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.
Subject(s)
Bodily Secretions/virology , Levivirus/isolation & purification , RNA, Viral/isolation & purification , Respiratory System , Virology/methods , Automation/methods , Equipment and Supplies , Glass , Humans , Levivirus/genetics , RNA, Viral/genetics , Sensitivity and SpecificityABSTRACT
We describe here a rapid assay for the detection of the tuf gene for the identification of Staphylococcus genus, the femB gene for the identification of Staphylococcus aureus species, and the mecA gene for the identification of methicillin resistance directly from BACTEC blood culture bottles showing Gram-positive cocci in clusters. The test, configured on a thin-film biosensor platform, allows for detection of genomic DNA from blood culture samples without the need for nucleic acid amplification. In an initial study to validate the technology, 107 consecutive positive blood cultures were tested on the thin-film biosensor, and the assay exhibited 100% concordance in comparison with standard microbiological methods for identifying methicillin-susceptible and methicillin-resistant S. aureus and for identifying methicillin-susceptible and methicillin-resistant coagulase-negative Staphylococcus. Results were obtained within 90 min directly from signal positive bottles with no instrumentation required.
Subject(s)
Biosensing Techniques/methods , Methicillin Resistance , Nucleic Acid Hybridization/methods , Staphylococcal Infections/microbiology , Staphylococcus/drug effects , Staphylococcus/isolation & purification , Bacterial Proteins/genetics , Humans , Penicillin-Binding Proteins , Peptide Elongation Factor Tu/genetics , Sensitivity and Specificity , Staphylococcus/geneticsABSTRACT
C. elegans spermatogenesis employs lysosome-related fibrous body-membranous organelles (FB-MOs) for transport of many cellular components. Previous work showed that spe-10 mutants contain FB-MOs that prematurely disassemble, resulting in defective transport of FB components into developing spermatids. Consequently, spe-10 spermatids are smaller than wild type and contain defective FB-MO derivatives. In this article, we show that spe-10 encodes a four-pass integral membrane protein that has a DHHC-CRD zinc-finger motif. The DHHC-CRD motif is found in a large, diverse family of proteins that have been implicated in palmitoyl transfer during protein lipidation. Seven spe-10 mutants were analyzed, including missense, nonsense, and deletion mutants. An antiserum to SPE-10 showed significant colocalization with a known marker for the FB-MOs during wild-type spermatogenesis. In contrast, the spe-10(ok1149) deletion mutant lacked detectable SPE-10 staining; this mutant lacks a spe-10 promoter and most coding sequence. The spe-10(eb64) missense mutation, which changes a conserved residue within the DHHC-CRD domain in all homologues, behaves as a null mutant. These results suggest that wild-type SPE-10 is required for the MO to properly deliver the FB to the C. elegans spermatid and the DHHC-CRD domain is essential for this function.
