GroEL PCR- RFLP – An efficient tool to discriminate closely related pathogenic Vibrio species

Silvester, Reshma; Alexander, Deborah; Antony, Ally C.; Hatha, Mohamed

doi:10.1016/j.micpath.2017.02.029

Cited by 14 publications

(10 citation statements)

References 15 publications

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“…Extracted gDNA was submitted to the UNC Chapel Hill High-Throughput Sequencing Facility for PCR amplification of the hsp60 universal target (UT) and V4V5 region of the 16S rRNA genes (see Table 4 for Vibrio Dynamics Revealed by hsp60 Amplicon Sequencing Applied and Environmental Microbiology primer sequences), library preparation, and amplicon sequencing. The hsp60 primers we used are universal for chaperonin sequences found in bacteria, some archaea, mitochondria, and plasmids and have been used as targets for the detection and identification of Vibrio and other clinically relevant bacteria (26,32,33,74). For the hsp60 PCR, primers were mixed at a 1:3 ratio of H279/H280 to H1612/H1613 to improve template representation of amplicon sequences as described by Hill et al (75).…”

Section: Methodsmentioning

confidence: 99%

“…The heat shock protein 60 gene (hsp60) is an excellent marker for differentiating closely related taxa, since it is protein encoding and thus more variable than 16S rRNA markers (31). This gene has been used to identify and differentiate Vibrio and other clinically relevant bacteria with high taxonomic resolution (32,33) and is a promising target for NGS of PCR amplicons, since reads as short as 200 bp can be used for species-level identification (31).…”

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confidence: 99%

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Vibrio Ecology in the Neuse River Estuary, North Carolina, Characterized by Next-Generation Amplicon Sequencing of the Gene Encoding Heat Shock Protein 60 ( hsp60 )

Jesser

Noble

2018

Appl Environ Microbiol

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Of marine eubacteria, the genus is intriguing because member species are relevant to both marine ecology and human health. Many studies have touted the relationships of to environmental factors, especially temperature and salinity, to predict total abundance but lacked the taxonomic resolution to identify the relationships among species and the key drivers of dynamics. To improve next-generation sequencing (NGS) surveys of, we have conducted both 16S small subunit rRNA and heat shock protein 60 () amplicon sequencing of water samples collected at two well-studied locations in the Neuse River Estuary, NC. Samples were collected between May and December 2016 with enhanced sampling efforts in response to two named storms. Using sequences, 21 species were identified, including the potential human pathogens ,, and Changes in the community mirrored seasonal and storm-related changes in the water column, especially in response to an influx of nutrient-rich freshwater to the estuary after Hurricane Matthew, which initiated dramatic changes in the overall community. Individual species dynamics were wide ranging, indicating that individual taxa have unique ecologies and that total abundance predictors are insufficient for risk assessments of potentially pathogenic species. Positive relationships between, dinoflagellates, and were identified, as were intraspecies associations, which further illuminated the interactions of cooccurring taxa along environmental gradients. The objectives of this research were to utilize a novel approach to improve sequence-based surveys of communities and to demonstrate the usefulness of this approach by presenting an analysis of dynamics in the context of environmental conditions, with a particular focus on species that cause disease in humans and on storm effects. The methods presented here enabled the analysis of dynamics with excellent taxonomic resolution and could be incorporated into future ecological studies and risk prediction strategies for potentially pathogenic species. Next-generation sequencing of and other innovative sequence-based approaches are valuable tools and show great promise for studying ecology and associated public health risks.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Vibrio Ecology in the Neuse River Estuary, North Carolina, Characterized by Next-Generation Amplicon Sequencing of the Gene Encoding Heat Shock Protein 60 ( hsp60 )

Jesser

Noble

2018

Appl Environ Microbiol

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“…The BLAST output was filtered to retain taxa assigned to the Vibrio genus using the filter_basta_fasta.py script also provided by BASTA (Kahlke and Ralph, 2019) and genes assigned as hsp60 and groEL were collected and added to our Vibrio-hsp60 dataset. Both of these genes were chosen because they have previously been assigned as the same gene, but are annotated differently (Silvester et al, 2017). The aforementioned steps generated 1926 Vibrio hsp60/groEL sequences.…”

Section: Primer and Vibrio Reference Dataset Constructionmentioning

confidence: 99%

“…Due to the inherent inadequacies of the 16S rRNA gene for correctly identifying Vibrio species, another gene target encoding the heat shock protein 60 (hsp60, also known as groEL or cpn60), a type I chaperonin protein that assists in protein folding (Hemmingsen et al, 1988;Farr et al, 2000;Brinker et al, 2001), has been proposed as a good candidate for Vibrio phylogenetic studies (Kwok et al, 2002). Previous studies utilizing hsp60 have primarily relied on Vibrio isolates for species identification (Preheim et al, 2011;Szabo et al, 2012;Silvester et al, 2017), but a recent study applied universal hsp60 primers to characterize Vibrio community diversity using high throughput amplicon sequencing (Jesser and Noble, 2018). While this approach delivered improved Vibrio species identification relative to 16S rRNA, it used universal, rather than Vibrio-centric, hsp60 primers and required bioinformatic filtering for Vibrio assigned species, as is often required for 16S rRNA sequencing.…”

Section: Introductionmentioning

confidence: 99%

A New High Throughput Sequencing Assay for Characterizing the Diversity of Natural Vibrio Communities and Its Application to a Pacific Oyster Mortality Event

et al. 2019

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The Vibrio genus is notable for including several pathogens of marine animals and humans, yet characterization of Vibrio diversity using routine 16S rRNA sequencing methods is often constrained by poor resolution beyond the genus level. Here, a new high throughput sequencing approach targeting the heat shock protein (hsp60) as a phylogenetic marker was developed to more precisely discriminate members of the Vibrio genus in environmental samples. The utility of this new assay was tested using mock communities constructed from known dilutions of Vibrio isolates. Relative to standard and Vibrio-specific 16S rRNA sequencing assays, the hsp60 assay delivered high levels of fidelity with the mock community composition at the species level, including discrimination of species within the Vibrio harveyi clade. This assay was subsequently applied to characterize Vibrio community composition in seawater and delivered substantially improved taxonomic resolution of Vibrio species compared to 16S rRNA analysis. Finally, this assay was applied to examine patterns in the Vibrio community within oysters during a Pacific oyster mortality event. In these oysters, the hsp60 assay identified species-level Vibrio community shifts prior to disease onset, pinpointing V. harveyi as a putative pathogen. Given that shifts in the Vibrio community can precede, cause, and follow disease onset in numerous marine organisms, there is a need for an accurate high throughput assay for defining Vibrio community composition in natural samples. This Vibrio-centric hsp60 sequencing assay offers the potential for precise high throughput characterization of Vibrio diversity, providing an enhanced platform for dissecting Vibrio dynamics in the environment.

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“…69 The ease of the use of PCR-RFLP was demonstrated in its successful implementation in species identification and discrimination in algae, bacteria, and fungi. [70][71][72]…”

Section: Parameter Of Comparison Pcr-rflp Pcr-sscpmentioning

confidence: 99%

Exploring the Potential and Limitations of PCR-RFLP and PCR-SSCP for SNP Detection: A Review

Hashim

Al-Shuhaib

2019

J Apple Biotechnol Rep

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Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP) are two independent methods used in the post-amplification genotyping of DNA variations. Both techniques are used in a wide range of screening applications to characterize single nucleotide polymorphisms (SNPs). The PCR-SSCP enables the identification of a potentially causative unknown SNP that could not be identified by PCR-RFLP. However, because complicated steps are not required to perform PCR-RFLP, it is used in many applications. On the other hand, PCR-RFLP is easier to process in terms of time and handover experience, the detection of a particular unknown SNP by PCR-SSCP has further chances. The simplicity of PCR-RFLP does not mean that it is better than PCR-SSCP. The reason is the limited ability of PCR-RFLP to detect nucleotide variations, which often go undetected because each restriction enzyme (RE) scans only a few recognition sequences, and other sequences are ignored. Furthermore, the efficacy of PCR-SSCP is sometimes hindered by many optimizations and also lack of experience. As PCR-SSCP allows other sequences within an amplicon to be separated and characterized, the choice between PCR-RFLP and PCR-SSCP is largely dependent on the reason for each genotyping experiment. This review provides a useful guide for comparing PCR-RFLP and PCR-SSCP in terms of their concepts, efficiency, ease of use, interpretation, and sensitivity as well as several other parameters. The comparison is extended to the practical applications of both techniques in terms of their utilization in molecular diagnostics and related applications.

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GroEL PCR- RFLP – An efficient tool to discriminate closely related pathogenic Vibrio species

Cited by 14 publications

References 15 publications

Vibrio Ecology in the Neuse River Estuary, North Carolina, Characterized by Next-Generation Amplicon Sequencing of the Gene Encoding Heat Shock Protein 60 ( hsp60 )

Vibrio Ecology in the Neuse River Estuary, North Carolina, Characterized by Next-Generation Amplicon Sequencing of the Gene Encoding Heat Shock Protein 60 ( hsp60 )

A New High Throughput Sequencing Assay for Characterizing the Diversity of Natural Vibrio Communities and Its Application to a Pacific Oyster Mortality Event

Exploring the Potential and Limitations of PCR-RFLP and PCR-SSCP for SNP Detection: A Review

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