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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.

BACKGROUND: Bacterial contamination or platelet (PLT) metabolism can change the pH of stored PLT concentrates (PCs). Measurement of pH for quality control is currently done on a limited basis. An easy noninvasive method was developed to obtain sequential pH measurements over time, without risking contamination and/or consuming PCs.

It is well known that DNA strands bind to silica surfaces in the presence of high concentrations of chaotropic salts. We developed simple methods to evaluate binding and recovery of DNA on flat glass microscope slides and compared their properties with commercially available silica "spin columns". Surprisingly, genomic DNA was recovered efficiently from untreated glass slides. Binding and elution times from glass slides were optimized in experiments with DNA samples of various sizes and defined buffers. Average DNA recovery from 500 ng of input genomic DNA varied from 25 to 53% for the glass slide protocol. Yields were comparable to those of spin columns. Human serum albumin and plasma components decreased DNA binding to glass several-fold in a concentration-dependent manner. These results support the concept of using flat glass slides as DNA purification surfaces in microfluidic devices for PCR sample preparation.

The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

Background Tuberculosis (TB) is difficult to diagnose in children using molecular tests, because children have difficulty providing respiratory samples. Stool could replace sputum for diagnostic TB testing if adequate sample processing techniques were available. Methods We developed a rapid method to process large volumes of stool for downstream testing by the Xpert MTB/RIF (Xpert) TB-detection assay. The method was tested and optimized on stool samples spiked with known numbers of M. tuberculosis colony forming units (CFU), and stools from M. tuberculosis-infected cynomolgus macaques (Macaca fascicularis). Performance was scored on number of positive Xpert tests, the cycle thresholds (Cts) of the Xpert sample-processing control (SPC), and the Cts of the M. tuberculosis-specific rpoB probes. The method was then validated on 20 confirmed TB cases and 20 controls in Durban, South Africa. Results The assay’s analytical limit of detection was 1,000 CFU/g of stool. As much as one gram of spiked stool could be tested without showing increased PCR inhibition. In analytical spiking experiments using human stool, 1g samples provided the best sensitivity compared to smaller amounts of sample. However, in Macaques with TB, 0.6g stool samples performed better than either 0.2g or 1.2g samples. Testing the stool of pediatric TB suspects and controls suggested an assay sensitivity of 85% (95% CI 0.6–0.9) and 84% (95% CI 0.6–0.96) for 0.6g and 1.2g stool samples, respectively, and a specificity of 100% (95% CI 0.77–1) and 94% (95% CI 0.7–0.99), respectively. Conclusion This novel approach may permit simple and rapid detection of TB using pediatric stool samples.

The Moloney murine leukemia virus (M-MuLV) repressor binding site (RBS) mediates cell-type-specific repression in embryonal carcinoma (EC) cells of expression from several different promoters, including the M-MuLV long terminal repeat promoter. Silencing has been shown to depend on an element normally located in the proviral 5' noncoding region and occurs at the DNA level in the absence of retroviral proteins. Using fragments of the RBS region, we now show that the minimal size of the silencer corresponds to M-MuLV nt 147-163 and overlaps with the retroviral primer binding site region by 17 of its 18 bp. A panel of point mutations within the RBS has been examined to yield a consensus RBS sequence which is consistent with the notion that a previously identified nuclear factor (binding factor A) mediates RBS repression. Viral vectors using neomycin, beta-galactosidase, and luciferase reporters have been employed to show that RBS-mediated repression occurs in EC and embryonal stem, but not in other tested cell types. Repression was observed to occur within 48 hr of infection, prior to when global methylation of proviruses has been reported to occur. Repression also occurred after azacytidine treatment of EC cells, supporting the notion that the RBS functions independently of provirus methylation. However, levels of provirus methylation in selected cells were increased in the presence of a wild-type RBS, and methylation correlated with a secondary stage of virus repression. Thus, the M-MuLV RBS acts directly to control expression in EC cells and also appears to trigger a secondary level of repression which is coincident with provirus methylation.

Bacterial contamination or platelet (PLT) metabolism can change the pH of stored PLT concentrates (PCs). Measurement of pH for quality control is currently done on a limited basis. An easy noninvasive method was developed to obtain sequential pH measurements over time, without risking contamination and/or consuming PCs.The objective was to measure pH profiles of bacterially contaminated PCs over 7 days of storage. Small-volume PC storage bags with incorporated pH sensor were prepared and in vitro variables were tested using aliquots of PCs. The pH sensors were used to delineate trends associated with the deterioration of these PCs upon inoculation with 19 different bacterial strains and one yeast.Monitoring the pH trends in real time in a noninvasive fashion, most bacterial strains were detected within 24 to 72 hours after spiking into the bag. At the time of detection, bacterial concentrations had reached levels between 1×10(3) and 1×10(8) colony-forming units/mL. Several strains had pH rebound after initial drop. Multiple noninvasive pH reads allowed bacterial detection whereas single pH reads could give false-negative results.The noninvasive pH sensor facilitated the detection of most strains of bacterial contaminants within 3 days with no potential for sampling error.

A previous genetic screen was designed to separate Hin recombinase mutants into distinct classes based on the stage in the recombination reaction at which they are blocked (O. Nanassy, Zoltan, and K. T. Hughes, Genetics 149:1649–1663, 1998). One class of DNA binding-proficient, recombination-deficient mutants was predicted by genetic classification to be defective in the step prior to invertasome formation. Based on the genetic criteria, mutants from this class were also inferred to be defective in interactions with Fis. In order to understand how the genetic classification relates to individual biochemical steps in the recombination reaction these mutants, R123Q, T124I, and A126T, were purified and characterized for DNA cleavage and recombination activities. Both the T124I and A126T mutants were partially active, whereas the R123Q mutant was inactive. The A126T mutant was not as defective for recombination as the T124I allele and could be partially rescued for recombination both in vivo and in vitro by increasing the concentration of Fis protein. Rescue of the A126T allele required the Fis protein to be DNA binding proficient. A model for a postsynaptic role for Fis in the inversion reaction is presented.

The Hin recombinase mediates the site-specific inversion of a segment of the Salmonella chromosome between two flanking 26 bp hix DNA recombination sites. Mutations in two amino acid residues, R43 and R69 of the catalytic domain of the Hin recombinase, were identified that can compensate for loss of binding resulting from elimination of certain major and minor groove contacts within the hix recombination sites. With one exception, the R43 and R69 mutants were also able to bind a hix sequence with an additional 4 bp added to the centre of the site, unlike wild-type Hin. Purified Hin mutants R43H and R69C had both partial cleavage and inversion activities in vitro while mutants R43L, R43C, R69S, and R69P had no detectable cleavage and inversion activities. These data support a model in which the catalytic domain plays a role in DNA-binding specificity, and suggest that the arginine residues at positions 43 and 69 function to position the Hin recombinase on the DNA for a step in the recombination reaction which occurs either at and/or prior to DNA cleavage.