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Challenges of detecting directional selection after a bottleneck: lessons from Sorghum bicolor.

Multilocus surveys of sequence variation can be used to identify targets of directional selection, which are expected to have reduced levels of variation. Following a population bottleneck, the signal of directional selection may be hard to detect because many loci may have low variation by chance and the frequency spectrum of variation may be perturbed in ways that resemble the effects of selection. Cultivated Sorghum bicolor contains a subset of the genetic diversity found in its wild ancestor(s) due to the combined effects of a domestication bottleneck and human selection on traits associated with agriculture. As a framework for distinguishing between the effects of demography and selection, we sequenced 204 loci in a diverse panel of 17 cultivated S. bicolor accessions. Genomewide patterns of diversity depart strongly from equilibrium expectations with regard to the variance of the number of segregating sites, the site frequency spectrum, and haplotype configuration. Furthermore, gene genealogies of most loci with an excess of low frequency variants and/or an excess of segregating sites do not show the characteristic signatures of directional and diversifying selection, respectively. A simple bottleneck model provides an improved but inadequate fit to the data, suggesting the action of other population-level factors, such as population structure and migration. Despite a known history of recent selection, we find little evidence for directional selection, likely due to low statistical power and lack of an appropriate null model.

Agriculture↗

Direct detection of Mycobacterium tuberculosis complex in respiratory specimens by Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test and Roche Amplicor Mycobacterium Tuberculosis Test.

Two hundred and fifty-six sputum, bronchoalveolar lavage, and bronchial and tracheal aspirate specimens from 243 patients were tested for the presence of Mycobacterium tuberculosis complex by auramine fluorochrome staining, rRNA target amplification (Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test [AMTD]), and PCR (Roche Amplicor Mycobacterium Tuberculosis Test [Amplicor PCR]. The results were compared with those of conventional Löwenstein-Jensen tube culture and BACTEC radiometric liquid culture. A total of 26 specimens from 18 patients were culture positive for M. tuberculosis. In addition, seven specimens were positive by staining and by culture for other Mycobacterium species but negative by nucleic acid amplification methods and were not included in the comparison. When compared with that for culture, the sensitivities of the techniques were as follows: for staining, 80.8%; for Gen-Probe AMTD, 84.6%; and for Roche Amplicor PCR, 84.6%. The specificities were 99.1, 98.7, and 99.1%, respectively. After resolution of discrepant results by review of the patients' clinical data, 29 specimens from 21 patients were considered positive, and the overall sensitivities, specificities, and positive and negative predictive values were 89.7, 100, 100, and 98.7% for culture; 75.9, 99.5, 95.7, and 96.9% for staining; 86.2, 100, 100, and 98.2% for Gen-Probe AMTD; and 82.8, 100, 100, and 97.9% for Roche Amplicor PCR, respectively. It is concluded that both nucleic acid amplification methods are rapid, sensitive, and specific methods for the detection of M. tuberculosis in respiratory specimens.

Bacteriological Techniques↗

Development and evaluation of a rapid, simple, sensitive, monoclonal antibody-based co-agglutination test for direct detection of Vibrio cholerae 01.

Cholera epidemics caused by Vibrio cholerae 01 continue to represent a major public health concern in many developing countries. A rapid and simple test kit for the detection of V. cholerae 01 has been developed. The kit, CholeraScreen is a monoclonal antibody-based, co-agglutination test and is used directly with stool specimens. It does not include culturing the specimen and is performed without the need for sophisticated laboratory equipment. Specificity of the test was demonstrated, using 118 reference cultures, to which cross-reactions were not observed. Preliminary results of field trials carried out in Guatemala and Bangladesh demonstrated that the test is equally sensitive as conventional culture methods in detecting V. cholerae and, in many cases, more sensitive. The CholeraScreen test is simple, specific, and does not require culturing procedures, making it suitable for direct detection of cells of V. cholerae in clinical specimens, even in the field. Also, the test requires less than five minutes to complete.

Agglutination Tests↗

Direct detection of repetitive, whole chromosome paint and telomere DNA probes by immunogold electron microscopy.

Biotinylated repetitive, whole chromosome paint and telomere DNA probes were investigated at the electron microscope level after non-isotopic in situ hybridization and direct immunogold detection. The protocol described allowed the visualization of a biotinylated chromosome 1 specific satellite DNA probe in the light microscope without silver intensification. This sensitive method was exploited to analyse factors contributing to signal strength in immunogold chromosome painting. Furthermore, it allowed us to investigate the distribution of (TTAGGG)n telomere repeats in human metaphase chromosomes and interphase nuclei. Telomeric and internal (TTAGGG)n repeats were detected at high spatial resolution in human metaphase chromosomes. In the periphery of lymphocyte interphase nuclei, two types of telomere hybridization signals were observed. They differed remarkably in compactness, indicating two types of chromatin conformation present at the interphase telomeres in situ.

Base Sequence↗

A DNA microarray platform based on direct detection of rRNA for characterization of freshwater sediment-related prokaryotic communities.

A DNA microarray platform for the characterization of bacterial communities in freshwater sediments based on a heterogeneous set of 70 16S rRNA-targeted oligonucleotide probes and directly labeled environmental RNA was developed and evaluated. Application of a simple protocol for the efficient background blocking of aminosilane-coated slides resulted in an improved signal-to-noise ratio and a detection limit of 10 ng for particular 16S rRNA targets. An initial specificity test of the system using RNA from pure cultures of different phylogenetic lineages showed a fraction of false-positive signals of approximately 5% after protocol optimization and a marginal loss of correct positive signals. Subsequent microarray analysis of sediment-related community RNA from four different German river sites suggested low diversity for the groups targeted but indicated distinct differences in community composition. The results were supported by parallel fluorescence in situ hybridization in combination with sensitive catalyzed reporter deposition (CARD-FISH). In comparisons of the data of different sampling sites, specific detection of populations with relative cellular abundances down to 2% as well as a correlation of microarray signal intensities and population size is suggested. Our results demonstrate that DNA microarray technology allows for the fast and efficient precharacterization of complex bacterial communities by the use of standard single-cell hybridization probes and the direct detection of environmental rRNA, also in methodological challenging habitats such as heterogeneous lotic freshwater sediments.

Bacteria↗

Direct detection of weakly interacting massive particles using non-cryogenic techniques.

A flux of weakly interacting neutral particles incident on the Earth would produce occasional nuclear-recoil events in any material. These could be detected as scintillation or ionization pulses in suitable target materials. Expected rates are in the range 1-10(-4) events kg(-1) d(-1). These must be distinguished from much higher rates due to backgrounds from gamma and beta background, even in deep underground locations. Methods of uniquely identifying nuclear recoils are described using crystal scintillators, scintillation and ionization processes in liquid xenon, and ionization tracks in gases. World progress on these techniques and future prospects are summarized. A possible future weakly interacting massive particle detector based on mechanical recoil of suspended microgranules is proposed which, with advances in nanotechnology, could eventually be extended to the detection of low-energy relic neutrinos.

Journal Article↗

Direct detection of angioinvasive pulmonary aspergillosis in immunosuppressed patients: preliminary results with high-resolution 16-MDCT angiography.

OBJECTIVE: The aim of this study was to optimize detection of angioinvasive pulmonary aspergillosis by showing direct vessel involvement at a peripheral level with high-resolution MDCT angiography in patients with antibiotic-resistant fever of unknown origin under immunosuppression. Twelve CT angiographic examinations were prospectively performed in 10 patients with an optimized CT angiography protocol with 16-MDCT after IV administration of contrast agent using care bolus (Siemens Medical Solutions). Axial images and maximum intensity projections were evaluated for vascular occlusion by an experienced radiologist blinded to the clinical histories. Results were correlated with histology and clinical follow-up data including follow-up CT. Fourteen focal pulmonary lesions were detected by CT in eight patients. Eight of 14 lesions were confirmed by histology. In the remaining six lesions, diagnosis was made by clinical and CT follow-up. In nine of nine lesions in which angioinvasive infection was excluded, CT angiography showed patent vessels. In four of five lesions with histologically proven fungal angioinvasion, vascular occlusion was detected on CT angiography. CONCLUSION: High-resolution MDCT angiography has been shown to be a feasible technique to depict directly vessel occlusion in the setting of suspected fungal infections, especially for early diagnosis of angioinvasive pulmonary aspergillosis in immunosuppressed patients.

Adult↗

Comparative evaluation of initial and new versions of the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test for direct detection of Mycobacterium tuberculosis in respiratory and nonrespiratory specimens.

We evaluated the initial version of the Amplified Mycobacterium Tuberculosis Direct Test (Gen-Probe) (AMTDT 1) and the new version of AMTDT (AMTDT 2) for the detection of Mycobacterium tuberculosis directly from respiratory and nonrespiratory samples and compared the results with those of culture and staining methods. The assays were applied to 410 respiratory and 272 nonrespiratory samples collected from 515 patients. The combination of the culture results and clinical diagnosis was considered to be the "gold standard." Ninety-five respiratory specimens were collected from 67 patients with a diagnosis of pulmonary tuberculosis (TB) and 68 nonrespiratory specimens were collected from 61 patients with a diagnosis of extrapulmonary TB. With respiratory specimens, the sensitivity, specificity, and positive and negative predictive values were 83, 100, 100, and 96%, respectively, for AMTDT 1 and 94.7, 100, 100, and 98.4%, respectively, for AMTDT 2. With nonrespiratory specimens, the sensitivity, specificity, and positive and negative predictive values were 83, 100, 100, and 94%, respectively, for AMTDT 1 and 86.8, 100, 100, and 98.4%, respectively, for AMTDT 2. The overall results of AMTDT 1 and AMTDT 2 were concordant for 97% (661 of 682) of the samples. Statistically significant differences in sensitivities were found between AMTDT 1 and AMTDT 2 with respiratory specimens. It was concluded that although both nucleic acid amplification methods are rapid, sensitive, and specific for the detection of M. tuberculosis complex in all types of clinical samples, AMTDT 2 appeared to be more sensitive than AMTDT 1 when applied to smear-negative specimens. In contrast AMTDT 2 is more susceptible than AMTDT 1 to inhibitory substances in the amplification reaction. The turnaround time of AMTDT 2 is shorter (3.5 h) than that for AMTDT 1 (5 h).

Bacteriological Techniques↗

Multiplet structure of 13C NMR signal from glutamate and direct detection of tricarboxylic acid (TCA) cycle intermediates.

For the first time, 13C NMR signals are shown from 13C-enriched, low-level tricarboxylic acid (TCA) cycle intermediates from extracts of normal cardiac tissue. As the low tissue content of the key intermediates alpha-ketoglutarate (alpha-KG) and succinate (SUC) in normal, well perfused tissues has until now precluded direct NMR detection from intact tissues and tissue extracts, 13C NMR signal from glutamate has generally been used to infer the isotopomer patterns of intermediates that are in chemical exchange with glutamate. However, the required assumptions regarding intracellular compartmentation for such indirect analysis have not been previously tested, as glutamate is largely cytosolic while the TCA cycle enzymes are located in the mitochondria. Chromatographic isolation of alpha-KG and SUC from heart tissue extracts allowed isotopomer analysis to be performed for comparison with that of glutamate. At steady state, a direct relationship between glutamate and alpha-ketoglutarate isotopomers was found, but succinate isotopomers matched those of glutamate only in hearts that displayed negligible contributions from the oxidation of unlabeled endogenous carbon sources.

Animals↗

Development of a rapid PCR assay specific for Staphylococcus saprophyticus and application to direct detection from urine samples.

Staphylococcus saprophyticus is one of the most frequently encountered microorganisms associated with acute urinary tract infections (UTIs) in young, sexually active female outpatients. Conventional identification methods based on biochemical characteristics can efficiently identify S. saprophyticus, but the rapidities of these methods need to be improved. Rapid and direct identification of this bacterium from urine samples would be useful to improve time required for the diagnosis of S. saprophyticus infections in the clinical microbiology laboratory. We have developed a PCR-based assay for the specific detection of S. saprophyticus. An arbitrarily primed PCR amplification product of 380 bp specific for S. saprophyticus was sequenced and used to design a set of S. saprophyticus-specific PCR amplification primers. The PCR assay was specific for S. saprophyticus when tested with DNA from 49 gram-positive and 31 gram-negative bacterial species. This assay was also able to amplify efficiently DNA from all 60 strains of S. saprophyticus from various origins tested. This assay was adapted for direct detection from urine samples. The sensitivity levels achieved with urine samples was 19 CFU with 30 cycles of amplification and 0.5 CFU with 40 cycles of amplification. This PCR assay for the specific detection of S. saprophyticus is simple and rapid (approximately 90 min, including the time for urine specimen preparation).

DNA, Bacterial↗

[The direct detection of volatile fatty acids by gas chromatography in microbiological diagnosis].

The gas chromatographic analysis of volatile fatty acids (VFA) is of great significance in the diagnosis of anaerobes and anaerobic infections, respectively. The ether extraction commonly used for this purpose is relatively consuming in time and material. In this relation, the presented method of detection of VFA in the aqueous phase directly shows essential simplifications but has a lower sensitivity. Therefore, this method cannot replace the ether extraction to investigate broth cultures of anaerobes for taxonomical purposes. But it enables the determination of VFA with sufficient sensitivity in clinical materials of patients. Herewith, the detection of acetic acid only indicates an infection without anaerobes if the concentration is higher than 20 mg/100 ml. On the contrary, a high correlation exists between the detection of n-butyric acid as well as isovaleric acid and the cultural isolation of anaerobic microorganisms.

Bacteria, Anaerobic↗

Monoclonal antibodies for the direct detection of influenza-A virus by ELISA in clinical specimens from patients with respiratory infections.

BACKGROUND: Monoclonal antibody technology provides antibody reagents of known specificity, high titres and unlimited availability, that form ideal reference antibodies for use in specific viral antigen-detection methods. OBJECTIVES: To produce mouse monoclonal antibodies against antigenic sites of influenza-A virus, and evaluate their use as diagnostic reagents in a sandwich ELISA. STUDY DESIGN: (1) Production and characterization of monoclonal antibodies against influenza-A virus; (2) application of these antibodies in an ELISA method for direct antigen detection; and (3) evaluation of the ELISA as routine procedure. RESULTS: Four monoclonal antibodies (A1-A4) from mice immunized intranasally with influenza-A virus were selected according to their specific reactivity with either nucleoprotein or matrix protein antigens as demonstrated by Western blot analysis. These antibodies lacked haemagglutination inhibition and neutralization properties and recognized both H1N1 and H3N2 strains of influenza-A virus equally. A sandwich ELISA using unlabelled antibodies for antigen capture and biotin-labelled antibodies for antigen detection was used to analyse nasopharyngeal secretions or nasal swabs from culture-confirmed influenza-A-infected patients and comparable specimens from patients with other viral respiratory infections. Only influenza-A virus (strains H1N1 and H3N2) could be detected in samples from patients with known influenza-A and influenza-B infections, and also after re-isolation of such viruses in conventional cultures of MDCK cells or embryonated hens' eggs. The antigen-detection assay showed a diagnostic sensitivity of 100% and a specificity of 98.3% compared with conventional culture methods. CONCLUSION: The reported ELISA appears to be a rapid and inexpensive method for diagnosis and epidemiological studies of influenza-A infections.

Journal Article↗

A new gel tube method for the direct detection, identification and susceptibility testing of bacteria in clinical samples.

We recently developed a simple new method which is designed to separate and concentrate bacteria from a sample by centrifugation in a gel system. Bacterial enzyme activity is then detected inside the gel without further manipulation using a colorimetric or fluorogenic substrate. The method provides a rapid, direct means of detecting bacteria in clinical samples, dispensing with the 24-h period normally required to isolate colonies on agar. Various applications of the method are described below, e.g. screening of negative urine samples, identification of Escherichia coli in urine samples, identification of Staphylococcus aureus in blood culture broths and detection of oxacillin-resistant S. aureus in blood culture broths. The advantages of the gel system and other applications are discussed.

Bacteriological Techniques↗

Direct detection of Neisseria gonorrhoeae with monoclonal antibodies characterized by serotyping reagents.

A panel of monoclonal antibodies (MAbs) directed to outer membrane protein I were generated with the ultimate aim of detecting Neisseria gonorrhoeae in patient samples by a direct immunofluorescence (IF) test. In an initial evaluation of the sensitivity of these reagents, a cocktail of six IF MAbs recognized 491 (91%) of 540 gonococci isolates from several centers in Sydney, Australia. IF MAbs designated 185 and 228 recognized serovars of WI serogroup and IF MAbs 208, 210, and 312 recognized serovars of WII/III serogroup. IF MAb 198 recognized serovars within both serogroups. Three additional IF MAbs, designated 322, 323, and 330, were then generated by using strains which failed to react with the original MAb cocktail and which belonged to particular serovars. The new cocktail of nine IF MAbs recognized 96% of the gonococcal isolates, which incidentally contained representatives of serovars shown to have a worldwide distribution in previous studies. Although subtle differences were apparent in the reaction patterns found with coagglutination (serotyping) and IF, there nonetheless seems to be merit in the approach of continually evaluating the sensitivity of diagnostic reagents such as MAbs. This is especially true with an organism such as N. gonorrhoeae, which has the capacity to regularly alter the antigenic structure of its outer membrane proteins.

Antibodies, Monoclonal↗

Enhancement of varicella-zoster virus detection in A-549 shell vials by use of freeze-thawed specimens, extended incubation, and "a centrifuged, not incubated" direct detection method.

A total of 95 clinical samples were cultured for periods of 2 and 7 days in centrifuged A-549 shell vials before and after freezing and thawing of specimens. In addition, centrifuged A-549 shell vials were tested directly for varicella-zoster virus without incubation using a direct fluorescent antibody (DFA) technique. Twenty-seven specimens were positive by at least one method. The sensitivity for DFA on unincubated A-549 shell vials was 85.2%; for unfrozen 2-day cultures, 88.9%; for unfrozen 7-day cultures, 92.6%; for freeze-thaw 2-day cultures, 92.6%; and for freeze-thaw 7-day cultures, 96.3%. Freeze-thawed specimens cultured for 7 days yielded the highest number of positive results with conspicuous cell-to-cell spread as a sign of viral replication.

Cell Line↗

Direct detection and quantification of Alternaria alternata lycopersici toxins using high-performance liquid chromatography-evaporative light-scattering detection.

Alternaria alternata lycopersici toxins are produced by the tomato pathogenic fungus A. alternata f. sp. lycopersici. They are primary determinants for stem canker disease of tomato and induce apoptosis in animals and plants. Due to the lack of a UV chromophore, their detection has relied on derivatizations or immunoassays, which require tedious pre-assay processes. Here, we describe a method for direct and quantitative detection of the toxins. The method employs an evaporative light-scattering detector (ELSD) coupled to HPLC. It gives a linear response in the range of 400-4000 ng toxin, with a detection limit of about 6 ng/mul. The detection of all major toxin congeners by HPLC-ESLD was confirmed by liquid chromatography-mass spectrometry. Thus, the method provides a simple and sensitive tool for the studies of this group of host-specific mycotoxins.

Alternaria↗

Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. We used LAMP for detection of Mycobacterium tuberculosis complex, Mycobacterium avium, and Mycobacterium intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) or on a solid medium (Ogawa's medium). Species-specific primers were designed by targeting the gyrB gene, and their specificities were validated on 24 mycobacterial species and 7 nonmycobacterial species. The whole procedure is quite simple, starting with the mixing of all reagents in a single tube, followed by an isothermal reaction during which the reaction mixture is held at 63 degrees C. The resulting amplicons are visualized by adding SYBR Green I to the reaction tube. The only equipment needed for the amplification reaction is a regular laboratory water bath or heat block that furnishes a constant temperature of 63 degrees C. The assay had a detection limit of 5 to 50 copies of purified DNA with a 60-min incubation time. The reaction time could be shortened to 35 min for the species identification of M. tuberculosis complex, M. avium, and M. intracellulare from a solid-medium culture. Residual DNA lysates prepared for the Amplicor assay (Roche Diagnostics GmbH) from 66 sputum specimens were tested in the LAMP assay. Although the sample size used for the latter assay was small, 2.75 micro l of the DNA lysates, it showed a performance comparable with that of the Amplicor assay, which required 50 micro l of the lysates. This LAMP-based assay is simple, rapid, and sensitive; a result is available in 35 min for a solid-medium culture and in 60 min for a liquid-medium culture or for a sputum specimen that contains a corresponding amount of DNA available for testing.

Animals↗