Rapid differentiation of bacterial species by high resolution melting curve analysis

Simenc, Janez; Potočnik, Uroš

doi:10.1134/s0003683811030136

Cited by 18 publications

(14 citation statements)

References 26 publications

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“…They were able to distinguish between 25 different pathogenic bacteria with 94% accuracy. Since then, others have used this technique to identify different species of Chlamydiaceae (9), Bartonella (10), and a wide array of pathogenic bacteria (11). Similar experiments have been done using denaturing high-performance liquid chromatography (DHPLC), which also succeeded in discriminating between a wide range of pure bacterial cultures (12).…”

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confidence: 69%

High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

Hjelmsø

Hansen

Bælum

et al. 2014

Appl Environ Microbiol

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In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.T he increased availability of sequencing facilities and the relative reduction in sequencing costs have made nucleotide sequencing a common tool in research. Despite this, the costs of sequencing and subsequent bioinformatic analyses still represent a substantive expense in many project budgets. A cheap and efficient initial screening of samples before sequencing is therefore an attractive way to discriminate samples and thus potentially reduce costs by focusing the effort on the most interesting ones. Traditional fingerprinting methods such as denaturing gradient gel electrophoresis (DGGE) (1) or terminal restriction fragment length polymorphism (T-RFLP) have previously been used extensively to study diversity changes in complex environmental samples. However, these methods require considerable technical experience and are laborious and time-consuming (2). An alternative to these gel-based methods is desirable, and we suggest here that high-resolution melt (HRM) analysis (3) as a screening tool could provide a superior alternative. The thermal stability of a PCR product is determined by its GC content, sequence length, and primary structure (4). This stability is used in melt curve analysis, in which specific PCR products are denatured under tightly controlled conditions and their melting behavior observed. Melt curve analysis of a PCR product was initially developed in conjunction with the quantitative PCR (qPCR) approach for quantifying a particular amplicon product (4). With this method it is possible to identify nonspecific PCR products or primer dimers. To examine PCR products in further detail, HRM analysis was developed (5). T...

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confidence: 69%

High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

Hjelmsø

Hansen

Bælum

et al. 2014

Appl Environ Microbiol

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“…[24]], single-strand conformational polymorphism (SSCP) [25], restriction fragment length polymorphism (RFLP) [e.g. [26]], denaturing gradient gel electrophoresis (DGGE) (e.g.…”

Section: Introductionmentioning

confidence: 99%

Using Next-Generation Sequencing to Analyse the Diet of a Highly Endangered Land Snail (Powelliphanta augusta) Feeding on Endemic Earthworms

et al. 2013

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Predation is often difficult to observe or quantify for species that are rare, very small, aquatic or nocturnal. The assessment of such species’ diet can be conducted using molecular methods that target prey DNA remaining in predators’ guts and faeces. These techniques do not require high taxonomic expertise, are applicable to soft-bodied prey and allow for identification at the species level. However, for generalist predators, the presence of mixed prey DNA in guts and faeces can be a major impediment as it requires development of specific primers for each potential prey species for standard (Sanger) sequencing. Therefore, next generation sequencing methods have recently been applied to such situations. In this study, we used 454-pyrosequencing to analyse the diet of Powelliphantaaugusta , a carnivorous landsnail endemic to New Zealand and critically endangered after most of its natural habitat has been lost to opencast mining. This species was suspected to feed mainly on earthworms. Although earthworm tissue was not detectable in snail faeces, earthworm DNA was still present in sufficient quantity to conduct molecular analyses. Based on faecal samples collected from 46 landsnails, our analysis provided a complete map of the earthworm-based diet of P . augusta . Predated species appear to be earthworms that live in the leaf litter or earthworms that come to the soil surface at night to feed on the leaf litter. This indicates that P . augusta may not be selective and probably predates any earthworm encountered in the leaf litter. These findings are crucial for selecting future translocation areas for this highly endangered species. The molecular diet analysis protocol used here is particularly appropriate to study the diet of generalist predators that feed on liquid or soft-bodied prey. Because it is non-harmful and non-disturbing for the studied animals, it is also applicable to any species of conservation interest.

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“…High-resolution melting analysis has already been successfully used for discriminating related species of bacteria (26,40). In addition, by measuring the T m of a small amplicon of the hsp65 gene, it has been found to be possible to differentiate Mycobacterium abscessus and M. chelonae, 2 very closely related, rapidly growing species that cannot be discriminated biochemically (41).…”

Section: Discussionmentioning

confidence: 99%

“…(22,23). High-resolution melting analysis (HRMA), further developed from real-time PCR, is an emerging technique in medical microbiology that may allow simultaneous detection and diagnosis of pathogens at the species and subspecies levels (24)(25)(26). This technique, first reported in 2002, is based on the difference in melting behaviors of DNA molecules, according to their sequence, lengths, and GC content (25,27).…”

mentioning

confidence: 99%

Rapid Detection and Identification of Nontuberculous Mycobacterial Pathogens in Fish by Using High-Resolution Melting Analysis

Phung

Caruso

Godreuil

et al. 2013

Appl Environ Microbiol

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e Mycobacterial infections in fish are commonly referred to as piscine mycobacteriosis, irrespectively of the specific identity of the causal organism. They usually cause a chronic disease and sometimes may result in high mortalities and severe economic losses. Nearly 20 species of Mycobacterium have been reported to infect fish. Among them, Mycobacterium marinum, M. fortuitum, and M. chelonae are generally considered the major agents responsible for fish mycobacteriosis. As no quick and inexpensive diagnostic test exists, we tested the potential of high-resolution melting analysis (HRMA) to rapidly identify and differentiate several Mycobacterium species involved in fish infections. By analyzing both the melting temperature and melting profile of the 16S-23S rRNA internal transcribed spacer (ITS), we were able to discriminate 12 different species simultaneously. Sensitivity tests conducted on purified M. marinum and M. fortuitum DNA revealed a limit of detection of 10 genome equivalents per reaction. The primers used in this procedure did not lead to any amplification signal with 16 control non-Mycobacterium species, thereby demonstrating their specificity for the genus Mycobacterium.

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Rapid differentiation of bacterial species by high resolution melting curve analysis

Cited by 18 publications

References 26 publications

High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

Using Next-Generation Sequencing to Analyse the Diet of a Highly Endangered Land Snail (Powelliphanta augusta) Feeding on Endemic Earthworms

Rapid Detection and Identification of Nontuberculous Mycobacterial Pathogens in Fish by Using High-Resolution Melting Analysis

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