Development of strain-specific PCR primers for the identification of Fusobacterium nucleatum subsp. fusiforme ATCC 51190T and subsp. vincentii ATCC 49256T

Shin, Hwan Seon; Kim, Minjung; Kim, Hwa-Sook; Park, Soon-Nang; Kim, Do Kyung; Baek, Dong-Heon; Kim, Chan; Kook, Joong-Ki

doi:10.1016/j.anaerobe.2009.04.003

Cited by 9 publications

(10 citation statements)

References 12 publications

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“…While all three kits were accurate without exception, the strain was not detected using the PCR primers. These results underscore the primers’ high specificity for the subspecies fusiforme , as indicated in the original publications [27]. Jervøe-Storm et al [8] and Verner et al [52] showed that, compared with the cultivation method, detection reliability using real-time PCR varies from pathogen to pathogen.…”

Section: Discussionsupporting

confidence: 60%

“…vincentii not available (208)Shin et al [27]Fv35-R1CCC AAG GAA AAT ACT AAFs17-F14GAT GAG GAT GAA AAG AAA CAA AGT A Fusobacterium nucleatum subsp. fusiforme not available (393)Shin et al [27]Fs17-R14CCA TTG AGA AGG GCT ATT GACPrInfw a TTT GTT GGG GAG TAA AGC GGG Prevotella intermedia 458-1,032 (404)Ashimoto et al [25]PrInrev a TCA ACA TCT CTG TAT CCT GCG TAgAcfw a AAA CCC ATC TCT GAG TTC TTC TTC Aggregatibacter actinomycetemcomitans 478-1,034 (557)Ashimoto et al [25]AgAcrev a ATG CCA ACT TGA CGT TAA ATEiCofw a CGA TTA GCT GTT GGG CAA CTT Eikenella corrodens not available (410)Furcht et al [7]EiCorev a ACC CTC TGT ACC GAC CAT TGT ATCaRefw a TTT CGG AGC GTA AAC TCC TTT TC Campylobacter rectus 415-1,012 (598)Slots et al [24]CaRerev a TTT CTG CAA GCA GAC ACT CTTSm479FTCG CGA AAA AGA TAA ACA AAC A Streptococcus mutans 599-1,077 (478)Chen et al [26]Sm479RGCC CCT TCA CAG TTG GTT AGLF1AAT ACC GCA TTA CAA CTT TG Lactobacillus fermentum 196-529 (337)(Dickson et al, 2005)LF2GGT TAA ATA CCG TCA ACG TA a Names assigned in this study …”

Section: Methodsmentioning

confidence: 99%

“…For the present study, such microbial mock communities were randomly composed from a pool of 16 different bacterial species that represent key periodontal pathogens and other oral or human-associated bacteria. These mock communities were used to test the capability of accurately identifying bacteria, as shown by (1) three commercial kits: ParoCheck®Kit20 (Greiner Bio-One GmbH, Frickenhausen, Germany), micro-IDent®plus11 (Hain Lifescience GmbH, Nehren, Germany), Carpegen® Perio Diagnostik (Carpegen GmbH, Münster, Germany) and (2) eleven previously published PCR primer pairs [7, 24–27]. …”

Section: Introductionmentioning

confidence: 99%

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Accuracy of commercial kits and published primer pairs for the detection of periodontopathogens

et al. 2016

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ObjectivesDespite the input of microbiome research, a group of 20 bacteria continues to be the focus of periodontal diagnostics and therapy. The aim of this study was to compare three commercial kits and laboratory-developed primer pairs for effectiveness in detecting such periodontopathogens.Materials and methodsFourteen bacterial mock communities, consisting of 16 randomly assembled bacterial strains, were used as reference standard for testing kits and primers. Extracted DNA from mock communities was analyzed by PCR in-house with specific primers and forwarded for analysis to the manufacturer’s laboratory of each of the following kits: ParoCheck®Kit 20, micro-IDent®plus11, and Carpegen® Perio Diagnostik.ResultsThe kits accurately detected Fusobacterium nucleatum, Prevotella intermedia/Prevotella nigrescens, Parvimonas micra, Aggregatibacter actinomycetemcomitans, Campylobacter rectus/showae, Streptococcus mitis, Streptococcus mutans, and Veillonella parvula. The in-house primers for F.nucleatum were highly specific to subtypes of the respective periopathogen. Other primers repeatedly detected oral pathogens not present in the mock communities, indicating reduced specificity.ConclusionsThe commercial kits used in this study are reliable tools to support periodontal diagnostics. Whereas the detection profile of the kits is fixed at a general specificity level, the design of primers can be adjusted to differentiate between highly specific strains. In-house primers are more error-prone. Bacterial mock communities can be established as a reference standard for any similar testing.Clinical relevanceThe tested kits render good results with selected bacterial species. Primers appear to be less useful for routine clinical diagnostics and of limited applicability in research. Basic information about the periodontopathogens identified in this study supports clinical decision-making.

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Section: Discussionsupporting

confidence: 60%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Accuracy of commercial kits and published primer pairs for the detection of periodontopathogens

et al. 2016

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“…Initially, all clinical isolates were confirmed as F. nucleatum by PCR (5) and RAPID32A (bioMérieux, France). To determine which subspecies we were working with, we used specific PCR primers described in the literature (17,29) to identify F. nucleatum subsp. vincentii and F. nucleatum subsp.…”

Section: Methodsmentioning

confidence: 99%

Isolation of a Novel Bacteriophage Specific for the Periodontal Pathogen Fusobacterium nucleatum

Machuca

Daille

Vinés

et al. 2010

Appl Environ Microbiol

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Fusobacterium nucleatum is a periodontal pathogen that has been directly associated with the development and progression of periodontal disease, a widespread pathology that affects the support tissues of the tooth. We isolated a new bacteriophage (Fnp⌽02) that specifically infects this bacterium. Transmission electron microscopy showed that the virion is composed of an icosahedral head and a segmented tail. The size of the phage genome was estimated to be approximately 59 kbp of double-stranded DNA. The morphological features and the genetic characteristics suggest that Fnp⌽02 is part of the Siphoviridae family. Using one-step growth and adsorption experiments, the latent period, burst size, and adsorption rate were estimated to be 15 h, 100 infectious units per cell, and 7.5 ؋ 10 ؊10 ml min ؊1 , respectively. A small fragment of phage DNA was cloned and sequenced, showing 93% nucleotide identity with the phage PA6 of Propionibacterium acnes and amino acid identity with fragments of two proteins (Gp3 and Gp4) of this phage. To our knowledge, Fnp⌽02 is the first phage described to infect Fusobacterium nucleatum and provides the base for future exploration of phages in the control of periodontal disease.

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“…The melting curve analysis showed that non-specific PCR products were not amplified (data not shown). According to the recommendation for determining the optimal annealing temperature (OAT) for PCR (Shin et al, 2010), gradient PCR was performed with the genomic DNAs of P. gingivalis ATCC 33277 T and P. endodontalis ATCC 35406 T (data not shown). According to the PrimerSelect program, the recommended OAT was 55.7°C but according to the gradient PCR data, the Pg-F/Pg-R primers can detect P. gingivalis up to 72°C while maintaining the signal intensity of the amplicons.…”

mentioning

confidence: 99%

Development of Porphyromonas gingivalis-specific quantitative real-time PCR primers based on the nucleotide sequence of rpoB

Park

Kook

2011

J Microbiol.

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Species-specific quantitative real-time PCR (qPCR) primers were developed for the detection of Porphyromonas gingivalis. These primers, Pg-F/Pg-R, were designed based on the nucleotide sequences of RNA polymerase β-subunit gene (rpoB). Species-specific amplicons were obtained from the tested P. gingivalis strains but not in any of the other strains (46 strains of 46 species). The qPCR primers could detect as little as 4 fg of P. gingivalis chromosomal DNA. These findings suggest that these qPCR primers are suitable for applications in epidemiological studies.

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Development of strain-specific PCR primers for the identification of Fusobacterium nucleatum subsp. fusiforme ATCC 51190T and subsp. vincentii ATCC 49256T

Cited by 9 publications

References 12 publications

Accuracy of commercial kits and published primer pairs for the detection of periodontopathogens

Accuracy of commercial kits and published primer pairs for the detection of periodontopathogens

Isolation of a Novel Bacteriophage Specific for the Periodontal Pathogen Fusobacterium nucleatum

Development of Porphyromonas gingivalis-specific quantitative real-time PCR primers based on the nucleotide sequence of rpoB

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