial infection. It may result from opportunistic colonization following immunosuppression by the primary pathogen and can be influenced by the time interval between infections, microbial physiology, or host resistance. Experimental challenge and in vitro models are sometimes used in virulence and infectivity studies.
Light absorbing proteins and protein prosthetic groups found in certain microorganisms. Some microbial photoreceptors initiate specific chemical reactions which signal a change in the environment, while others generate energy by pumping specific ions across a cellular membrane.
Live microbial DIETARY SUPPLEMENTS which beneficially affect the host animal by improving its intestinal microbial balance. Antibiotics and other related compounds are not included in this definition. In humans, lactobacilli are commonly used as probiotics, either as single species or in mixed culture with other bacteria. Other genera that have been used are bifidobacteria and streptococci. (J. Nutr. 1995;125:1401-12)
Polymerase Chain Reaction (PCR)
PCR (Polymerase Chain Reaction) uses the ability of DNA polymerase (enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to create two ident...
Immunoassay - ELISA
Immunoassays are quick and accurate tests to detect specific molecules. Immunoassays rely on an antibody to bind to the specific structure of a molecule. Antibodies are proteins generated by animals in response to the invasion of a foreign molecule (anti...