Restriction analysis and partial sequencing of the 16S rRNA gene as index for rapid identification of Bacillus species

ABSTRACT The diversity of the methanotrophic community in mildly acidic landfill cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4.8 versus 6.8), air/CH4/CO2 headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1× nitrate minimal salts [NMS] versus 0.2× NMS). Screening of the isolates showed that the nutrient-rich 1× NMS selected for type I methanotrophs, while the nutrient-poor 0.2× NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the Methylobacter-Methylomicrobiumgroup, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly publishedMethylosinus and Methylocystis genera, and two members of this cluster were also successfully cultured. Partial sequencing of the pmoA gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placement of the four isolates. Finally, not all of the bands separated by DGGE could be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described.

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Molecular identification of pufferfish species using PCR amplification and restriction analysis of a segment of the 16S rRNA gene

Comparative Biochemistry and Physiology Part D Genomics and Proteomics ◽

10.1016/j.cbd.2005.09.004 ◽

2006 ◽

Vol 1 (1) ◽

pp. 139-144 ◽

Cited By ~ 9

Author(s):

Shoichiro Ishizaki ◽

Yasuhiro Yokoyama ◽

Naomasa Oshiro ◽

Natsuko Teruya ◽

Yuji Nagashima ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Molecular Identification ◽

Restriction Analysis ◽

Pcr Amplification ◽

Rrna Gene ◽

The 16S Rrna Gene

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

Journal of Microbiological Methods ◽

10.1016/0167-7012(96)00933-5 ◽

1996 ◽

Vol 27 (1) ◽

pp. 89-95 ◽

Cited By ~ 7

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

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Molecular analysis of the bacterial diversity in a specialized consortium for diesel oil degradation

Revista Brasileira de Ciência do Solo ◽

10.1590/s0100-06832010000300019 ◽

2010 ◽

Vol 34 (3) ◽

pp. 773-781 ◽

Cited By ~ 10

Author(s):

Douglas Antonio Alvaredo Paixão ◽

Mauricio Rocha Dimitrov ◽

Rodrigo Matheus Pereira ◽

Fábio Raphael Accorsini ◽

Maria Benincasa Vidotti ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Diesel Oil ◽

Molecular Techniques ◽

Rrna Gene ◽

Oil Degradation ◽

Partial Sequencing ◽

Oligonucleotide Primers ◽

The 16S Rrna Gene ◽

Diesel Oil Degradation

Diesel oil is a compound derived from petroleum, consisting primarily of hydrocarbons. Poor conditions in transportation and storage of this product can contribute significantly to accidental spills causing serious ecological problems in soil and water and affecting the diversity of the microbial environment. The cloning and sequencing of the 16S rRNA gene is one of the molecular techniques that allows estimation and comparison of the microbial diversity in different environmental samples. The aim of this work was to estimate the diversity of microorganisms from the Bacteria domain in a consortium specialized in diesel oil degradation through partial sequencing of the 16S rRNA gene. After the extraction of DNA metagenomics, the material was amplified by PCR reaction using specific oligonucleotide primers for the 16S rRNA gene. The PCR products were cloned into a pGEM-T-Easy vector (Promega), and Escherichia coli was used as the host cell for recombinant DNAs. The partial clone sequencing was obtained using universal oligonucleotide primers from the vector. The genetic library obtained generated 431 clones. All the sequenced clones presented similarity to phylum Proteobacteria, with Gammaproteobacteria the most present group (49.8 % of the clones), followed by Alphaproteobacteira (44.8 %) and Betaproteobacteria (5.4 %). The Pseudomonas genus was the most abundant in the metagenomic library, followed by the Parvibaculum and the Sphingobium genus, respectively. After partial sequencing of the 16S rRNA, the diversity of the bacterial consortium was estimated using DOTUR software. When comparing these sequences to the database from the National Center for Biotechnology Information (NCBI), a strong correlation was found between the data generated by the software used and the data deposited in NCBI.

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Development of a rapid identification method forAeromonasspecies by multiplex-PCR

Canadian Journal of Microbiology ◽

10.1139/w05-089 ◽

2005 ◽

Vol 51 (11) ◽

pp. 957-966 ◽

Cited By ~ 22

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.

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PCR-based Specific Detection of Bacillus in Liquid Organic Fertilizer

Journal of Microbial Systematics and Biotechnology ◽

10.37604/jmsb.v1i1.21 ◽

2019 ◽

Vol 1 (1) ◽

pp. 44-47

Author(s):

Iman Hidayat ◽

Nur Laili ◽

Dwi Agustiyani ◽

Sarjiya Antonius

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Sequence Data ◽

Organic Fertilizer ◽

Bacillus Species ◽

Rrna Gene ◽

16S Rrna Gene Sequences ◽

Pcr Assay ◽

Nucleotide Sequence Data ◽

The 16S Rrna Gene

Rapid molecular PCR-based detection method for Bacillus species used in the production of Beyonic® liquid organic fertilizer was carried out based on nucleotide sequence data from the 16S rRNA gene. The method involved sequencing the 16S rRNA gene of several Bacillus species and identifying around 16-22 specific nucleotide bases from 5' and 3' ends in the Bacillus 16S rRNA gene sequences. One specific primer pair for Bacillus detection was determined as follow: 5' - CAT AAG ACT GGG ATA ACT CCG GG - 3' (forward) from positions of 85-107 bp, and 5’ - CCA GGC GGA GTG CTT AAT GC - 3’ (reverse) from positions of 836-854 bp. PCR assay and gel electrophoresis analysis showed that the primer pair was specific to the genus Bacillus.

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