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Automated TruTip nucleic acid extraction and purification from raw sputum.

ABSTRACT: Automated nucleic acid extraction from primary (raw) sputum continues to be a significant technical challenge for molecular diagnostics. In this work, we developed a prototype open-architecture, automated nucleic acid workstation that includes a mechanical homogenization and lysis function integrated with heating and TruTip purification; optimized an extraction protocol for raw sputum; and evaluated system performance on primary clinical specimens. Eight samples could be processed within 70 min. The system efficiently homogenized primary sputa and doubled nucleic acid recovery relative to an automated protocol that did not incorporate sample homogenization. Nucleic acid recovery was at least five times higher from raw sputum as compared to that of matched sediments regardless of smear or culture grade, and the automated workstation reproducibly recovered PCR-detectable DNA to at least 80 CFU mL-1 raw sputum. M. tuberculosis DNA was recovered and detected from 122/123 (99.2%) and 124/124 (100%) primary sputum and sediment extracts, respectively. There was no detectable cross-contamination across 53 automated system runs and amplification or fluorescent inhibitors (if present) were not detectable. The open fluidic architecture of the prototype automated workstation yields purified sputum DNA that can be used for any molecular diagnostic test. The ability to transfer TruTip protocols between personalized, on-demand pipetting tools and the fully automated workstation also affords public health agencies an opportunity to standardize sputum nucleic acid sample preparation procedures, reagents, and quality control across multiple levels of the health care system.

SUBMITTER: Thakore N

PROVIDER: S-EPMC6033430 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Automated TruTip nucleic acid extraction and purification from raw sputum.

Thakore Nitu N Norville Ryan R Franke Molly M Calderon Roger R Lecca Leonid L Villanueva Michael M Murray Megan B MB Cooney Christopher G CG Chandler Darrell P DP Holmberg Rebecca C RC

PloS one 20180705 7

Automated nucleic acid extraction from primary (raw) sputum continues to be a significant technical challenge for molecular diagnostics. In this work, we developed a prototype open-architecture, automated nucleic acid workstation that includes a mechanical homogenization and lysis function integrated with heating and TruTip purification; optimized an extraction protocol for raw sputum; and evaluated system performance on primary clinical specimens. Eight samples could be processed within 70 min. ...[more]

PMID: 29975759

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Project description:BackgroundBlood plasma, one of the most studied liquid biopsies, contains various molecules that have biomarker potential for cancer detection, including cell-free DNA (cfDNA) and cell-free RNA (cfRNA). As the vast majority of cell-free nucleic acids in circulation are non-cancerous, a laboratory workflow with a high detection sensitivity of tumor-derived nucleic acids is a prerequisite for precision oncology. One way to meet this requirement is by the combined analysis of cfDNA and cfRNA from the same liquid biopsy sample. So far, no study has systematically compared the performance of cfDNA and cfRNA co-purification to increase sensitivity.ResultsFirst, we set up a framework using digital PCR (dPCR) technology to quantify cfDNA and cfRNA from human blood plasma in order to compare cfDNA/cfRNA co-purification kit performance. To that end, we optimized two dPCR duplex assays, designed to quantify both cfDNA and cfRNA with the same assays, by ensuring that primers and probes are located within a highly abundant exon. Next, we applied our optimized workflow to evaluate the co-purification performance of two manual and two semi-automated methods over a range of plasma input volumes (0.06-4 mL). Some kits result in higher nucleic acid concentrations in the eluate, while consuming only half of the plasma volume. The combined nucleic acid quantification systematically results in higher nucleic acid concentrations as compared to a parallel quantification of cfDNA and cfRNA in the eluate.ConclusionsWe provide a framework to evaluate the performance of cfDNA/cfRNA co-purification kits and have tested two manual and two semi-automated co-purification kits in function of the available plasma input amount and the intended use of the nucleic acid eluate. We demonstrate that the combined quantification of cfDNA and cfRNA has a benefit compared to separate quantification. We foresee that the results of this study are instrumental for clinical applications to help increase mutation detection sensitivity, allowing improved disease detection and monitoring.

Dataset Information

Automated TruTip nucleic acid extraction and purification from raw sputum.

Publications

Automated TruTip nucleic acid extraction and purification from raw sputum.

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