Melting of the Ribosomal RNA Gene Reveals Bacterial Species Identity: A Step toward a New Rapid Test in Clinical Microbiology

Reischl, Udo

doi:10.1373/clinchem.2006.076240

Cited by 7 publications

(7 citation statements)

References 15 publications

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“…Recent trends in the application of HRM analysis for microorganism identification (4,12,21) led us to explore a new avenue for influenza A virus subtyping. This novel approach is based on the same concept used for HMA and has been applied to mutation scanning and genotyping (30,37).…”

Section: Discussionmentioning

confidence: 99%

Rapid Differentiation of Influenza A Virus Subtypes and Genetic Screening for Virus Variants by High-Resolution Melting Analysis

et al. 2008

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Section: Discussionmentioning

confidence: 99%

Rapid Differentiation of Influenza A Virus Subtypes and Genetic Screening for Virus Variants by High-Resolution Melting Analysis

et al. 2008

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“…Detection of strains within species should also be possible using this methodology since melt curve analysis can identify SNPs in the amplicon, as has been shown previously in human studies (13,24,49). A DNA strand of approximately 200 bp has 4 200 different possible sequences, but the melting temperature (T m ) varies over only a 40°C range.…”

mentioning

confidence: 96%

“…Recent improvements in PCR methods have shown melt curve analysis to be useful for single nucleotide polymorphism (SNP) detection and differentiation of hetero-and homozygotes, as well as species detection (13,24,49). Small changes in sequence of the ITS-1, 5.8S, and ITS-2 rRNA gene regions between different strains of the same species should also be detectable and facilitate species as well as strain identification of Pseudonitzschia spp.…”

mentioning

confidence: 99%

Quantitative PCR Coupled with Melt Curve Analysis for Detection of Selected Pseudo-nitzschia spp. (Bacillariophyceae) from the Northwestern Mediterranean Sea

Andrée

Fernández-Tejedor

Elandaloussi

et al. 2011

Appl Environ Microbiol

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The frequency and intensity of Pseudo-nitzschia spp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells of Pseudo-nitzschia from clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for all Pseudo-nitzschia spp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. Total Pseudo-nitzschia cell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eight Pseudo-nitzschia species-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the total Pseudo-nitzschia cell counts from optical microscopy.

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“…Additionally, mussels containing only metacercarial cysts of H. quissetensis were not detected through qPCR. The melt curve analysis, used to assess qPCR assay specificity (Reischl 2006, Andree et al 2011, returned a single peak, confirming the presence of only one amplification product. This is important since H. quissetensis can be common in mussel samples (Stunkard 1938).…”

Section: Discussionmentioning

confidence: 88%

Development of quantitative PCR assay for detection of the trematode parasite Proctoeces maculatus in the blue mussel Mytilus edulis

Markowitz

Williams²,

Krause³

2016

Dis. Aquat. Org.

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The digenean trematode Proctoeces maculatus is an important parasite of the blue mussel Mytilus edulis. The parasite reduces mussel quality and yield, negatively impacting mussel aquaculture efforts. Typically, the trematode is detected by visual observation. To provide a better diagnostic tool able to detect this parasite at any life stage and at low intensities, we designed a species-specific molecular assay to detect P. maculatus in M. edulis tissue. Primers targeting the 18S nuclear ribosomal DNA (rDNA) from P. maculatus were used to develop an endpoint polymerase chain reaction assay and a quantitative polymerase chain reaction (qPCR) assay. Analytical specificity of the assays was demonstrated using DNA from 4 other digenean trematodes. The qPCR assay was linear from 6.79 × 10 2 to 6.79 × 10 7 copies of the cloned target DNA and had a conservative detection limit of 68 copies. The qPCR assay detected single cercariae, and the number of isolated cercariae was linearly correlated with the threshold cycle (C T ). Diagnostic sensitivity of the PCR-based methods was 100%. The assays also detected the parasite in 6 additional samples from the 57 tested through microscopy. We used the assays to verify the presence of encapsulated sporocysts in the mantle and to document infected mussels from Dover, New Hampshire, extending the previously described northern range of the species. Thus, this work has important implications for detection of the parasite in aquaculture and in monitoring its potential spread with climate change.KEY WORDS: Digenean · Aquaculture · Bivalve · Marine · Northwest Atlantic Resale or republication not permitted without written consent of the publisherDis Aquat Org 122: [125][126][127][128][129][130][131][132][133][134][135][136] 2016 and visible abscesses (Sunila et al. 2004). The parasites deplete glycogen and triglyceride levels in the mantle and hepatopancreas of infected mussels (Dennis et al. 1974, Machkevsky & Shchepkina 1985 and cause major organ damage as they spread throughout the mussel (Machkevsky & Gaevskaja 2008). Intense infections cause mussels to gape and detach from the substrate (Machkevsky & Gaevskaja 2008). Thus, infected mussels may be unable to handle the stress of harvesting and processing involved in aquaculture (Cole 1935, Canzonier 1972. Infection slows mussel growth (Machkevsky 1988) and at the highest intensity levels, ultimately leads to mortality (Machkevsky 1982, Machkevsky & Gaevskaja 2008. P. maculatus was implicated in mass mortalities of Mytilus galloprovincialis Lamarck, 1819 in Laguna Vaneta, Italy (Munford et al. 1981), and in the Black Sea (Machkevsky & Parukhin 1981), where the disease is referred to as 'proctecosis' (Machkevsky & Gaevs kaja 2008).P. maculatus has been found along the Gulf Coast (Wardle 1980) and east coast of the USA as far north as Woods Hole, MA. After numerous attempts by previous resear chers to find P. maculatus at sites north of Woods Hole were unsuccessful (Uzmann 1953, Pondick 1983, it was concluded that Woods Hol...

show abstract

Melting of the Ribosomal RNA Gene Reveals Bacterial Species Identity: A Step toward a New Rapid Test in Clinical Microbiology

Cited by 7 publications

References 15 publications

Rapid Differentiation of Influenza A Virus Subtypes and Genetic Screening for Virus Variants by High-Resolution Melting Analysis

Rapid Differentiation of Influenza A Virus Subtypes and Genetic Screening for Virus Variants by High-Resolution Melting Analysis

Quantitative PCR Coupled with Melt Curve Analysis for Detection of Selected Pseudo-nitzschia spp. (Bacillariophyceae) from the Northwestern Mediterranean Sea

Development of quantitative PCR assay for detection of the trematode parasite Proctoeces maculatus in the blue mussel Mytilus edulis

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