Development and Analytical Validation of a DNA Dual-Strand Approach for the US Food and Drug Administration-Approved Next-Generation Sequencing-Based Praxis Extended RAS Panel for Metastatic Colorectal Cancer Samples.

A next-generation sequencing method was developed that can distinguish single-stranded modifications from low-frequency somatic mutations present on both strands of DNA in formalin-fixed paraffin-embedded colorectal cancer samples. We applied this method for analytical validation of the Praxis Extended RAS Panel, a US Food and Drug Administration-approved companion diagnostic for panitumumab, on the Illumina MiSeqDx platform. With the use of the TruSeq amplicon workflow, both strands of DNA from the starting material were interrogated independently. Mutations were reported only if found on both strands; artifacts usually present on only one strand would not be reported. A total of 56 mutations were targeted within the KRAS and NRAS genes. A minimum read depth of 1800× per amplicon is required per sample but averaged >30,000× at maximum multiplexing levels. Analytical validation studies were performed to determine the simultaneous detection of mutations on both strands, reproducibility, assay detection level, precision of the assay across various factors, and the impact of interfering substances. In conclusion, this assay can clearly distinguish single-stranded artifacts from low-frequency mutations. Furthermore, the assay is accurate, precise, and reproducible, can achieve consistent detection of a mutation at 5% mutation frequency, exhibits minimal impact from tested interfering substances, and can simultaneously detect 56 mutations in a single run using 10 samples plus controls.

Detection of 11 common viral and bacterial pathogens causing community-acquired pneumonia or sepsis in asymptomatic patients by using a multiplex reverse transcription-PCR assay with manual (enzyme hybridization) or automated (electronic microarray) detection.

Community-acquired pneumonia (CAP) and sepsis are important causes of morbidity and mortality. We describe the development of two molecular assays for the detection of 11 common viral and bacterial agents of CAP and sepsis: influenza virus A, influenza virus B, respiratory syncytial virus A (RSV A), RSV B, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Legionella micdadei, Bordetella pertussis, Staphylococcus aureus, and Streptococcus pneumoniae. Further, we report the prevalence of carriage of these pathogens in respiratory, skin, and serum specimens from 243 asymptomatic children and adults. The detection of pathogens was done using both a manual enzyme hybridization assay and an automated electronic microarray following reverse transcription and PCR amplification. The analytical sensitivities ranged between 0.01 and 100 50% tissue culture infective doses, cells, or CFU per ml for both detection methods. Analytical specificity testing demonstrated no significant cross-reactivity among 19 other common respiratory organisms. One hundred spiked "surrogate" clinical specimens were all correctly identified with 100% specificity (95% confidence interval, 100%). Overall, 28 (21.7%) of 129 nasopharyngeal specimens, 11 of 100 skin specimens, and 2 of 100 serum specimens from asymptomatic subjects tested positive for one or more pathogens, with S. pneumoniae and S. aureus giving 89% of the positive results. Our data suggest that asymptomatic carriage makes the use of molecular assays problematic for the detection of S. pneumoniae or S. aureus in upper respiratory tract secretions; however, the specimens tested showed virtually no carriage of the other nine viral and bacterial pathogens, and the detection of these pathogens should not be a significant diagnostic problem. In addition, slightly less sensitive molecular assays may have better correlation with clinical disease in the case of CAP.

An integrated, stacked microlaboratory for biological agent detection with DNA and immunoassays.

An integrated, stacked microlaboratory for performing automated electric-field-driven immunoassays and DNA hybridization assays was developed. The stacked microlaboratory was fabricated by orderly laminating several different functional layers (all 76 x 76 mm(2)) including a patterned polyimide layer with a flip-chip bonded CMOS chip, a pressure sensitive acrylic adhesive (PSA) layer with a fluidic cutout, an optically transparent polymethyl methacrylate (PMMA) film, a PSA layer with a via, a patterned polyimide layer with a flip-chip bonded silicon chip, a PSA layer with a fluidic cutout, and a glass cover plate layer. Versatility of the stacked microlaboratory was demonstrated by various automated assays. Escherichia coli bacteria and Alexa-labeled protein toxin staphylococcal enterotoxin B (SEB) were detected by electric-field-driven immunoassays on a single chip with a specific-to-nonspecific signal ratios of 4.2:1 and 3.0:1, respectively. Furthermore, by integrating the microlaboratory with a module for strand displacement amplification (SDA), the identification of the Shiga-like toxin gene (SLT1) from E. coli was accomplished within 2.5 h starting from a dielectrophoretic concentration of intact E. coli bacteria and finishing with an electric-field-driven DNA hybridization assay, detected by fluorescently labeled DNA reporter probes. The integrated microlaboratory can be potentially used in a wide range of applications including detection of bacteria and biowarfare agents, and genetic identification.

Microelectronic array devices and techniques for electric field enhanced DNA hybridization in low-conductance buffers.

A variety of electronic DNA array devices and techniques have been developed that allow electric field enhanced hybridization to be carried out under special low-conductance conditions. These devices include both planar microelectronic DNA array/chip devices as well as electronic microtiter plate-like devices. Such "active" electronic devices are able to provide controlled electric (electrophoretic) fields that serve as a driving force to move and concentrate nucleic acid molecules (DNA/RNA) to selected microlocation test-sites on the device. In addition to ionic strength, pH, temperature and other agents, the electric field provides another controllable parameter that can affect and enhance DNA hybridization. With regard to the planar microelectronic array devices, special low-conductance buffers were developed in order to maintain rapid transport of DNA molecules and to facilitate hybridization within the constrained low current and voltage ranges for this type of device. With regard to electronic microtiter plate type devices (which do not have the low current/voltage constraints), the use of mixed buffers (low conductance upper chamber/high conductance lower chamber) can be used in a unique fashion to create favorable hybridization conditions in a microzone within the test site location. Both types of devices allow DNA molecules to be rapidly and selectively hybridized at the array test sites under conditions where the DNA in the bulk solution can remain substantially denatured.