A bench-top automated workstation for nucleic acid isolation from clinical sample types

Abstract

Systems that automate extraction of nucleic acid from cells or viruses in complex clinical matrices have tremendous value even in the absence of an integrated downstream detector. We describe our bench-top automated workstation that integrates our previously-reported extraction method - TruTip - with our newly-developed mechanical lysis method. This is the first report of this method for homogenizing viscous and heterogeneous samples and lysing difficult-to-disrupt cells using "MagVor": a rotating magnet that rotates a miniature stir disk amidst glass beads confined inside of a disposable tube. Using this system, we demonstrate automated nucleic acid extraction from methicillin-resistant Staphylococcus aureus (MRSA) in nasopharyngeal aspirate (NPA), influenza A in nasopharyngeal swabs (NPS), human genomic DNA from whole blood, and Mycobacterium tuberculosis in NPA. The automated workstation yields nucleic acid with comparable extraction efficiency to manual protocols, which include commercially-available Qiagen spin column kits, across each of these sample types. This work expands the scope of applications beyond previous reports of TruTip to include difficult-to-disrupt cell types and automates the process, including a method for removal of organics, inside a compact bench-top workstation.

Performance and workflow assessment of six nucleic acid extraction technologies for use in resource limited settings

“…automated and utilizes their TruTip technology; a silica coated frit housed in a 1 mL micropipettor tip [37]. Negative and positive air pressure is used to manipulate liquid lysed material and buffers across the frit to sequentially capture, wash and elute NAs from a sample using an automated pipettor [33,40]. Molbio and developer E ranked second and third, respectively, but did not consistently detect all NA replicates in the lowest spiked samples.…”

“…The inclusion of turnaround times (TAT) and manual steps into the ASSUR dataset were the underlying reasons for Molbio (F) moving ahead of Akonni Biosystems (A) A. Molbio also had good performance but also a more rapid TAT (35 minutes versus 55 minutes) and fewer user steps than the Akonni methodology and so was awarded higher ranking via the radar plot analyses (Fig 1). Both of these developers use systems that are automated to differing degrees with the Molbio platform hosting a cassette that processes the sample preparation steps [33,34]. The data from other developers such as B also improved, primarily due to the incorporation of the fastest TATs to augment their relatively poor performance data ( Table 2 and Fig 1).…”

“…As with any laboratory procedure, it is possible that contamination of specimens or extracts occurred during preparation, processing, handling, or testing, as observed with MS2 in extracts from developer B. The risk of operatorintroduced contamination is lower when automated systems are used and was observed with the both Akonni and Molbio platforms that have varying degrees of automation [33,34]. Good laboratory practices for molecular testing laboratories were followed by all laboratories to minimize the potential for contamination of specimens, extracts, or PCR reactions in order to ensure integrity of results.…”

Detection of Mycobacterium tuberculosis in pediatric stool samples using TruTip technology

“…127 128 For lysis and DNA extraction procedures using the TruTip workstation we followed methods 129 similar to previously described for sputum samples (18). In brief, 500 milligrams of thawed stool 130 was homogenized in TE buffer, in a magnetically-induced vortexing (MagVor) element, and 131 after a 20 minute heating step at 56°C, the samples were transferred to the TruTip workstation 132 for DNA extraction (16…”

An automated, high throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma

“…For large sample numbers, the column-based method has been the most commonly used, as precipitation methods that involve multiple centrifugation steps create bottlenecks in the workflow. Several reports have established that MB-based isolation methods yield high-quality DNA in similar quantities to column-based methods (25,(31)(32)(33)(34). It has also been reported that MB-based extractions were the only method able to isolate key sequences when compared to other precipitation-and column-based methods (31).…”