Identification of Pathogenic
            <i>Nocardia</i>
            Species by Reverse Line Blot Hybridization Targeting the 16S rRNA and 16S-23S rRNA Gene Spacer Regions

Xiao, Meng; Kong, Fanrong; Sorrell, Tania C.; Cao, Yongyan; Lee, Ok Cha; Liu, Ying; Sintchenko, Vitali; Chen, Wen

doi:10.1128/jcm.01761-09

Cited by 21 publications

(29 citation statements)

References 34 publications

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“…MLST methods, however, are costly and not readily available in all laboratories. The PCR-based reverse line blot (RLB) assay is a simple method to identify microbial pathogens using PCR followed by hybridization with species-specific oligonucleotide probes (39,41). This has the capacity to analyze simultaneously up to 43 strains against multiple probes (13).…”

mentioning

confidence: 99%

Accurate and Practical Identification of 20 Fusarium Species by Seven-Locus Sequence Analysis and Reverse Line Blot Hybridization, and an In Vitro Antifungal Susceptibility Study

Xiao

Kong

et al. 2011

J Clin Microbiol

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Eleven reference and 25 clinical isolates of Fusarium were subject to multilocus DNA sequence analysis to determine the species and haplotypes of the fusarial isolates from Beijing and Shandong, China. Seven loci were analyzed: the translation elongation factor 1 alpha gene (EF-1␣); the nuclear rRNA internal transcribed spacer (ITS), large subunit (LSU), and intergenic spacer (IGS) regions; the second largest subunit of the RNA polymerase gene (RPB2); the calmodulin gene (CAM); and the mitochondrial small subunit (mtSSU) rRNA gene. We also evaluated an IGS-targeted PCR/reverse line blot (RLB) assay for species/haplotype identification of Fusarium. Twenty Fusarium species and seven species complexes were identified. Of 25 clinical isolates (10 species), the Gibberella (Fusarium) fujikuroi species complex was the commonest (40%) and was followed by the Fusarium solani species complex (FSSC) (36%) and the F. incarnatum-F. equiseti species complex (12%). Six FSSC isolates were identified to the species level as FSSC-3؉4, and three as FSSC-5. Twenty-nine IGS, 27 EF-1␣, 26 RPB2, 24 CAM, 18 ITS, 19 LSU, and 18 mtSSU haplotypes were identified; 29 were unique, and haplotypes for 24 clinical strains were novel. By parsimony informative character analysis, the IGS locus was the most phylogenetically informative, and the rRNA gene regions were the least. Results by RLB were concordant with multilocus sequence analysis for all isolates. Amphotericin B was the most active drug against all species. Voriconazole MICs were high (>8 g/ml) for 15 (42%) isolates, including FSSC. Analysis of larger numbers of isolates is required to determine the clinical utility of the seven-locus sequence analysis and RLB assay in species classification of fusaria.

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confidence: 99%

Accurate and Practical Identification of 20 Fusarium Species by Seven-Locus Sequence Analysis and Reverse Line Blot Hybridization, and an In Vitro Antifungal Susceptibility Study

Xiao

Kong

et al. 2011

J Clin Microbiol

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“…remains a challenge with the increasing recognition of novel species and taxonomic reassignment (1,14,19). The present study demonstrates the potential of combining a novel IGS-directed PCR with CGE to gen- erate Nocardia species-specific CGE electropherograms to identify clinically relevant Nocardia isolates, including uncommon species.…”

Section: Discussionmentioning

confidence: 94%

“…This approximated the sensitivity of a combined 16S and 16S-23S IGS PCR-based reverse line blot (RLB) assay previously described by our group (19). While a minority (about 1 in 5) of isolates in our collection were found to have a unique or "signature" fragment size, it was usually the combination of fragments that provided discriminating power due to the fact that the majority of Nocardia species yielded at least two different fragment lengths.…”

Section: Discussionmentioning

confidence: 99%

“…The number of fragments/peaks, their length(s), and their general pattern as represented by the GeneMapper software, including whether smaller peaks (Ͻ10% of the highest peak) were present, were studied to determine the correlation of species identification by CGE pattern (electropherogram) with that of 16S rRNA gene sequencing (7,19). Isolates of one species matching those of another species based on the above-described criteria were analyzed further to determine if they could be separated based on the exact value to two decimal places of their fragments and pattern as depicted by the GeneMapper software, with a difference of Ն0.5 bp considered significant based on the results of subsequent reproducibility testing.…”

Section: Methodsmentioning

confidence: 99%

“…DNA extracts of 145 Nocardia isolates, previously characterized by standard phenotypic methods (1) and identified to the species level by 16S rRNA gene sequencing (7,8,18,19), were studied (Table 1). Extracts were retrieved from Ϫ80°C storage.…”

Section: Methodsmentioning

confidence: 99%

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A PCR-Based Intergenic Spacer Region-Capillary Gel Electrophoresis Typing Method for Identification and Subtyping of Nocardia Species

et al. 2012

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c While 16S rRNA sequence-based identification of Nocardia species has become the gold standard, it is not without its limitations. We evaluated a novel approach encompassing the amplification of the Nocardia 16S-23S rRNA intergenic spacer (IGS) region followed by fragment analysis by capillary gel electrophoresis (CGE) of the amplified product for species identification of Nocardia. One hundred forty-five Nocardia isolates (19 species) and four non-Nocardia aerobic actinomycetes were studied. Reproducibility testing was performed in a subset (21%) of isolates. Ninety-five different electropherograms were identified, with heterogeneity within species being a general observation. Among common Nocardia species (e.g., Nocardia cyriacigeorgica, N. nova, N. farcinica), 2 or 3 dominant electropherogram subgroups were typical. While only a minority (8/19; 42%) of the different Nocardia species contained isolates displaying unique fragment sizes that were predictive of a particular species, virtually all isolates (142/145; 98%) could be assigned to the correct species using IGS-CGE typing based on the number and size of amplified fragments. The median number of fragments for each isolate was 2 (range, 1 to 5) with only a minority (17%) having a single fragment detected. The majority (93%) of amplified fragments were between 408 and 461 bp. The technique was also non-operator dependent, highly reproducible, and quicker and less expensive than 16S sequencing. In summary, PCR-based IGS-CGE typing is relatively simple, accurate, reproducible, and cost-effective and offers a potential alternative to 16S rRNA sequencing for identifying and subtyping Nocardia isolates.

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Identification and quantification of Thiothrix eikelboomii using qPCR for early detection of bulking incidents in a full-scale water reclamation plant

Asvapathanagul

Olson

Gedalanga

et al. 2015

Appl Microbiol Biotechnol

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This study found that the ratio of Thiothrix eikelboomii to total bacterial concentrations (TH/TB) (%) was a better indicator of bulking incidents affecting effluent quality compared to absolute T. eikelboomii abundance alone. This was determined using a genus-specific Thiothrix quantitative PCR primer and probe set, which was developed in this study to monitor specific Thiothrix populations over a 1-year period. T. eikelboomii was identified as the source of bulking incidents based on sequencing of the 16S rRNA gene at a nitrifying-denitrifying wastewater treatment plant. Peak T. eikelboomii concentrations observed in March, April, and July 2009 were 2.32 × 10(10), 2.64 × 10(10), and 1.84 × 10(10) cells/l, respectively. The highest fraction of T. eikelboomii to total bacterial population was measured at 0.24% in March, and a ratio >0.19% caused increases of suspended solids and biochemical oxygen demand in the secondary effluent. Additionally, food/mass ratios, dissolved oxygen concentrations in the anoxic selector, and ammonium ion concentrations in the primary effluent were three parameters displaying statistically significant correlations (r = 0.40, r = 0.50, and r = 0.32, respectively) to Thiothrix spp. abundance in an aeration tank. No bulking events caused by T. eikelboomii occurred when the dissolved oxygen concentrations in the anoxic selector was maintained at lower than 0.12 mg/l and the TH/TB ratios were <0.10%.

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Identification of Pathogenic Nocardia Species by Reverse Line Blot Hybridization Targeting the 16S rRNA and 16S-23S rRNA Gene Spacer Regions

Cited by 21 publications

References 34 publications

Accurate and Practical Identification of 20 Fusarium Species by Seven-Locus Sequence Analysis and Reverse Line Blot Hybridization, and an In Vitro Antifungal Susceptibility Study

Accurate and Practical Identification of 20 Fusarium Species by Seven-Locus Sequence Analysis and Reverse Line Blot Hybridization, and an In Vitro Antifungal Susceptibility Study

A PCR-Based Intergenic Spacer Region-Capillary Gel Electrophoresis Typing Method for Identification and Subtyping of Nocardia Species

Identification and quantification of Thiothrix eikelboomii using qPCR for early detection of bulking incidents in a full-scale water reclamation plant

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