Extraction of MS2 Phage RNA from Upper Respiratory Tract Specimens by Use of Flat Glass Devices

Abstract

The isolation of pure nucleic acids from clinical samples is a crucial step in the molecular diagnosis of viral infections by nucleic acid testing (NAT). In this study, novel flat glass devices (cards) were demonstrated to support the rapid and efficient extraction of nucleic acids from upper respiratory tract specimens (nasal washes and swabs). The performance of the nucleic acid extraction cards was directly compared to an existing standardized and automated platform for viral extraction from these types of specimens. The flowthrough card method improved the speed of nucleic acid purification and accommodated larger sample volumes in extraction of bacteriophage MS2 RNA from the various specimen matrices. The dynamic range and estimated sensitivity of the card extraction method for reverse transcriptase quantitative real-time PCR (RT-qPCR)-based detection approximate those of the standardized magnetic glass bead extraction method used in this study.Molecular diagnostic tests are widely used in clinical virology to detect a range of respiratory tract viral infections. Methods for nucleic acid (NA) extraction of upper respiratory tract samples have recently been standardized with the availability of FDA-510(k)-approved tests for respiratory viruses such as the Nanosphere Verigene SP respiratory virus nucleic acid test and the Prodesse ProFlu, Luminex RVP xTAG, and CDC multiplex flu tests. Approved extraction methods for these highly complex tests (with the exception of the moderately complex Nanosphere test) are slow, and they tend to process lower clinical sample volumes than do other tests. The NucliSens EasyMag extraction system (bioMérieux, Marcy l'Etoile, France) has been compared to the BioRobot 9604 (Qiagen GmbH, Hilden, Germany) and to the manual QIAamp RNA/DNA extraction kit (Qiagen GmbH) for the detection of NAs from viral respiratory tract pathogens using reverse transcriptase quantitative real-time PCR (RT-qPCR) assays (7). The performances of these systems for extracting (0.14-to 0.25-ml) volumes of respiratory tract samples were shown to be equivalent, but the advantages of the automated systems were speed and better cross-contamination control, whereas the manual method was less expensive but required more manipulation than did the automated systems.Extraction kits and systems commonly used to extract respiratory tract samples (7) often use finely divided glass particles or magnetic glass beads as the nucleic acid binding matrix and chaotropic salt binding and wash buffers (4, 5). These silica particles offer a greater surface area for binding of NAs (18,19), but the porous matrix can release trapped contaminants that may be inhibitory to NA testing (NAT) or even sequester target NAs (14,19). In addition, these types of columns do not bind small DNA molecules (up to about 150 bp) efficiently (9, 16; M. W. Reed et al., U.S. patent application 12/348,244).We have previously demonstrated that glass microscope slides could be used to purify genomic DNA from various sample types in an open system (12), ...

Molecular Viability Testing of Bacterial Pathogens from a Complex Human Sample Matrix

“…The detection protocol used automated nucleic acid extraction cards [29] and the single-step RT-qPCR method described in Materials and Methods. It required 5.5 hours to complete, including a 1.5-hour nutritional stimulation.…”

“…Nucleic acid was extracted from frozen bacterial pellets by using the Blood Cell Storage Inc. (BCSI) Nucleic Acid Extraction System [29]. Pellets were briefly thawed at room temperature, and 380 µL of TE plus 1% SDS (lysis buffer) and 10 µL of 20 mg/mL Proteinase K (VWR IB05406) were added to each pellet, vortexed for 20 seconds, and placed in a 60°C incubator for 10 minutes.…”

Biosynthetic Enhancement of the Detection of Bacteria by the Polymerase Chain Reaction

“…In some experiments, total nucleic acid was extracted from frozen bacterial cell pellets by using the BCSI Nucleic Acid Extraction System [15] . Pellets were briefly thawed at room temperature, and 380 µL of TE + 1% SDS (lysis buffer) and 10 µL of 20 mg/mL Proteinase K (VWR IB05406) were added to each pellet.…”