A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva

A microfluidic chip integrating DNA extraction, amplification, and detection for the identification of bacteria in saliva is described. The chip design integrated a monolithic aluminum oxide membrane (AOM) for DNA extraction with seven parallel reaction wells for real-time polymerase chain reaction (rtPCR) amplification of the extracted DNA. Samples were first heated to lyse target organisms and then added to the chip and filtered through the nanoporous AOM to extract the DNA. PCR reagents were added to each of the wells and the chip was thermocycled. Identification of Streptococcus mutans in a saliva sample is demonstrated along with the detection of 300 fg (100–125 copies) of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) genomic DNA (gDNA) spiked into a saliva sample. Multiple target species and strains of bacteria can be simultaneously identified in the same sample by varying the primers and probes used in each of the seven reaction wells. In initial tests, as little as 30 fg (8–12 copies) of MSSA gDNA in buffer has been successfully amplified and detected with this device

An automated integrated platform for rapid and sensitive multiplexed protein profiling using human saliva samples

During the last decade, saliva has emerged as a potentially ideal diagnostic biofluid for noninvasive testing. In this paper, we present an automated, integrated platform useable by minimally trained personnel in the field for the diagnosis of respiratory diseases using human saliva as a sample specimen. In this platform, a saliva sample is loaded onto a disposable microfluidic chip containing all the necessary reagents and components required for saliva analysis. The chip is then inserted into the automated analyzer, the SDReader, where multiple potential protein biomarkers for respiratory diseases are measured simultaneously using a microsphere-based array via fluorescence sandwich immunoassays. The results are read optically, and the images are analyzed by a custom-designed algorithm. The fully automated assay requires as little as 10 μL of saliva sample, and the results are reported in 70 min. The performance of the platform was characterized by testing protein standard solutions, and the results were comparable to those from the 3.5-h lab bench assay that we have previously reported. The device was also deployed in two clinical environments where 273 human saliva samples collected from different subjects were successfully tested, demonstrating the device’s potential to assist clinicians with the diagnosis of respiratory diseases by providing timely protein biomarker profiling information. This platform, which combines non-invasive sample collection and fully automated analysis, can also be utilized in point-of-care diagnostics

Genome-wide association study identifies acyl-lipid metabolism candidate genes involved in the genetic control of natural variation for seed fatty acid traits in Brassica napus L.

Brassica napus L. represents a potential plant feedstock for the sustainable production of hydrotreated renewable fuels needed to support carbon-based energy production. However, to increase the use of plant-derived oils for energy needs, breeding efforts are required to optimize the amount and profile of fatty acids (FAs) contained in the oil extracted from B. maims seed to meet demands of the various market categories. To this end, we analyzed the genetic basis of FA content and composition of seed from a diverse panel of spring-type B. napus accessions evaluated at four US locations across multiple years. The extent of phenotypic variations for total oil content, nine FA compounds, and 14 derivative traits were found, in general, to be highly heritable. A genome-wide association study (GWAS) was conducted that detected 53 SNPs significantly associated with one or more of the 24 FA seed traits, resulting in the implicated genetic role of 12 candidate genes, four of which had two homologs each, from the acyl-lipid pathway. To our knowledge, the two detected homologs of 3-Ketoacyl-CoA thiolase (KAT), have never been associated with seed oil traits in B. napus. Through the application of whole-genome prediction, the 24 FA seed traits were generally found to have moderately high predictive abilities (70% of traits with abilities > 0.70), suggesting that these traits are highly amenable to genomic selection. Overall, our results contribute to the expanding body of knowledge regarding key enzymes in the acyl-lipid pathway at the quantitative genetic level and illustrate how genomics-assisted breeding could be leveraged to genetically improve FA seed traits in B. napus.24 month embargo; published online: 6 January 2020This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva

A microfluidic chip integrating DNA extraction, amplification, and detection for the identification of bacteria in saliva is described. The chip design integrated a monolithic aluminum oxide membrane (AOM) for DNA extraction with seven parallel reaction wells for real-time polymerase chain reaction (rtPCR) amplification of the extracted DNA. Samples were first heated to lyse target organisms and then added to the chip and filtered through the nanoporous AOM to extract the DNA. PCR reagents were added to each of the wells and the chip was thermocycled. Identification of Streptococcus mutans in a saliva sample is demonstrated along with the detection of 300 fg (100–125 copies) of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) genomic DNA (gDNA) spiked into a saliva sample. Multiple target species and strains of bacteria can be simultaneously identified in the same sample by varying the primers and probes used in each of the seven reaction wells. In initial tests, as little as 30 fg (8–12 copies) of MSSA gDNA in buffer has been successfully amplified and detected with this device

An automated integrated platform for rapid and sensitive multiplexed protein profiling using human saliva samples

During the last decade, saliva has emerged as a potentially ideal diagnostic biofluid for noninvasive testing. In this paper, we present an automated, integrated platform useable by minimally trained personnel in the field for the diagnosis of respiratory diseases using human saliva as a sample specimen. In this platform, a saliva sample is loaded onto a disposable microfluidic chip containing all the necessary reagents and components required for saliva analysis. The chip is then inserted into the automated analyzer, the SDReader, where multiple potential protein biomarkers for respiratory diseases are measured simultaneously using a microsphere-based array via fluorescence sandwich immunoassays. The results are read optically, and the images are analyzed by a custom-designed algorithm. The fully automated assay requires as little as 10 μL of saliva sample, and the results are reported in 70 min. The performance of the platform was characterized by testing protein standard solutions, and the results were comparable to those from the 3.5-h lab bench assay that we have previously reported. The device was also deployed in two clinical environments where 273 human saliva samples collected from different subjects were successfully tested, demonstrating the device’s potential to assist clinicians with the diagnosis of respiratory diseases by providing timely protein biomarker profiling information. This platform, which combines non-invasive sample collection and fully automated analysis, can also be utilized in point-of-care diagnostics