Development of a rapid identification method forAeromonasspecies by multiplex-PCR

2005 ◽  
Vol 51 (11) ◽  
pp. 957-966 ◽  
Author(s):  
Keya Sen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Multiplex Pcr ◽  
Rapid Identification ◽  
Complete Agreement ◽  
Aeromonas Species ◽  
Rrna Gene ◽  
Primer Sets ◽  
Species Specific ◽  
The 16S Rrna Gene

Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.Key words: Aeromonas, multiplex-PCR, identification.

Rapid identification of Saccharomonospora strains by multiplex PCR using species-specific primers within the 16S rRNA gene

1996 ◽  
Vol 27 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Jung-Hoon Yoon ◽  
Sung Taik Lee ◽  
Yong Kook Shin ◽  
Sam-Bong Kim ◽  
Hong-Joong Kim ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Multiplex Pcr ◽  
Rapid Identification ◽  
Rrna Gene ◽  
Specific Primers ◽  
Species Specific ◽  
The 16S Rrna Gene

Rapid identification of bacteria by real-time amplification and sequencing of the 16S rRNA gene

2006 ◽  
Vol 66 (1) ◽  
pp. 156-164 ◽  
Author(s):  
Inge Vliegen ◽  
Jan A. Jacobs ◽  
Erik Beuken ◽  
Cathrien A. Bruggeman ◽  
Cornelis Vink
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Rapid Identification ◽  
Rrna Gene ◽  
Identification Of Bacteria ◽  
Time Amplification ◽  
The 16S Rrna Gene

scholarly journals Impact of DNA purification method and primer selection on 16S rRNA gene metabarcoding on wine

OENO One ◽  
2019 ◽  
Vol 53 (3) ◽  
Author(s):  
Francesco Cerutti ◽  
Diego Cravero ◽  
Antonella Costantini ◽  
Laura Pulcini ◽  
Paola Modesto ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Alternative Methods ◽  
Dna Purification ◽  
Rrna Gene ◽  
Purification Method ◽  
Main Species ◽  
Purification Methods ◽  
Primer Sets ◽  
The 16S Rrna Gene

Aim: The high-throughput sequencing methods have revolutionized the study of the microbiota in different matrices including those of the grapevine production chain. DNA extraction is a crucial step in the sample processing. In this study, we compared different DNA purification methods and two primer sets for 16S rRNA gene metabarcoding to evaluate the best protocol to explore the wine microbiota by metabarcoding.Methods and results: We collected a wine from Barbera grapes after malolactic fermentation previously inoculated by Oenococcus oeni starter. The same sample was used to evaluate the best performing protocol to study the wine microbiota. DNA was purified using nine different methods and then amplified for the 16S rRNA gene with two primer sets (according to Illumina or Earth Microbiome Project protocols). The obtained amplicons were then sequenced in a single sequencing session on an Illumina MiSeq. We evaluated the best protocol considering DNA concentration and purity, alpha (Observed species) and beta diversity from metabarcoding analysis.The sequencing generated 36,031,756 reads in total. Although no statistically significant difference was observed between purification methods or primer sets, better results were obtained with phenol-chloroform DNA purification combined to Earth Microbiome Project primers.Metabarcoding was able to highlight the domination of the inoculum, O. oeni, representing the main species of the analyzed wine microbiota.Conclusion: Our data show that, for the tested wine, metabarcoding output is more influenced by the primer set than by the DNA purification method. Moreover, the metabarcoding detected that O. oeni represents the main species, evidencing the domination of the inoculum done with lyophilized commercial preparation of this species. Other lactic acid bacteria are present at a much lower abundance.Significance and impact of the study: This is the first report applying the 16S rRNA gene metabarcoding to study the microbiota of wine. For this reason, the evaluation of alternative methods for DNA processing is essential for future research using this innovative methodology.

Identification of non-tuberculous mycobacteria: utility of the GenoType Mycobacterium CM/AS assay compared with HPLC and 16S rRNA gene sequencing

2009 ◽  
Vol 58 (7) ◽  
pp. 900-904 ◽  
Author(s):  
Andie S. Lee ◽  
Peter Jelfs ◽  
Vitali Sintchenko ◽  
Gwendolyn L. Gilbert
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Gene Sequencing ◽  
Rapid Identification ◽  
Mycobacterium Abscessus ◽  
Clinical Isolates ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Clinically Significant ◽  
Species Specific

Non-tuberculous mycobacteria (NTM) causing clinical disease have become increasingly common and more diverse. A new reverse line probe assay, GenoType Mycobacterium CM/AS (Hain Lifescience), was evaluated for identification of a broad range of NTM. It was compared with phenotypic (HPLC) and molecular (DNA probes, in-house real-time multiplex species-specific PCR, 16S rRNA gene PCR and sequencing) identification techniques, which together provided the reference ‘gold standard’. A total of 131 clinical isolates belonging to 31 Mycobacterium species and 19 controls, including 5 non-Mycobacterium species, was used. Concordant results between the GenoType Mycobacterium assay and the reference identification were obtained in 119/131 clinical isolates (90.8 %). Identification of Mycobacterium abscessus and Mycobacterium lentiflavum by the assay was problematic. The GenoType Mycobacterium assay enables rapid identification of a broad range of potentially clinically significant Mycobacterium species, but some species require further testing to differentiate or confirm ambiguous results.

scholarly journals Hydrazine Synthase, a Unique Phylomarker with Which To Study the Presence and Biodiversity of Anammox Bacteria

2011 ◽  
Vol 78 (3) ◽  
pp. 752-758 ◽  
Author(s):  
Harry R. Harhangi ◽  
Mathilde Le Roy ◽  
Theo van Alen ◽  
Bao-lan Hu ◽  
Joost Groen ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Environmental Samples ◽  
Anammox Bacteria ◽  
Rrna Gene ◽  
A Subunit ◽  
Primer Sets ◽  
Marine Environmental ◽  
The 16S Rrna Gene ◽  
Wastewater Treatment Systems

ABSTRACTAnaerobic ammonium-oxidizing (anammox) bacteria play an important role in the biogeochemical cycling of nitrogen. They derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. Several methods have been used to detect the presence and activity of anammox bacteria in the environment, including 16S rRNA gene-based approaches. The use of the 16S rRNA gene to study biodiversity has the disadvantage that it is not directly related to the physiology of the target organism and that current primers do not completely capture the anammox diversity. Here we report the development of PCR primer sets targeting a subunit of the hydrazine synthase (hzsA), which represents a unique phylogenetic marker for anammox bacteria. The tested primers were able to retrievehzsAgene sequences from anammox enrichment cultures, full-scale anammox wastewater treatment systems, and a variety of freshwater and marine environmental samples, covering all known anammox genera.

scholarly journals Analysis of the 16S rRNA Gene Sequence of Anaplasma centrale and Its Phylogenetic Relatedness to Other Ehrlichiae

2001 ◽  
Vol 8 (2) ◽  
pp. 241-244 ◽  
Author(s):  
Hisashi Inokuma ◽  
Yutaka Terada ◽  
Tugihiko Kamio ◽  
Didier Raoult ◽  
Philippe Brouqui
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Distance Analysis ◽  
Specific Pcr ◽  
Species Specific ◽  
Anaplasma Centrale ◽  
The 16S Rrna Gene

ABSTRACT The nucleotide sequence of the Anaplasma centrale 16S rRNA gene was determined and compared with the sequences of ehrlichial bacteria. The sequence of A. centrale was closely related to Anaplasma marginale by both level-of-similarity (98.08% identical) and distance analysis. A species-specific PCR was developed based upon the alignment data. The PCR can detect A. centrale DNA extracted from 10 infected bovine red blood cells in a reaction mixture. A. centrale DNA was amplified in the reaction, but not other related ehrlichial species.

Sign in / Sign up

Export Citation Format

Share Document