Evaluation of Q Gene Mycobacteria: A novel and easy nucleic acid chromatography method for mycobacterial species identification

ABSTRACTUse of whole-genome sequencing (WGS) for routine mycobacterial species identification and drug susceptibility testing (DST) is becoming a reality. We compared the performances of WGS and standard laboratory workflows prospectively, by parallel processing at a major mycobacterial reference service over the course of 1 year, for species identification, first-lineMycobacterium tuberculosisresistance prediction, and turnaround time. Among 2,039 isolates with line probe assay results for species identification, 74 (3.6%) failed sequencing or WGS species identification. Excluding these isolates, clinically important species were identified for 1,902 isolates, of which 1,825 (96.0%) were identified as the same species by WGS and the line probe assay. A total of 2,157 line probe test results for detection of resistance to the first-line drugs isoniazid and rifampin were available for 728M. tuberculosiscomplex isolates. Excluding 216 (10.0%) cases where there were insufficient sequencing data for WGS to make a prediction, overall concordance was 99.3% (95% confidence interval [CI], 98.9 to 99.6%), sensitivity was 97.6% (91.7 to 99.7%), and specificity was 99.5% (99.0 to 99.7%). A total of 2,982 phenotypic DST results were available for 777M. tuberculosiscomplex isolates. Of these, 356 (11.9%) had no WGS comparator due to insufficient sequencing data, and in 154 (5.2%) cases the WGS prediction was indeterminate due to discovery of novel, previously uncharacterized mutations. Excluding these data, overall concordance was 99.2% (98.7 to 99.5%), sensitivity was 94.2% (88.4 to 97.6%), and specificity was 99.4% (99.0 to 99.7%). Median processing times for the routine laboratory tests versus WGS were similar overall, i.e., 20 days (interquartile range [IQR], 15 to 31 days) and 21 days (15 to 29 days), respectively (P= 0.41). In conclusion, WGS predicts species and drug susceptibility with great accuracy, but work is needed to increase the proportion of predictions made.

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Evaluation of the Ribosomal Protein S1 Gene (rpsA) as a Novel Biomarker forMycobacteriumSpecies Identification

BioMed Research International ◽

10.1155/2015/271728 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 8

Author(s):

Hongfei Duan ◽

Guan Liu ◽

Xiaobo Wang ◽

Yuhong Fu ◽

Qian Liang ◽

...

Keyword(s):

Sequence Analysis ◽

Phylogenetic Tree ◽

Ribosomal Protein ◽

Species Identification ◽

Comparative Sequence Analysis ◽

Clinical Isolates ◽

Species Differentiation ◽

Alignment Algorithm ◽

Mycobacterial Species ◽

Ribosomal Protein S1

Objectives. To evaluate the resolution and reliability of therpsAgene, encoding ribosomal protein S1, as a novel biomarker for mycobacteria species identification.Methods. A segment of therpsAgene (565 bp) was amplified by PCR from 42 mycobacterial reference strains, 172 nontuberculosis mycobacteria clinical isolates, and 16M. tuberculosiscomplex clinical isolates. The PCR products were sequenced and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method.Results. Comparative sequence analysis of therpsAgene provided the basis for species differentiation within the genusMycobacterium. Slow- and rapid-growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. The sequences discrepancy was obvious betweenM. kansasiiandM. gastri, M. chelonaeandM. abscessus, M. aviumandM. intracellulare, andM. szulgaiandM. malmoense, which cannot be achieved by 16S ribosomal DNA (rDNA) homologue genes comparison. 183 of the 188 (97.3%) clinical isolates, consisting of 8 mycobacterial species, were identified correctly byrpsAgene blast.Conclusions. Our study indicates thatrpsAsequencing can be used effectively for mycobacteria species identification as a supplement to 16S rDNA sequence analysis.

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Nucleic acids in the last stages of maturation of two varieties

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.1977.056 ◽

2015 ◽

Vol 46 (4) ◽

pp. 691-700

Author(s):

R. Górecki ◽

S. Grzesiuk ◽

A. Rejowski

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Column Chromatography ◽

Acid Metabolism ◽

Nucleic Acid Metabolism ◽

Wheat Grain ◽

5S Rna ◽

Chromatography Method ◽

Wheat Embryos

Using the MAK column chromatography method the process of wheat grain maturation was found to be closely correlated with nucleic acid metabolism. The amount of nucleic acids in the embryos increased until the end of the maturation of the grain. This increase was the result of an intensive synthesis mainly of rRNA fractions and to some extent also of sRNA. The quantitative proportion of various fractions of nucleic acids also changed in the last stages of grain maturation. In the period of wax maturity in wheat embryos a higher content of 4S and 5S RNA was found which partly explains a higher viability of these seeds.

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Evaluation of a Low-Density Hydrogel Microarray Technique for Mycobacterial Species Identification

Journal of Clinical Microbiology ◽

10.1128/jcm.02579-14 ◽

2015 ◽

Vol 53 (4) ◽

pp. 1103-1114 ◽

Cited By ~ 14

Author(s):

Danila V. Zimenkov ◽

Elena V. Kulagina ◽

Olga V. Antonova ◽

Maria A. Krasnova ◽

Ekaterina N. Chernyaeva ◽

...

Keyword(s):

Species Identification ◽

Clinical Samples ◽

Probe Design ◽

Clinical Microbiology Laboratory ◽

Low Density ◽

Pathogenic Species ◽

Mycobacterial Species ◽

Accurate Identification ◽

Content Type ◽

Species Specific

In addition to the obligatory pathogenic species of theMycobacterium tuberculosiscomplex andMycobacterium leprae, the genusMycobacteriumalso includes conditionally pathogenic species that in rare cases can lead to the development of nontuberculous mycobacterial diseases. Because tuberculosis and mycobacteriosis have similar clinical signs, the accurate identification of the causative agent in a clinical microbiology laboratory is important for diagnostic verification and appropriate treatment. This report describes a low-density hydrogel-based microarray containing oligonucleotide probes based on the species-specific sequences of thegyrBgene fragment for mycobacterial species identification. The procedure included the amplification of a 352-nucleotide fragment of the gene and its hybridization on a microarray. The triple-species-specific probe design and the algorithm for hybridization profile recognition based on the calculation of Pearson correlation coefficients, followed by the construction of a profile database, allowed for the reliable and accurate identification of mycobacterial species, including mixed-DNA samples. The assay was used to evaluate 543 clinical isolates from two regions of Russia, demonstrating its ability to detect 35 mycobacterial species, with 99.8% sensitivity and 100% specificity when usinggyrB, 16S, and internal transcribed spacer (ITS) fragment sequencing as the standard. The testing of clinical samples showed that the sensitivity of the assay was 89% to 95% for smear-positive samples and 36% for smear-negative samples. The large number of identified species, the high level of sensitivity, the ability to detect mycobacteria in clinical samples, and the up-to-date profile database make the assay suitable for use in routine laboratory practice.

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Introduction

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100070266 ◽

1978 ◽

Vol 36 (3) ◽

pp. 543-544

Author(s):

W. Bernard

Keyword(s):

Molecular Weight ◽

Electron Microscope ◽

Nucleic Acid ◽

Gene Mapping ◽

Molecular Genetics ◽

Cell Biology ◽

Gene Activity ◽

Close Connection ◽

Basic Information ◽

Simple Systems

In comparison to many other fields of ultrastructural research in Cell Biology, the successful exploration of genes and gene activity with the electron microscope in higher organisms is a late conquest. Nucleic acid molecules of Prokaryotes could be successfully visualized already since the early sixties, thanks to the Kleinschmidt spreading technique - and much basic information was obtained concerning the shape, length, molecular weight of viral, mitochondrial and chloroplast nucleic acid. Later, additonal methods revealed denaturation profiles, distinction between single and double strandedness and the use of heteroduplexes-led to gene mapping of relatively simple systems carried out in close connection with other methods of molecular genetics.

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Infection of Escherichia coli with bacteriophages

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100063767 ◽

1969 ◽

Vol 27 ◽

pp. 370-371

Author(s):

Manfred E. Bayer

Keyword(s):

Escherichia Coli ◽

Nucleic Acid ◽

Cell Surface ◽

Virus Type ◽

Infection Process ◽

Adsorption Site ◽

The Other ◽

Specific Receptors ◽

Bacterial Receptors

The first step in the infection of a bacterium by a virus consists of a collision between cell and bacteriophage. The presence of virus-specific receptors on the cell surface will trigger a number of events leading eventually to release of the phage nucleic acid. The execution of the various "steps" in the infection process varies from one virus-type to the other, depending on the anatomy of the virus. Small viruses like ØX 174 and MS2 adsorb directly with their capsid to the bacterial receptors, while other phages possess attachment organelles of varying complexity. In bacteriophages T3 (Fig. 1) and T7 the small conical processes of their heads point toward the adsorption site; a welldefined baseplate is attached to the head of P22; heads without baseplates are not infective.

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