DNA extraction was performed using the EZ1 DNA Tissue Kit and BioRobot EZ1 Advanced XL (Qiagen, Courtaboeuf, France). DNA extracts were used for 16S rRNA amplification using the fD1 and rP2 primers (Eurogentec, Angers, France). Amplicon sequencing was performed using the Big Dye® Terminator v1.1 Cycle Sequencing Kit and an ABI Prism 3130xl Genetic Analyzer capillary sequencer (Applied Biosystems), as previously described42 (link). The obtained 16S rRNA sequences were compared with those available in GenBank (http://www.ncbi.nlm.nih.gov/genbank ). Identification at the species level was defined by a 16S rRNA gene sequence similarity ≥98.65% with the sequence of the prototype strain of a species with standing in nomenclature. When this percentage of identity was lower than the generally accepted thresholds of 98.65% or 95%, the strain studied was considered a putative new species or genus, respectively43 ,44 (link).
Abi prism 3130xl genetic analyzer capillary sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
The ABI Prism 3130xl Genetic Analyzer is a capillary sequencer used for DNA sequencing. It is a multi-capillary electrophoresis instrument designed for high-throughput genetic analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Geneamp pcr system 2720, by Thermo Fisher Scientific (2 mentions)
Fd1 and rp2 universal primers, by Eurogentec (2 mentions)
Maldi biotyper software, by Bruker (2 mentions)
Ez1 dna tissue kit, by Qiagen (1 mentions)
Ez1 advanced xl biorobot, by Qiagen (1 mentions)
Chromaspro 1, by Technelysium (1 mentions)
Microflex lt maldi tof mass spectrometer, by Bruker (1 mentions)
7 protocols using abi prism 3130xl genetic analyzer capillary sequencer
1
16S rRNA Sequence Analysis for Microbial Identification
2
Bacterial Identification via MALDI-TOF and 16S rRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Using a MSP 96 MALDI‐TOF target plate, bacterial colonies were spotted and identified by the means of MALDI‐TOF MS using a Microflex LT spectrometer as previously described (Seng et al., 2009 ). In case of MALDI‐TOF's identification failure due to lack of a reference strain in the database, 16S rRNA sequencing was used for further analysis using the GeneAmp PCR System 2720 thermal cyclers (Applied Biosystems, Foster City, CA, USA) and the ABI Prism 3130xl Genetic Analyzer capillary sequencer (Applied Biosystems) (Morel et al., 2015 ). Sequences were assembled and modified using CodonCode Aligner software (http://www.codoncode.com ) and finally blasted against the online database of National Center for Biotechnology Information (NCBI) database (http://blast.ncbi.nlm.nih.gov.gate1.inist.fr/Blast.cgi ). Once blasted, a sequence similarity of less than 98.65% with the closest species was used to define a new species and 95% for defining a new genus (Kim, Oh, Park, & Chun, 2014 ). Subsequently, the mass spectrum of the new species was added to the URMITE [Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes] database (http://www.mediterranee-infection.com/article.php?laref=256&titre=urms-database ) and its 16S rRNA gene sequence was submitted to EMBL‐EBI with an accession number of LT598568 .
3
Bacterial Identification via MALDI-TOF and 16S rRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to identify bacteria, MALDI-TOF was used following the same protocol as previously described [5] (link), [6] (link), [7] (link), [8] (link). Identification of bacteria continued with a 16S rRNA standard PCR coupled with sequencing. The apparatuses used were GeneAmp PCR System 2720 thermal cyclers (Applied Biosystems, Foster City, CA, USA) and the ABI Prism 3130xl Genetic Analyzer capillary sequencer (Applied Biosystems), respectively [10] (link). The 16S rRNA nucleotide sequence obtained after amplification and sequencing was corrected using Chromas Pro 1.34 software (Technelysium, Tewantin, Australia). Then a BLASTN was systematically performed in the online PubMed National Center for Biotechnology Information (NCBI) database (http://blast.ncbi.nlm.nih.gov.gate1.inist.fr/Blast.cgi ). The spectrum of K. gabonensis strain GM4 was entered in the Bruker database, and its 16S rRNA sequence was deposited in GenBank under accession number LN849790. Sequences were aligned using CLUSTALW, and phylogenetic inferences were obtained using the maximum-likelihood method within MEGA software [5] (link), [6] (link), [7] (link), [8] (link).
4
16S rRNA Gene Amplification and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 16S rRNA gene amplification PCR and sequencing were performed using GeneAmp PCR System 2720 thermal cyclers (Applied Bio systems, Bedford, MA, USA) and ABI Prism 3130xl Genetic Analyzer capillary sequencer (Applied Bio systems), respectively, as described by Drancourt et al. [6] . The CodonCode Aligner was used to correct sequences and BLASTn searches were performed on the NCBI (National Centre for Biotechnology Information) web server at http://blast .ncbi.nlm.nih.gov.gate1 .inist .fr/Blast .cgi for the taxonomic assignation. Pairwise sequence similarities were calculated using the method recommended by Meier-Kolthoff et al. [23] and as described previously [33] . Sequences were aligned using ClustalW with default parameters and phylogenies were inferred using the GGDC web server available at http://ggdc.dsmz.de/ using the DSMZ phylogenomics pipeline.
5
Bacterial 16S rRNA Gene Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DNA of the bacterial strain was extracted using the EZ1 (Qiagen, Venlo, The Netherlands) DNA tissue kit on the EZ1 (Qiagen) automate. The universal primers fD1 and rP2 (Eurogentec, Angers, France) were used to amplify the16S rRNA gene sequence. Sequencing was performed using the BigDye® Terminator v1.1 Cycle Sequencing Kit and ABI Prism 3130xl Genetic Analyzer capillary sequencer (Thermo Fisher, Saint-Aubin, France), as previously described [14 (link)]. Using Codon Code Aligner software (http://www.codoncode.com ), the 16S rRNA nucleotide sequences were assembled and corrected. Consensus sequence from 16S rRNA gene sequencing was compared by BLASTn within the NCBI 16S rRNA database (https://blast.ncbi.nlm.nih.gov/ ). In order to create a robust phylogenetic tree, the 16S rRNA sequences of species with a validly published name were downloaded from the LPSN website (https://lpsn.dsmz.de/ ). Using MEGA X software [15 (link)], the sequences were aligned and a phylogenetic tree was constructed with 1,000 bootstrap replicates.
6
Identification and Phylogenetic Analysis of Bacterial Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Identification of strains Marseille-Q5893 and Marseille-Q5883 was carried out using a Microflex LT MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) (Seng et al. 2013 (link)). The spectra of the two strains were imported into the MALDI Biotyper software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings). Interpretation of the scores was performed as previously described (Hadjadj et al. 2016 (link)).
The 16S rRNA gene of strains Marseille-Q5893 and Marseille-Q5883 was sequenced using the fD1 and rP2 universal primers (Eurogentec, Angers, France) as previously described (Drancourt et al. 2000 (link)), using an ABI Prism 3130xl Genetic Analyzer capillary sequencer (Thermo Fisher, Saint-Aubin, France). If the 16S rRNA sequence similarity with closely related species was between 95 and 98.65%, the strain was suggested as belonging to a new species (Stackebrandt and Goebel 1994 (link); Kim et al. 2014 (link)). If the similarity was below 95%, the strain would be considered a new genus (Tindall et al. 2010 (link); Rossi-Tamisier et al. 2015 (link)).
Using the MEGA-X (Kumar et al. 2018 (link)), the resulting 16S rRNA gene sequences were aligned and a phylogenetic tree was obtained with 1000 bootstrap replicates, based on the maximum likelihood (ML) and the Kimura 2-parameter methods (Kimura 1980 (link)).
The 16S rRNA gene of strains Marseille-Q5893 and Marseille-Q5883 was sequenced using the fD1 and rP2 universal primers (Eurogentec, Angers, France) as previously described (Drancourt et al. 2000 (link)), using an ABI Prism 3130xl Genetic Analyzer capillary sequencer (Thermo Fisher, Saint-Aubin, France). If the 16S rRNA sequence similarity with closely related species was between 95 and 98.65%, the strain was suggested as belonging to a new species (Stackebrandt and Goebel 1994 (link); Kim et al. 2014 (link)). If the similarity was below 95%, the strain would be considered a new genus (Tindall et al. 2010 (link); Rossi-Tamisier et al. 2015 (link)).
Using the MEGA-X (Kumar et al. 2018 (link)), the resulting 16S rRNA gene sequences were aligned and a phylogenetic tree was obtained with 1000 bootstrap replicates, based on the maximum likelihood (ML) and the Kimura 2-parameter methods (Kimura 1980 (link)).
7
Strain Identification via MALDI-TOF and 16S rRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Identi cation of strains Marseille-Q5893 and Marseille-Q5883 was carried out using a Micro ex LT MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) (Seng et al. 2013 (link)). The spectra of the two strains were imported into the MALDI Biotyper software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings). Interpretation of the scores was performed as previously described (Hadjadj et al. 2016 (link)).
The 16S rRNA gene of strains Marseille-Q5893 and Marseille-Q5883 was sequenced using the fD1 and rP2 universal primers (Eurogentec, Angers, France) as previously described (Drancourt et al. 2000) , using an ABI Prism 3130xl Genetic Analyzer capillary sequencer (Thermo Fisher, Saint-Aubin, France). Using the MEGA X (Kumar et al. 2018 (link)), the resulting 16S rRNA gene sequences were aligned and a phylogenetic tree was obtained with 1,000 bootstrap replicates, based on the maximum likelihood (ML) and the Kimura 2-parameter methods (Kimura 1980) (link).
The 16S rRNA gene of strains Marseille-Q5893 and Marseille-Q5883 was sequenced using the fD1 and rP2 universal primers (Eurogentec, Angers, France) as previously described (Drancourt et al. 2000) , using an ABI Prism 3130xl Genetic Analyzer capillary sequencer (Thermo Fisher, Saint-Aubin, France). Using the MEGA X (Kumar et al. 2018 (link)), the resulting 16S rRNA gene sequences were aligned and a phylogenetic tree was obtained with 1,000 bootstrap replicates, based on the maximum likelihood (ML) and the Kimura 2-parameter methods (Kimura 1980) (link).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!