scholarly journals Design of PCR assays to specifically detect and identify 37 Lactobacillus species in a single 96 well plate

2020 ◽  
Author(s):  
Eiseul Kim ◽  
Seung-Min Yang ◽  
Bora Lim ◽  
Si Hong Park ◽  
Bryna Rackerby ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Genomic Analysis ◽  
23S Rrna ◽  
Lactobacillus Species ◽  
Comparative Genomic ◽  
Rrna Gene ◽  
Gene Sequences ◽  
23S Rrna Gene ◽  
Species Specific

Abstract Background Lactobacillus species are used as probiotics and play an important role in fermented food production. However, use of 16S rRNA gene sequences as standard markers for the differentiation of Lactobacillus species offers a very limited scope, as several species of Lactobacillus share similar 16S rRNA gene sequences. In this study, we developed a rapid and accurate method based on comparative genomic analysis for the simultaneous identification of 37 Lactobacillus species that are commonly used in probiotics and fermented foods. Results To select species-specific sequences or genes, a total of 180 Lactobacillus genome sequences were compared using Python scripts. In 14 out of 37 species, species-specific sequences could not be found due to the similarity of the 16S–23S rRNA gene. Selected unique genes were obtained using comparative genomic analysis and all genes were confirmed to be specific for 52,478,804 genomes via in silico analysis; they were found not to be strain-specific, but to exist in all strains of the same species. Species-specific primer pairs were designed from the selected 16S–23S rRNA gene sequences or unique genes of species. The specificity of the species-specific primer pairs was confirmed using reference strains, and the accuracy and efficiency of the polymerase chain reaction (PCR) with the standard curve were confirmed. The PCR method developed in this study is able to accurately differentiate species that were not distinguishable using the 16S rRNA gene alone. This PCR assays were designed to detect and identify 37 Lactobacillus species. The developed method was then applied in the monitoring of 19 probiotics and 12 dairy products. The applied tests confirmed that the species detected in 17 products matched those indicated on their labels, whereas the remaining products contained species other than those appearing on the label. Conclusions The method developed in this study is able to rapidly and accurately distinguish different species of Lactobacillus , and can be used to monitor specific Lactobacillus species in foods such as probiotics and dairy products.

scholarly journals Design of PCR assays to specifically detect and identify 37 Lactobacillus species in a single 96 well plate

2020 ◽  
Author(s):  
Eiseul Kim ◽  
Seung-Min Yang ◽  
Bora Lim ◽  
Si Hong Park ◽  
Bryna Rackerby ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Genomic Analysis ◽  
23S Rrna ◽  
Lactobacillus Species ◽  
Comparative Genomic ◽  
Rrna Gene ◽  
Gene Sequences ◽  
23S Rrna Gene ◽  
Species Specific

Abstract Background Lactobacillus species are used as probiotics and play an important role in fermented food production. However, use of 16S rRNA gene sequences as standard markers for the differentiation of Lactobacillus species offers a very limited scope, as several species of Lactobacillus share similar 16S rRNA gene sequences. In this study, we developed a rapid and accurate method based on comparative genomic analysis for the simultaneous identification of 37 Lactobacillus species that are commonly used in probiotics and fermented foods. Results To select species-specific sequences or genes, a total of 180 Lactobacillus genome sequences were compared using Python scripts. In 14 out of 37 species, species-specific sequences could not be found due to the similarity of the 16S–23S rRNA gene. Selected unique genes were obtained using comparative genomic analysis and all genes were confirmed to be specific for 52,478,804 genomes via in silico analysis; they were found not to be strain-specific, but to exist in all strains of the same species. Species-specific primer pairs were designed from the selected 16S–23S rRNA gene sequences or unique genes of species. The specificity of the species-specific primer pairs was confirmed using reference strains, and the accuracy and efficiency of the polymerase chain reaction (PCR) with the standard curve were confirmed. The PCR method developed in this study is able to accurately differentiate species that were not distinguishable using the 16S rRNA gene alone. This PCR assays were designed to detect and identify 37 Lactobacillus species. The developed method was then applied in the monitoring of 19 probiotics and 12 dairy products. The applied tests confirmed that the species detected in 17 products matched those indicated on their labels, whereas the remaining products contained species other than those appearing on the label. Conclusions The method developed in this study is able to rapidly and accurately distinguish different species of Lactobacillus , and can be used to monitor specific Lactobacillus species in foods such as probiotics and dairy products.

scholarly journals Simultaneous detection of 37 Lactobacillus species using a real-time PCR assay based on whole-genome sequence analysis

2020 ◽  
Author(s):  
Eiseul Kim ◽  
Seung-Min Yang ◽  
Bora Lim ◽  
Si Hong Park ◽  
Bryna Rackerby ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Genomic Analysis ◽  
23S Rrna ◽  
Comparative Genomic ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Species Specific

Abstract BackgroundLactobacillus species are used as probiotics and play an important role in fermented food production. However, use of 16S rRNA gene sequences as standard markers for the differentiation of Lactobacillus species offers a very limited scope, as several species of Lactobacillus share similar 16S rRNA gene sequences. In this study, we developed a rapid and accurate method based on comparative genomic analysis for the simultaneous identification of 37 Lactobacillus species that are commonly used in probiotics and fermented foods. ResultsTo select species-specific sequences or genes, a total of 143 Lactobacillus complete genome sequences were compared using Python scripts. In 14 out of 37 species, species-specific sequences could not be found due to the similarity of the 16S–23S rRNA gene. Selected unique genes were obtained using comparative genomic analysis and all genes were confirmed to be specific for 52,478,804 genomes via in silico analysis; they were found not to be strain-specific, but to exist in all strains of the same species. Species-specific primer pairs were designed from the selected 16S–23S rRNA gene sequences or unique genes of species. The specificity of the species-specific primer pairs was confirmed using reference strains, and the accuracy and efficiency of the real-time polymerase chain reaction (PCR) with the standard curve were confirmed. The real-time PCR method developed in this study is able to accurately differentiate species that were not distinguishable using the 16S rRNA gene alone. This Real-time PCR method was designed to detect 37 Lactobacillus species in a single reaction. The developed method was then applied in the monitoring of 19 probiotics and 12 dairy products. The applied tests confirmed that the species detected in 17 products matched those indicated on their labels, whereas the remaining products contained species other than those appearing on the label. ConclusionsThe method developed in this study is able to rapidly and accurately distinguish different species of Lactobacillus, and can be used to monitor specific Lactobacillus species in foods such as probiotics and dairy products.

scholarly journals Simultaneous detection of 37 Lactobacillus species using a real-time PCR assay based on whole-genome sequence analysis

2020 ◽  
Author(s):  
Eiseul Kim ◽  
Seung-Min Yang ◽  
Bora Lim ◽  
Si Hong Park ◽  
Bryna Rackerby ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
23S Rrna ◽  
Lactobacillus Species ◽  
Comparative Genomic ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Species Specific

Abstract Background Lactobacillus species are used as probiotics and play an important role in fermented food production. However, use of 16S rRNA gene sequences as standard markers for the differentiation of Lactobacillus species offers a very limited scope, as several species of Lactobacillus share similar 16S rRNA gene sequences. In this study, we developed a rapid and accurate method based on comparative genomic analysis for the simultaneous identification of 37 Lactobacillus species that are commonly used in probiotics and fermented foods. Results To select species-specific sequences or genes, a total of 180 Lactobacillus genome sequences were compared using Python scripts. In 14 out of 37 species, species-specific sequences could not be found due to the similarity of the 16S–23S rRNA gene. Selected unique genes were obtained using comparative genomic analysis and all genes were confirmed to be specific for 52,478,804 genomes via in silico analysis; they were found not to be strain-specific, but to exist in all strains of the same species. Species-specific primer pairs were designed from the selected 16S–23S rRNA gene sequences or unique genes of species. The specificity of the species-specific primer pairs was confirmed using reference strains, and the accuracy and efficiency of the real-time polymerase chain reaction (PCR) with the standard curve were confirmed. The real-time PCR method developed in this study is able to accurately differentiate species that were not distinguishable using the 16S rRNA gene alone. This Real-time PCR method was designed to detect 37 Lactobacillus species in a single reaction. The developed method was then applied in the monitoring of 19 probiotics and 12 dairy products. The applied tests confirmed that the species detected in 17 products matched those indicated on their labels, whereas the remaining products contained species other than those appearing on the label. Conclusions The method developed in this study is able to rapidly and accurately distinguish different species of Lactobacillus, and can be used to monitor specific Lactobacillus species in foods such as probiotics and dairy products.

Psychrobacter sanguinis sp. nov., recovered from four clinical specimens over a 4-year period

2012 ◽  
Vol 62 (1) ◽  
pp. 49-54 ◽  
Author(s):  
Samantha E. Wirth ◽  
Héctor L. Ayala-del-Río ◽  
Jocelyn A. Cole ◽  
Donna J. Kohlerschmidt ◽  
Kimberlee A. Musser ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Novel Species ◽  
Sequence Similarity ◽  
23S Rrna ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
23S Rrna Gene ◽  
Reference Specimens

An analysis of 16S rRNA gene sequences from archived clinical reference specimens identified a novel species of the genus Psychrobacter, of which four strains have been independently isolated from human blood. On the basis of 16S rRNA gene sequence similarity, the closest relatives with validly published names were Psychrobacter arenosus R7T (98.7 %), P. pulmonis CECT 5989T (97.7 %), P. faecalis Iso-46T (97.6 %) and P. lutiphocae IMMIB L-1110T (97.2 %). Maximum-likelihood phylogenetic analysis of 16S rRNA gene sequences showed that the isolates belonged to the genus Psychrobacter and were members of a cluster associated with Psychrobacter sp. PRwf-1, isolated from a silk snapper fish. DNA–DNA relatedness and partial 23S rRNA gene sequences also supported the finding that the isolates belonged to a species distinct from its closest phylogenetic neighbours. The predominant cellular fatty acids were C18 : 1ω9c, C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), summed feature 5 (C18 : 2ω6,9c and/or anteiso-C18 : 0) and C18 : 0. Biochemical and morphological analysis further supported the assignment of the four isolates to a novel species. The name Psychrobacter sanguinis sp. nov. is proposed. The type strain is 13983T ( = DSM 23635T = CCUG 59771T).

scholarly journals Neisseria wadsworthii sp. nov. and Neisseria shayeganii sp. nov., isolated from clinical specimens

2011 ◽  
Vol 61 (1) ◽  
pp. 91-98 ◽  
Author(s):  
William J. Wolfgang ◽  
Andrea N. Carpenter ◽  
Jocelyn A. Cole ◽  
Sabine Gronow ◽  
Andrea Habura ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Strain ◽  
23S Rrna ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Nearest Neighbours ◽  
23S Rrna Gene ◽  
Additional Support

An analysis of 16S rRNA gene sequences from archived clinical reference specimens has identified two novel Neisseria species. For each species, two strains from independent sources were identified. Amongst species with validly published names, the closest species to the newly identified organisms were Neisseria canis, N. dentiae, N. zoodegmatis, N. animaloris and N. weaveri. DNA–DNA hybridization studies demonstrated that the newly identified isolates represent species that are distinct from these nearest neighbours. Analysis of partial 23S rRNA gene sequences for the newly identified strains and their nearest neighbours provided additional support for the species designation. Bayesian analysis of 16S rRNA gene sequences suggested that the newly identified isolates belong to distinct but related species of the genus Neisseria, and are members of a clade that includes N. dentiae, N. bacilliformis and N. canis. The predominant cellular fatty acids [16 : 0, summed feature 3 (16 : 1ω7c and/or iso-15 : 0 2-OH) and 18 : 1ω7c], as well as biochemical and morphological analyses further support the designation of Neisseria wadsworthii sp. nov. (type strain 9715T =DSM 22247T =CIP 109934T) and Neisseria shayeganii sp. nov. (type strain 871T =DSM 22246T =CIP 109933T).

Reclassification of Parvularcula flava as Aquisalinus luteolus nom. nov. and emended description of the genus Aquisalinus

2021 ◽  
Vol 71 (10) ◽  
Author(s):  
Jun-Jie Ying ◽  
Zhi-Cheng Wu ◽  
Yuan-Chun Fang ◽  
Lin Xu ◽  
Cong Sun
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Species ◽  
Comparative Genomic ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Content Type ◽  
Link Type ◽  
Type Strains

Parvularcula flava was proposed as a novel member of genus Parvularcula in 2016. Some time earlier, Aquisalinus flavus has been proposed as a novel species of a novel genus named Aquisalinus . When comparing the 16S rRNA gene sequences of type strains P. flava NH6-79T and A. flavus D11M-2T, they showed 97.9 % sequence identity, much higher than the sequence identities 92.7–94.3 % between P. flava NH6-79T and type strains in the genus Parvularcula , indicating that the later proposed novel taxon Parvularcula flava need reclassification. The phylogenetic trees based on 16S rRNA gene sequences and genome sequences both showed that P. flava NH6-79T and A. flavus D11M-2T formed a separated branch away from strains in the genera Parvularcula , Marinicaulis and Amphiplicatus . The average amino acid identity and average nucleotide identity values of P. flava NH6-79T and A. flavus D11M-2T were 87.9 and 85.0 %, respectively, much higher than the values between P. flava NH6-79T and other closely related type strains (54.3 %–58.1 % and 68.6–70.4 %, respectively). P. flava NH6-79T and A. flavus D11M-2T also contained summed feature 8 (C18 : 1  ω6c and/or C18 : 1  ω7c) and C16 : 0 as major fatty acids, distinguishing them from other closely related taxa. Based on the results of the phylogenetic, comparative genomic and phenotypic analyses, Parvularcula flava should be reclassified as Aquisalinus luteolus nom. nov. and the description of genus Aquisalinus is emended.

scholarly journals Asgard archaea are diverse, ubiquitous, and transcriptionally active microbes

2018 ◽  
Author(s):  
Mingwei Cai ◽  
Yang Liu ◽  
Zhichao Zhou ◽  
Yuchun Yang ◽  
Jie Pan ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Genomic Analysis ◽  
Phylogenetic Position ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Group D ◽  
Origin Of Eukaryotes

AbstractAsgard is a newly proposed archaeal superphylum. Phylogenetic position of Asgard archaea and its relationships to the origin of eukaryotes is attracting increasingly research interest. However, in-depth knowledge of their diversity, distribution, and activity of Asgard archaea remains limited. Here, we used phylogenetic analysis to cluster the publicly available Asgard archaeal 16S rRNA gene sequences into 13 subgroups, including five previously unknown subgroups. These lineages were widely distributed in anaerobic environments, with the majority of 16S rRNA gene sequences (92%) originating from sediment habitats. Co-occurrence analysis revealed potential relationships between Asgard, Bathyarchaeota, and Marine Benthic Group D archaea. Genomic analysis suggested that Asgard archaea are potentially mixotrophic microbes with divergent metabolic capabilities. Importantly, metatranscriptomics confirmed the versatile lifestyles of Lokiarchaeota and Thorarchaeota, which can fix CO2using the tetrahydromethanopterin Wood-Ljungdahl pathway, perform acetogenesis, and degrade organic matters. Overall, this study broadens the understandings of Asgard archaea ecology, and also provides the first evidence to support a transcriptionally active mixotrophic lifestyle of Asgard archaea, shedding light on the potential roles of these microorganisms in the global biogeochemical cycling.

Taxonomic study of the genus Salinicola: transfer of Halomonas salaria and Chromohalobacter salarius to the genus Salinicola as Salinicola salarius comb. nov. and Salinicola halophilus nom. nov., respectively

2010 ◽  
Vol 60 (4) ◽  
pp. 963-971 ◽  
Author(s):  
Rafael R. de la Haba ◽  
Cristina Sánchez-Porro ◽  
M. Carmen Márquez ◽  
Antonio Ventosa
Keyword(s):  
16S Rrna ◽  
Type Strain ◽  
Phylogenetic Trees ◽  
23S Rrna ◽  
Rrna Gene ◽  
Gene Sequences ◽  
The Family ◽  
23S Rrna Gene ◽  
Single Genus ◽  
Type Strains

We have carried out a polyphasic taxonomic characterization of the type strains of the species with the recently validated name Salinicola socius, together with two species that were phylogenetically closely related, Halomonas salaria and Chromohalobacter salarius. 16S rRNA gene sequence analyses showed that they constituted a coherent cluster, with sequence similarities between 98.7 and 97.7 %. We have determined the almost complete 23S rRNA gene sequences of these three type strains, and the percentage of similarity between them was 99.2–97.6 %. Phylogenetic trees based on the 16S rRNA and 23S rRNA gene sequences, obtained by using three different algorithms, were consistent and showed that these three species constituted a cluster separated from the other species of the genera of the family Halomonadaceae, supporting their placement in a single genus. All three species have ubiquinone 9 as the major respiratory quinone, and showed similar fatty acid and polar lipid profiles. The level of DNA–DNA hybridization between Salinicola socius DSM 19940T, Halomonas salaria DSM 18044T and Chromohalobacter salarius CECT 5903T was 41–21 %, indicating that they are different species of the genus Salinicola. A comparative phenotypic study of these strains following the proposed minimal standards for describing new taxa of the family Halomonadaceae has been carried out. The phenotypic data are consistent with the placement of these three species in a single genus and support their differentiation at the species level. On the basis of these data we have emended the description of the species Salinicola socius and we propose to transfer the species Halomonas salaria and Chromohalobacter salarius to the genus Salinicola, as Salinicola salarius comb. nov. (type strain M27T =KCTC 12664T =DSM 18044T) and Salinicola halophilus nom. nov. (type strain CG4.1T =CECT 5903T =LMG 23626T), respectively.

scholarly journals Sporosarcina newyorkensis sp. nov. from clinical specimens and raw cow’s milk

2012 ◽  
Vol 62 (2) ◽  
pp. 322-329 ◽  
Author(s):  
William J. Wolfgang ◽  
An Coorevits ◽  
Jocelyn A. Cole ◽  
Paul De Vos ◽  
Michelle C. Dickinson ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
New York State ◽  
23S Rrna ◽  
Rrna Gene ◽  
The Novel ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Clinical Specimens ◽  
The 16S Rrna Gene

Twelve independent isolates of a Gram-positive, endospore-forming rod were recovered from clinical specimens in New York State, USA, and from raw milk in Flanders, Belgium. The 16S rRNA gene sequences for all isolates were identical. The closest species with a validly published name, based on 16S rRNA gene sequence, is Sporosarcina koreensis (97.13 % similarity). DNA–DNA hybridization studies demonstrate that the new isolates belong to a species distinct from their nearest phylogenetic neighbours. The partial sequences of the 23S rRNA gene for the novel strains and their nearest neighbours also provide support for the novel species designation. Maximum-likelihood phylogenetic analysis of the 16S rRNA gene sequences confirmed that the new isolates are in the genus Sporosarcina. The predominant menaquinone is MK-7, the peptidoglycan has the type A4α l-Lys–Gly–d-Glu, and the polar lipids consist of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The predominant fatty acids are iso-C14 : 0, iso-C15 : 0 and anteiso-C15 : 0. In addition, biochemical and morphological analyses support designation of the twelve isolates as representatives of a single new species within the genus Sporosarcina, for which the name Sporosarcina newyorkensis sp. nov. (type strain 6062T  = DSM 23544T  = CCUG 59649T  = LMG 26022T) is proposed.

scholarly journals Gluconobacter nephelii sp. nov., an acetic acid bacterium in the class Alphaproteobacteria

2011 ◽  
Vol 61 (9) ◽  
pp. 2117-2122 ◽  
Author(s):  
Jintana Kommanee ◽  
Somboon Tanasupawat ◽  
Pattaraporn Yukphan ◽  
Taweesak Malimas ◽  
Yuki Muramatsu ◽  
...  
Keyword(s):  
Acetic Acid ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Phylogenetic Trees ◽  
23S Rrna ◽  
Rrna Gene ◽  
Phenotypic Data ◽  
Dna Relatedness ◽  
23S Rrna Gene ◽  
Type Strains

Three strains, RBY-1T, PHD-1 and PHD-2, were isolated from fruits in Thailand. The strains were Gram-negative, aerobic rods with polar flagella, produced acetic acid from ethanol and did not oxidize acetate or lactate. In phylogenetic trees based on 16S rRNA gene sequences and 16S–23S rRNA gene internal transcribed spacer (ITS) sequences, the strains formed a cluster separate from the type strains of recognized species of the genus Gluconobacter. The calculated 16S rRNA gene sequence and 16S–23S rRNA gene ITS sequence similarities were respectively 97.7–99.7 % and 77.3–98.1 %. DNA G+C contents ranged from 57.2 to 57.6 mol%. The strains showed high DNA–DNA relatedness of 100 % to one another, but low DNA–DNA relatedness of 11–34 % to the tested type strains of recognized Gluconobacter species. Q-10 was the major quinone. On the basis of the genotypic and phenotypic data obtained, the three strains clearly represent a novel species, for which the name Gluconobacter nephelii sp. nov. is proposed. The type strain is RBY-1T ( = BCC 36733T = NBRC 106061T = PCU 318T), whose DNA G+C content is 57.2 mol%.

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