Journal articles: '16S rRNA gene amplification'

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Suryani, Laksmi Ambarsari, and Efi Sanfitri Harahap. "AMPLIFIKASI GEN 16S-rRNA BAKTERI TERMOFILIK DARI SUMBER AIR PANAS, GUNUNG PANCAR BOGOR." Jurnal Riset Kimia 3, no. 1 (February 12, 2015): 83. http://dx.doi.org/10.25077/jrk.v3i1.97.

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ABSTRACT Exploration of thermophilic bacteria that produce thermostable enzyme is most useful in application for enzyme base industrial. The aim of of this research is to isolate and amplificate the 16S-rRNA gene from thermophilic bacteria isolate at hotspring, Mount of Pancar, Bogor. The research steps consist of bacteria isolation, chromosomal DNA extraction, and amplification of 16S-rRNA gene. The water sample as source for bacteria was collected from four cauldrons. Temperature and pH for each cauldron are red cauldron 75-80°C, pH 7; black cauldron 55°C, pH 7; white cauldron 57°C, pH 7; and saline cauldron 25°C, pH 6, respectively. The bacteria were cultivated at Luria Bertani (LB) and Thermus media. The chromosomal DNA have been extracted. Gene amplification of 16 S-rRNA have been carried out by using universal primer (Bac F1 and Uni B1). The size of amplicon is ± 1.5kb. Keywords : thermophilic bacteria, chromosomal DNA extraction, amplification of 16S-rRNA gene

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Hubu, Herlin S., Stenly Wullur, Veibe Warouw, Elvy L. Ginting, Robert A. Bara, and Adnan S. Wantasen. "FILOGENI MOLEKULER BAKTERI DARI MEDIA PEMELIHARAAN ROTIFER YANG DIBERI OLAHAN LIMBAH IKAN SEBAGAI SUMBER NUTRISI." JURNAL PESISIR DAN LAUT TROPIS 9, no. 1 (March 29, 2021): 38. http://dx.doi.org/10.35800/jplt.9.1.2021.33574.

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This study aims to identify and construct molecular phylogeny of an isolate bacteria from culture media of rotifer Brachionus rotudiforis supplied with processed fishery waste feed as nutritional source. The use of fish waste-based food for rotifer showed positive effects on growth and nutrient content of the rotifers. Genomic DNA of the isolate bacteria BRLI- 01 was extracted and the 16S rRNA gene was amplified using primers (8F and 1492F) and further sequenced using Sanger sequence technique. The 16S rRNA gene was analysed using SeqScanner® and MEGA® followed with BLAST (Basic Local Alignment Search Tool) analyses in the NCBI (National Centre for Biotechnology Information). Amplification result of 16S rRNA gene bacteria s NCBI site as a reference for identification and phylogeny of bacterial species. BRLI-01 was successfully cultured on rotifer rearing media. The results of the 16S rRNA gene amplification of the isolate bacteria showed a DNA band with a length of 1400 bp. The BLAST result on the NCBI showed that the isolate bacteria BRLI-01 had a percent identity (98.46%) and is in the same phylogony branching position with Vibrio rotiferianus Keywords: Rotifers, Bacteria, Fish waste, 16S rRNA Genes, Phylogeny identification

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Marchesi, Julian R., Takuichi Sato, Andrew J. Weightman, Tracey A. Martin, John C. Fry, Sarah J. Hiom, and William G. Wade. "Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA." Applied and Environmental Microbiology 64, no. 2 (February 1, 1998): 795–99. http://dx.doi.org/10.1128/aem.64.2.795-799.1998.

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ABSTRACT We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16S rRNA genes from bacteria. Their specificity and efficacy were tested systematically with a variety of bacterial species and environmental samples. They were found to be more useful for 16S rRNA gene amplification in ecological and systematic studies than PCR amplimers that are currently more generally used.

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O’Farrell, Katrina A., and Peter H. Janssen. "Detection of Verrucomicrobia in a Pasture Soil by PCR-Mediated Amplification of 16S rRNA Genes." Applied and Environmental Microbiology 65, no. 9 (September 1, 1999): 4280–84. http://dx.doi.org/10.1128/aem.65.9.4280-4284.1999.

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ABSTRACT Oligonucleotide primers were designed and used to amplify, by PCR, partial 16S rRNA genes of members of the bacterial divisionVerrucomicrobia in DNA extracted from a pasture soil. By applying most-probable-number theory to the assay, verrucomicrobia appeared to contribute some 0.2% of the soil DNA. Amplified ribosomal DNA restriction analysis of 53 cloned PCR-amplified partial 16S rRNA gene fragments and comparative sequence analysis of 21 nonchimeric partial 16S rRNA genes showed that these primers amplified only 16S rRNA genes of members of the Verrucomicrobia in DNA extracted from the soil.

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Frank, Jeremy A., Claudia I. Reich, Shobha Sharma, Jon S. Weisbaum, Brenda A. Wilson, and Gary J. Olsen. "Critical Evaluation of Two Primers Commonly Used for Amplification of Bacterial 16S rRNA Genes." Applied and Environmental Microbiology 74, no. 8 (February 22, 2008): 2461–70. http://dx.doi.org/10.1128/aem.02272-07.

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ABSTRACT rRNA-based studies, which have become the most common method for assessing microbial communities, rely upon faithful amplification of the corresponding genes from the original DNA sample. We report here an analysis and reevaluation of commonly used primers for amplifying the DNA between positions 27 and 1492 of bacterial 16S rRNA genes (numbered according to the Escherichia coli rRNA). We propose a formulation for a forward primer (27f) that includes three sequences not usually present. We compare our proposed formulation to two common alternatives by using linear amplification—providing an assessment that is independent of a reverse primer—and in combination with the 1492 reverse primer (1492r) under the PCR conditions appropriate for making community rRNA gene clone libraries. For analyses of DNA from human vaginal samples, our formulation was better at maintaining the original rRNA gene ratio of Lactobacillus spp. to Gardnerella spp., particularly under stringent amplification conditions. Because our 27f formulation remains relatively simple, having seven distinct primer sequences, there is minimal loss of overall amplification efficiency and specificity.

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Kimura, Hiroyuki, Maki Sugihara, Kenji Kato, and Satoshi Hanada. "Selective Phylogenetic Analysis Targeted at 16S rRNA Genes of Thermophiles and Hyperthermophiles in Deep-Subsurface Geothermal Environments." Applied and Environmental Microbiology 72, no. 1 (January 2006): 21–27. http://dx.doi.org/10.1128/aem.72.1.21-27.2006.

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ABSTRACT Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76°C) and river water (14°C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82°C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84°C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84°C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained.

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Conville, Patricia S., Steven H. Fischer, Charles P. Cartwright, and Frank G. Witebsky. "Identification of Nocardia Species by Restriction Endonuclease Analysis of an Amplified Portion of the 16S rRNA Gene." Journal of Clinical Microbiology 38, no. 1 (January 2000): 158–64. http://dx.doi.org/10.1128/jcm.38.1.158-164.2000.

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ABSTRACT Identification of clinical isolates of Nocardia to the species level is important for defining the spectrum of disease produced by each species and for predicting antimicrobial susceptibility. We evaluated the usefulness of PCR amplification of a portion of the Nocardia 16S rRNA gene and subsequent restriction endonuclease analysis (REA) for species identification. Unique restriction fragment length polymorphism (RFLP) patterns were found for Nocardia sp. type strains (except for the N. asteroides type strain) and representative isolates of the drug pattern types of Nocardia asteroides (except for N. asteroides drug pattern type IV, which gave inconsistent amplification). A variant RFLP pattern for Nocardia nova was also observed. Twenty-eight clinical isolates were evaluated both by traditional biochemical identification and by amplification and REA of portions of the 16S rRNA gene and the 65-kDa heat shock protein (HSP) gene. There was complete agreement among the three methods on identification of 24 of these isolates. One isolate gave a 16S rRNA RFLP pattern consistent with the biochemical identification but was not identifiable by its HSP gene RFLP patterns. Three isolates gave 16S rRNA RFLP patterns which were inconsistent with the identification obtained by both biochemical tests and HSP gene RFLP; sequence analysis suggested that two of these isolates may belong to undefined species. The PCR and REA technique described appears useful both for the identification of clinical isolates of Nocardia and for the detection of new or unusual species.

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Korczak, Bożena M., Regina Stieber, Stefan Emler, André P. Burnens, Joachim Frey, and Peter Kuhnert. "Genetic relatedness within the genus Campylobacter inferred from rpoB sequences." International Journal of Systematic and Evolutionary Microbiology 56, no. 5 (May 1, 2006): 937–45. http://dx.doi.org/10.1099/ijs.0.64109-0.

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The genus Campylobacter comprises 17 species, some of which are important animal and human pathogens. To gain more insight into the genetic relatedness of this genus and to improve the molecular tools available for diagnosis, a universal sequencing approach was established for the gene encoding the beta-subunit of RNA polymerase (rpoB) for the genus Campylobacter. A total of 59 strains, including the type strains of currently recognized species as well as field isolates, were investigated in the study. A primer set specific for Campylobacter species enabled straightforward amplification and sequencing of a 530 bp fragment of the rpoB gene. The 16S rRNA gene sequences of all of the strains were determined in parallel. A good congruence was obtained between 16S rRNA and rpoB gene sequence-based trees within the genus Campylobacter. The branching of the rpoB tree was similar to that of the 16S rRNA gene tree, even though a few discrepancies were observed for certain species. The resolution of the rpoB gene within the genus Campylobacter was generally much higher than that of the 16S rRNA gene sequence, resulting in a clear separation of most species and even some subspecies. The universally applicable amplification and sequencing approach for partial rpoB gene sequence determination provides a powerful tool for DNA sequence-based discrimination of Campylobacter species.

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Takeshi, Kouichi, Souichi Makino, Tetsuya Ikeda, Noriko Takada, Atsushi Nakashiro, Kazunori Nakanishi, Keiji Oguma, Yoshinobu Katoh, Hiroyuki Sunagawa, and Tohru Ohyama. "Direct and Rapid Detection by PCR ofErysipelothrix sp. DNAs Prepared from Bacterial Strains and Animal Tissues." Journal of Clinical Microbiology 37, no. 12 (1999): 4093–98. http://dx.doi.org/10.1128/jcm.37.12.4093-4098.1999.

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A PCR method for rapid screening of Erysipelothrix spp. in the slaughterhouse was carried out by using four species-specific sets of oligonucleotide primers after initial amplification with the primer set MO101-MO102, which amplifies the 16S rRNA sequences of all four Erysipelothrix species. The DNA sequences coding for the rRNA gene cluster, including 16S rRNA, 23S rRNA, and the noncoding region downstream of 5S rRNA, were determined in order to design primers for the species-specific PCR detection system. The homology among the 4.5-kb DNA sequences of the rRNA genes ofErysipelothrix rhusiopathiae serovar 2 (DNA Data Bank of Japan accession no. AB019247), E. tonsillarum serovar 7 (accession no. AB019248), E. rhusiopathiae serovar 13 (accession no. AB019249), and E. rhusiopathiae serovar 18 (accession no. AB019250) ranged from 96.0 to 98.4%. The PCR amplifications were specific and were able to distinguish the DNAs from each of the four Erysipelothrix species. The results of PCR tests performed directly with tissue specimens from diseased animals were compared with the results of cultivation tests, and the PCR tests were completed within 5 h. The test with this species-specific system based on PCR amplification with the DNA sequences coding for the rRNA gene cluster was an accurate, easy-to-read screening method for rapid diagnosis of Erysipelothrix sp. infection in the slaughterhouse.

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Jensen, J. S., M. B. Borre, and B. Dohn. "Detection of Mycoplasma genitalium by PCR Amplification of the 16S rRNA Gene." Journal of Clinical Microbiology 41, no. 1 (January 1, 2003): 261–66. http://dx.doi.org/10.1128/jcm.41.1.261-266.2003.

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Gaydos, C. A., T. C. Quinn, and J. J. Eiden. "Identification of Chlamydia pneumoniae by DNA amplification of the 16S rRNA gene." Journal of Clinical Microbiology 30, no. 4 (1992): 796–800. http://dx.doi.org/10.1128/jcm.30.4.796-800.1992.

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Teixeira, Diva do Carmo, Colette Saillard, Sandrine Eveillard, Jean Luc Danet, Paulo Inácio da Costa, Antonio Juliano Ayres, and Joseph Bové. "‘Candidatus Liberibacter americanus’, associated with citrus huanglongbing (greening disease) in São Paulo State, Brazil." International Journal of Systematic and Evolutionary Microbiology 55, no. 5 (September 1, 2005): 1857–62. http://dx.doi.org/10.1099/ijs.0.63677-0.

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Symptoms of huanglongbing (HLB) were reported in São Paulo State (SPS), Brazil, in March 2004. In Asia, HLB is caused by ‘Candidatus Liberibacter asiaticus' and in Africa by ‘Candidatus Liberibacter africanus’. Detection of the liberibacters is based on PCR amplification of their 16S rRNA gene with specific primers. Leaves with blotchy mottle symptoms characteristic of HLB were sampled in several farms of SPS and tested for the presence of liberibacters. ‘Ca. L. asiaticus' was detected in a small number of samples but most samples gave negative PCR results. Therefore, a new HLB pathogen was suspected. Evidence for an SPS-HLB bacterium in symptomatic leaves was obtained by PCR amplification with universal primers for prokaryotic 16S rRNA gene sequences. The amplified 16S rRNA gene was cloned and sequenced. Sequence analysis and phylogeny studies showed that the 16S rRNA gene possessed the oligonucleotide signatures and the secondary loop structure characteristic of the α-Proteobacteria, including the liberibacters. The 16S rRNA gene sequence phylogenetic tree showed that the SPS-HLB bacterium clustered within the α-Proteobacteria, the liberibacters being its closest relatives. For these reasons, the SPS-HLB bacterium is considered a member of the genus ‘Ca. Liberibacter’. However, while the 16S rRNA gene sequences of ‘Ca. L. asiaticus' and ‘Ca. L. africanus' had 98·4 % similarity, the 16S rRNA gene sequence of the SPS-HLB liberibacter had only 96·0 % similarity with the 16S rRNA gene sequences of ‘Ca. L. asiaticus' or ‘Ca. L. africanus’. This lower similarity was reflected in the phylogenetic tree, where the SPS-HLB liberibacter did not cluster within the ‘Ca. L asiaticus’/‘Ca. L. africanus group’, but as a separate branch. Within the genus ‘Candidatus Liberibacter’ and for a given species, the 16S/23S intergenic region does not vary greatly. The intergenic regions of three strains of ‘Ca. L. asiaticus’, from India, the People's Republic of China and Japan, were found to have identical or almost identical sequences. In contrast, the intergenic regions of the SPS-HLB liberibacter, ‘Ca. L. asiaticus' and ‘Ca. L. africanus' had quite different sequences, with similarity between 66·0 and 79·5 %. These results confirm that the SPS-HLB liberibacter is a novel species for which the name ‘Candidatus Liberibacter americanus' is proposed. Like the African and the Asian liberibacters, the ‘American’ liberibacter is restricted to the sieve tubes of the citrus host. The liberibacter could also be detected by PCR amplification of the 16S rRNA gene in Diaphorina citri, the psyllid vector of ‘Ca. L. asiaticus’, suggesting that this psyllid is also a vector of ‘Ca. L. americanus' in SPS. ‘Ca. L. americanus' was detected in 216 of 218 symptomatic leaf samples from 47 farms in 35 municipalities, while ‘Ca. L. asiaticus' was detected in only 4 of the 218 samples, indicating that ‘Ca. L. americanus' is the major cause of HLB in SPS.

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Webster, Gordon, R. John Parkes, John C. Fry, and Andrew J. Weightman. "Widespread Occurrence of a Novel Division of Bacteria Identified by 16S rRNA Gene Sequences Originally Found in Deep Marine Sediments." Applied and Environmental Microbiology 70, no. 9 (September 2004): 5708–13. http://dx.doi.org/10.1128/aem.70.9.5708-5713.2004.

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ABSTRACT Phylogenetic analysis of 16S rRNA gene sequences from deep marine sediments identified a deeply branching clade, designated candidate division JS1. Primers for PCR amplification of partial 16S rRNA genes that target the JS1 division were developed and used to detect JS1 sequences in DNA extracted from various sedimentary environments, including, for the first time, coastal marine and brackish sediments.

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Alfaruqi, Hamiyawati Qoimatu Dini, Nosa Septiana Anindita, and Arif Bimantara. "KAJIAN MOLEKULER PADA PROBIOTIK ASAL AIR SUSU IBU DALAM SINTESIS EKSOPOLISAKARIDA (EPS)." Jurnal Bioteknologi & Biosains Indonesia (JBBI) 8, no. 1 (June 22, 2021): 114–23. http://dx.doi.org/10.29122/jbbi.v8i1.4554.

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Molecular Studies on Probiotic of Human Breast Milk in the Synthesis of Exopolysaccharide (EPS) The glucosyltransferase (gtf) gene has an important role in exopolysaccharide (EPS) synthesis in probiotic bacteria. The EPS produced is associated with the adhesion ability of bacteria to the intestinal mucosa. Therefore, the gtf gene can be used as a parameter in the selection of potential probiotic through a molecular approach. This study was conducted to determine the presence of the gtf gene in probiotic from human breast milk using PCR technique. The methods in this study include the following: reculture of probiotic isolates, DNA isolation, amplification of the 16S rRNA gene using universal primers (pA and pB), amplification of specific LAB primers (LABfw and LABrv), specific primary design for the gtf gene, and the amplification of the gtf gene. The results of 16S rRNA gene amplification using universal primers obtained the amplicons of 500-1,000 bp in size. The results of amplification using specific LAB primers obtained an amplicon of about 700 bp in all isolates. The results of amplification of the gtf gene using a specific primer produced an amplicon of 325 bp in all isolates. Based on this study, it was concluded that 16 probiotic isolates from human breast milk were proven to have the gtf gene. Gen glukosiltransferase (gtf) memiliki peran penting dalam sintesis eksopolisakarida (EPS) pada bakteri probiotik. EPS yang diproduksi berhubungan dengan kemampuan adhesi bakteri pada mukosa usus. Oleh karena itu, gen gtf dapat dijadikan sebagai salah satu parameter dalam seleksi probiotik potensial melalui pendekatan molekuler. Penelitian ini dilakukan untuk mengetahui adanya gen gtf pada probiotik asal air susu ibu (ASI) menggunakan teknik PCR. Metode pada penelitian ini meliputi: reculture isolat probiotik, isolasi DNA, amplifikasi gen 16S rRNA menggunakan primer universal (pA dan pB), amplifikasi primer spesifik BAL (LABfw dan LABrv), desain primer spesifik untuk gen gtf dan amplifikasi gen gtf. Hasil amplifikasi gen 16S rRNA menggunakan primer universal diperoleh amplikon berukuran antara 500-1.000 bp. Adapun hasil amplifikasi menggunakan primer spesifik BAL diperoleh amplikon berukuran sekitar 700 bp pada seluruh isolat. Hasil amplifikasi gen gtf menggunakan primer spesifik menghasilkan amplikon berukuran sekitar 325 bp pada seluruh isolat. Berdasarkan penelitian ini dapat disimpulkan bahwa 16 isolat probiotik asal ASI terbukti memiliki gen gtf.

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Mancabelli, Leonardo, Christian Milani, Gabriele Andrea Lugli, Federico Fontana, Francesca Turroni, Douwe van Sinderen, and Marco Ventura. "The Impact of Primer Design on Amplicon-Based Metagenomic Profiling Accuracy: Detailed Insights into Bifidobacterial Community Structure." Microorganisms 8, no. 1 (January 17, 2020): 131. http://dx.doi.org/10.3390/microorganisms8010131.

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Next Generation Sequencing (NGS) technologies have overcome the limitations of cultivation-dependent approaches and allowed detailed study of bacterial populations that inhabit the human body. The consortium of bacteria residing in the human intestinal tract, also known as the gut microbiota, impacts several physiological processes important for preservation of the health status of the host. The most widespread microbiota profiling method is based on amplification and sequencing of a variable portion of the 16S rRNA gene as a universal taxonomic marker among members of the Bacteria domain. Despite its popularity and obvious advantages, this 16S rRNA gene-based approach comes with some important limitations. In particular, the choice of the primer pair for amplification plays a major role in defining the accuracy of the reconstructed bacterial profiles. In the current study, we performed an in silico PCR using all currently described 16S rRNA gene-targeting primer pairs (PP) in order to assess their efficiency. Our results show that V3, V4, V5, and V6 were the optimal regions on which to design 16S rRNA metagenomic primers. In detail, PP39 (Probio_Uni/Probio_Rev), PP41 (341F/534R), and PP72 (970F/1050R) were the most suitable primer pairs with an amplification efficiency of >98.5%. Furthermore, the Bifidobacterium genus was examined as a test case for accurate evaluation of intra-genus performances at subspecies level. Intriguingly, the in silico analysis revealed that primer pair PP55 (527f/1406r) was unable to amplify the targeted region of any member of this bacterial genus, while several other primer pairs seem to rather inefficiently amplify the target region of the main bifidobacterial taxa. These results highlight that selection of a 16S rRNA gene-based PP should be done with utmost care in order to avoid biases in microbiota profiling results.

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Puspitasari, Dhewanti, Hendro Pramono, and Oedjijono Oedjijono. "IDENTIFIKASI BAKTERI PENGOKSIDASI BESI DAN SULFUR BERDASARKAN GEN 16S rRNA DARI LAHAN TAMBANG TIMAH DI BELITUNG." Scripta Biologica 1, no. 1 (March 25, 2014): 10. http://dx.doi.org/10.20884/1.sb.2014.1.1.12.

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Heavy metals contamination disturb balance and diversity of microorganism in soil. Microorganisms which can able to survive in those conditions are bacteria capable of oxidizing heavy metals. Identification based on 16S rRNA was used to determine characteristics and phylogenetic relationship of bacteria which can oxidize iron and sulphur in tin mining areas. The aim of this research was able to determine the bacterias characteristics isolated from tin mining areas and determine the phylogenetic relation of iron-sulphur oxidizing bacteria on tin mining soil in Belitung based on 16S rRNA sequences. This research was done using descriptive method, including isolation, morphological characterization, and identification based on 16S rRNA sequences. Morphology characterization includes colony and cell morphology through Gram staining. Molecular characterization includes amplification of 16S rRNA gene (Polymerase Chain Reaction/ PCR), electrophoresis amplicon and sequencing. Bacteria identification was done by comparing the 16S rRNA gene sequence in GenBank. The result showed three bacterias were identified by 16S rRNA have a similarity with Bacillus anthracis strain Ames, Bacillus cereus ATCC 14579, Staphylococcus sciuri subsp. Sciuri strains DSM 20345 and Micrococcus luteus NCTC 2665.

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Nasare, Kanchan, Amit Yadav, Anil K. Singh, K. B. Shivasharanappa, Y. S. Nerkar, and V. S. Reddy. "Molecular and Symptom Analysis Reveal the Presence of New Phytoplasmas Associated with Sugarcane Grassy Shoot Disease in India." Plant Disease 91, no. 11 (November 2007): 1413–18. http://dx.doi.org/10.1094/pdis-91-11-1413.

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A total of 240 sugarcane (Saccharum officinarum) plants showing phenotypic symptoms of sugarcane grassy shoot (SCGS) disease were collected from three states of India, Maharashtra, Karnataka, and Uttar Pradesh. Phytoplasmas were detected in all symptomatic samples by the polymerase chain reaction (PCR) amplification of phytoplasma-specific 16S rRNA gene and 16S-23S rRNA spacer region (SR) sequences. No amplification was observed when DNA from asymptomatic plant samples was used as a template. Sixteen samples were selected on the basis of phenotypic symptoms and geographic location, and cloning and sequencing of the 16S rRNA and spacer regions were performed. Multiple sequence alignments of the 16S rRNA sequences revealed that they share very high sequence similarity with phytoplasmas of rice yellow dwarf, 16SrXI. However, the 16S-23S rRNA SR sequence analysis revealed that while the majority of phytoplasmas shared very high (>99%) sequence similarity with previously reported sugarcane phytoplasmas, two of them, namely BV2 (DQ380342) and VD7 (DQ380343), shared relatively low sequence similarity (79 and 84%, respectively). Therefore, these two phytoplasmas may be previously unreported ones that cause significant yield losses in sugarcane in India.

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Hakovirta, Janetta R., Samantha Prezioso, David Hodge, Segaran P. Pillai, and Linda M. Weigel. "Identification and Analysis of Informative Single Nucleotide Polymorphisms in 16S rRNA Gene Sequences of the Bacillus cereus Group." Journal of Clinical Microbiology 54, no. 11 (August 31, 2016): 2749–56. http://dx.doi.org/10.1128/jcm.01267-16.

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Analysis of 16S rRNA genes is important for phylogenetic classification of known and novel bacterial genera and species and for detection of uncultivable bacteria. PCR amplification of 16S rRNA genes with universal primers produces a mixture of amplicons from all rRNA operons in the genome, and the sequence data generally yield a consensus sequence. Here we describe valuable data that are missing from consensus sequences, variable effects on sequence data generated from nonidentical 16S rRNA amplicons, and the appearance of data displayed by different software programs. These effects are illustrated by analysis of 16S rRNA genes from 50 strains of theBacillus cereusgroup, i.e.,Bacillus anthracis,Bacillus cereus,Bacillus mycoides, andBacillus thuringiensis. These species have 11 to 14 rRNA operons, and sequence variability occurs among the multiple 16S rRNA genes. A single nucleotide polymorphism (SNP) previously reported to be specific toB. anthraciswas detected in someB. cereusstrains. However, a different SNP, at position 1139, was identified as being specific toB. anthracis, which is a biothreat agent with high mortality rates. Compared with visual analysis of the electropherograms, basecaller software frequently missed gene sequence variations or could not identify variant bases due to overlapping basecalls. Accurate detection of 16S rRNA gene sequences that include intragenomic variations can improve discrimination among closely related species, improve the utility of 16S rRNA databases, and facilitate rapid bacterial identification by targeted DNA sequence analysis or by whole-genome sequencing performed by clinical or reference laboratories.

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Morales, Sergio E., and William E. Holben. "Empirical Testing of 16S rRNA Gene PCR Primer Pairs Reveals Variance in Target Specificity and Efficacy Not Suggested by In Silico Analysis." Applied and Environmental Microbiology 75, no. 9 (February 27, 2009): 2677–83. http://dx.doi.org/10.1128/aem.02166-08.

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ABSTRACT Phylogenetic and “fingerprinting” analyses of the 16S rRNA genes of prokaryotes have been a mainstay of microbial ecology during the last two decades. However, many methods and results from studies that rely on the 16S rRNA gene for detection and quantification of specific microbial taxa have seemingly received only cursory or even no validation. To directly examine the efficacy and specificity of 16S rRNA gene-based primers for phylum-, class-, and operational taxonomic unit-specific target amplification in quantitative PCR, we created a collection of primers based solely on an extensive soil bacterial 16S rRNA gene clone library containing ∼5,000 sequences from a single soil sample (i.e., a closed site-specific library was used to create PCR primers for use at this site). These primers were initially tested in silico prior to empirical testing by PCR amplification of known target sequences and of controls based on disparate phylogenetic groups. Although all primers were highly specific according to the in silico analysis, the empirical analyses clearly exhibited a high degree of nonspecificity for many of the phyla or classes, while other primers proved to be highly specific. These findings suggest that significant care must be taken when interpreting studies whose results were obtained with target specific primers that were not adequately validated, especially where population densities or dynamics have been inferred from the data. Further, we suggest that the reliability of quantification of specific target abundance using 16S rRNA-based quantitative PCR is case specific and must be determined through rigorous empirical testing rather than solely in silico.

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Angelo, Rwarinda U., and Samiraj Ramesh Ramesh. "ANTIBIOGRAM AND MOLECULAR CHARACTERIZATION OF VIBRIO CHOLERAE ISOLATED FROM MARINE FISH." Asian Journal of Pharmaceutical and Clinical Research 10, no. 6 (June 1, 2017): 146. http://dx.doi.org/10.22159/ajpcr.2017.v10i6.17883.

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Objective: Molecular identification and antibiotic susceptibility evaluation of Vibrio cholerae from marine fish available in local fish market Thanjavur, Tamil nadu, India.Methods: inoculation was done by using nutrient agar as general media and TCBS agar as selective media and confirmed as V.cholerae by Gram stain (Microscopic Observation), Growth characteristics of different media, Biochemical tests like Methyl Red Test, Nitrate Reduction Tests, Indole Test etc. Sensitivity (drug sensitivity) was done in Muller Hinton Agar (MH Agar) using disc diffusion method ten different antibiotics were used to evaluate the antibiogram profile, molecular detection was done by targeting 16S rRNA gene by using a universal primer.Results: V.cholerae is present in marine fish samples, as showed by culture method and microscopic observation as well biochemical tests. PCR amplification of 16S rRNA gene showed the amplification of targeted gene and antibiogram profile showed that isolates are more sensitive to Ampicillin in comparison with others antibiotics used in this study. Ampicillin can be used for V.cholerae infection by the physicians and amoxicillin must be avoided which is resistant.Conclusion: Molecular detection is safe and rapid methods for bacteria identification as revealed by PCR amplification of 16S rRNA gene. As the isolates are more sensitive to Ampicillin in comparison with others antibiotics used in this study. Ampicillin can be used for V.cholerae infection by the physicians and amoxicillin and nitrofurantoin must be avoided.

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Li, Yong Feng, Yi Xuan Wang, Lu Wang, and Zhan Qing Wang. "Sequence Length Variation of Internal Genic Space of 16S rDNA-23S rDNA in Bacterium with High Yield of Hydrogen Production." Advanced Materials Research 183-185 (January 2011): 1413–16. http://dx.doi.org/10.4028/www.scientific.net/amr.183-185.1413.

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To develop the identification of species for fermentative biohydrogen-producing bacterium, scholars have found a method which is based on PCR amplification of the 16S rRNA gene (rDNA)-23S rDNA intergenic regions. In the study, a large fragment of the rDNA operon, including the 16S rDNA, the intergenic spacer region (ISR) and approximately 2000 bases of the 23S rDNA, were polymerasechain reaction (PCR) amplified. The PCR amplification of the genomic DNA of Leptonema ilk strain 3055 using primers directed against conserved regions of the rRNA operon provided evidence that the 16S and 23S rRNA genes were linked via an intergenic spacer region. The sequencing of the intergenic spacer region indicated that it was 435 nucleotides in length and sequence similarity searches revealed that it bore no homology to any known sequences including tRNA available in databases.

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La Scola, Bernard, and Didier Raoult. "Third Human Isolate of a Desulfovibriosp. Identical to the Provisionally Named Desulfovibrio fairfieldensis." Journal of Clinical Microbiology 37, no. 9 (1999): 3076–77. http://dx.doi.org/10.1128/jcm.37.9.3076-3077.1999.

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Valiunas, Deividas, Rasa Jomantiene, and Robert Edward Davis. "Evaluation of the DNA-dependent RNA polymerase β-subunit gene (rpoB) for phytoplasma classification and phylogeny." International Journal of Systematic and Evolutionary Microbiology 63, Pt_10 (October 1, 2013): 3904–14. http://dx.doi.org/10.1099/ijs.0.051912-0.

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Phytoplasmas are classified into 16Sr groups and subgroups and ‘Candidatus Phytoplasma ’ species, largely or entirely based on analysis of 16S rRNA gene sequences. Yet, distinctions among closely related ‘Ca. Phytoplasma ’ species and strains based on 16S rRNA genes alone have limitations imposed by the high degree of rRNA nucleotide sequence conservation across diverse phytoplasma lineages and by the presence in a phytoplasma genome of two, sometimes sequence-heterogeneous, copies of the 16S rRNA gene. Since the DNA-dependent RNA polymerase (DpRp) β-subunit gene (rpoB) exists as a single copy in the phytoplasma genome, we explored the use of rpoB for phytoplasma classification and phylogenetic analysis. We sequenced a clover phyllody (CPh) phytoplasma genetic locus containing ribosomal protein genes, a complete rpoB gene and a partial rpoC gene encoding the β′-subunit of DpRp. Primers and reaction conditions were designed for PCR-mediated amplification of rpoB gene fragments from diverse phytoplasmas. The rpoB gene sequences from phytoplasmas classified in groups 16SrI, 16SrII, 16SrIII, 16SrX and 16SrXII were subjected to sequence similarity and phylogenetic analyses. The rpoB gene sequences were more variable than 16S rRNA gene sequences, more clearly distinguishing among phytoplasma lineages. Phylogenetic trees based on 16S rRNA and rpoB gene sequences had similar topologies, and branch lengths in the rpoB tree facilitated distinctions among closely related phytoplasmas. Virtual RFLP analysis of rpoB gene sequences also improved distinctions among closely related lineages. The results indicate that the rpoB gene provides a useful additional marker for phytoplasma classification that should facilitate studies of disease aetiology and epidemiology.

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Shukla, Sanjay K., and Kurt D. Reed. "Desulfovibrio desulfuricans Bacteremia in a Dog." Journal of Clinical Microbiology 38, no. 4 (2000): 1701–2. http://dx.doi.org/10.1128/jcm.38.4.1701-1702.2000.

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Callahan, Benjamin J., Joan Wong, Cheryl Heiner, Steve Oh, Casey M. Theriot, Ajay S. Gulati, Sarah K. McGill, and Michael K. Dougherty. "High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution." Nucleic Acids Research 47, no. 18 (July 3, 2019): e103-e103. http://dx.doi.org/10.1093/nar/gkz569.

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AbstractTargeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

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SHU, QINGLONG, and NIANZHI JIAO. "NEW PRIMERS FOR AMPLIFICATION OF THE PLANCTOMYCETES 16S rRNA GENE FROM ENVIRONMENTAL SAMPLES." Journal of Rapid Methods & Automation in Microbiology 16, no. 4 (December 2008): 330–36. http://dx.doi.org/10.1111/j.1745-4581.2008.00139.x.

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Giongo, Adriana, Adriana Ambrosini, João Ruy Jardim Freire, Maria Helena Bodanese Zanettini, and Luciane Maria Pereira Passaglia. "Amplification of 16S rRNA gene sequences to differentiate two highly related bradyrhizobia species." Pesquisa Agropecuária Brasileira 42, no. 9 (September 2007): 1361–64. http://dx.doi.org/10.1590/s0100-204x2007000900019.

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A 16S rRNA gene PCR-based assay was developed aiming at a fast molecular diagnostic method to differentiate the two phylogenetically closely related species Bradyrhizobium japonicum and B. elkanii, isolated from soybean nodules, in order to identify those more competitive and comprising greater nitrogen fixation ability for use in the formulation of commercial inoculants. The assay used was able to discriminate ten reference strains belonging to these two Bradyrhizobium species, as well as to efficiently identify 37 strains isolated from fields cultivated with soybean.

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Chalupova, Miroslava, Anna Skalova, Tomas Hajek, Lenka Geigerova, Dana Kralova, Pavel Liska, Hana Hecova, Jiri Molacek, and Jaroslav Hrabak. "Bacterial DNA detected on pathologically changed heart valves using 16S rRNA gene amplification." Folia Microbiologica 63, no. 6 (May 22, 2018): 707–11. http://dx.doi.org/10.1007/s12223-018-0611-6.

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Parker, Alex, and Irv Kornfield. "An improved amplification and sequencing strategy for phylogenetic studies using the mitochondrial large subunit rRNA gene." Genome 39, no. 4 (August 1, 1996): 793–97. http://dx.doi.org/10.1139/g96-099.

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Numerous molecular systematic studies have employed variation in the mitochondrial large subunit (16s) rRNA gene to infer patterns of relationship among species and higher taxa. The primers most commonly employed in 16s rRNA amplification and sequencing bracket an approximately 600 bp portion of this gene. However, most of the informative variation occurs within a 200 bp subset of this segment. We describe a novel primer pair designed to amplify this variable region in a wide range of taxa, allowing broader application and considerable streamlining of data acquisition for studies using this gene. Key words : molecular phylogenetics, polymerase chain reaction, mtDNA, large ribosomal subunit, control region.

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Raghunathan, Arumugham, Harley R. Ferguson, Carole J. Bornarth, Wanmin Song, Mark Driscoll, and Roger S. Lasken. "Genomic DNA Amplification from a Single Bacterium." Applied and Environmental Microbiology 71, no. 6 (June 2005): 3342–47. http://dx.doi.org/10.1128/aem.71.6.3342-3347.2005.

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ABSTRACT Genomic DNA was amplified about 5 billion-fold from single, flow-sorted bacterial cells by the multiple displacement amplification (MDA) reaction, using φ 29 DNA polymerase. A 662-bp segment of the 16S rRNA gene could be accurately sequenced from the amplified DNA. MDA methods enable new strategies for studying nonculturable microorganisms.

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Mohamad, Rohaslinda, Mohd Rafatullah, Tengku Yusof, Yi Sim, Norli Ismail, and Japareng Lalung. "Detection of Microcystin (Mcye) Gene in Recreational Lakes in Miri, Sarawak, Malaysia." Current World Environment 11, no. 3 (December 25, 2016): 690–99. http://dx.doi.org/10.12944/cwe.11.3.02.

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Toxic cyanobacteria blooms became a worldwide problems as many countries encounter the presence of the blooms in most of water bodies. As part to develop monitoring of cyanobacterial toxins in Malaysia, samples taken in twelve points in five different lakes in Miri, Sarawak. Polymerase chain reaction (PCR) amplification of cyanobacterial 16S rRNA were carried out to detect the presence of cyanobacteria in the water samples. Cyanobacterial 16S rRNA were detected in all the samples collected. While molecular analysis for detection of cyanobacterial toxin encoding gene were done using specific primers. PCR amplification of cyanobacterial toxin-encoding gene were carried using the combination of forward primer; mcyE-F2 and reverse primer; mcyE-R4 to amplify generic microcystin (mcyE) gene in the samples. Out of twelve samples collected, microcystin (mcyE) producing gene was detected in one of the samples tested. Presence of microcystin encoding gene indicates the risk of cyanobacterial toxins in Miri, Sarawak.

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Nübel, Ulrich, Ferran Garcia-Pichel, Michael Kühl, and Gerard Muyzer. "Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats." Applied and Environmental Microbiology 65, no. 2 (February 1, 1999): 422–30. http://dx.doi.org/10.1128/aem.65.2.422-430.1999.

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ABSTRACT We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied.

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Bertilsson, Stefan, Colleen M. Cavanaugh, and Martin F. Polz. "Sequencing-Independent Method To Generate Oligonucleotide Probes Targeting a Variable Region in Bacterial 16S rRNA by PCR with Detachable Primers." Applied and Environmental Microbiology 68, no. 12 (December 2002): 6077–86. http://dx.doi.org/10.1128/aem.68.12.6077-6086.2002.

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ABSTRACT Oligonucleotide probes targeting the small-subunit rRNA are commonly used to detect and quantify bacteria in natural environments. We developed a PCR-based approach that allows synthesis of oligonucleotide probes targeting a variable region in the 16S rRNA without prior knowledge of the target sequence. Analysis of all 16S rRNA gene sequences in the Ribosomal Database Project database revealed two universal primer regions bracketing a variable, population-specific region. The probe synthesis is based on a two-step PCR amplification of this variable region in the 16S rRNA gene by using three universal bacterial primers. First, a double-stranded product is generated, which then serves as template in a linear amplification. After each of these steps, products are bound to magnetic beads and the primers are detached through hydrolysis of a ribonucleotide at the 3′ end of the primers. This ultimately produces a single-stranded oligonucleotide of about 30 bases corresponding to the target. As probes, the oligonucleotides are highly specific and could discriminate between nucleic acids from closely and distantly related bacterial strains, including different species of Vibrio. The method will facilitate rapid generation of oligonucleotide probes for large-scale hybridization assays such as screening of clone libraries or strain collections, ribotyping microarrays, and in situ hybridization. An additional advantage of the method is that fluorescently or radioactively labeled nucleotides can be incorporated during the second amplification, yielding intensely labeled probes.

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Martini, Marta, Carmine Marcone, Jelena Mitrović, Michael Maixner, Duška Delić, Arben Myrta, Paolo Ermacora, Assunta Bertaccini, and Bojan Duduk. "‘Candidatus Phytoplasma convolvuli’, a new phytoplasma taxon associated with bindweed yellows in four European countries." International Journal of Systematic and Evolutionary Microbiology 62, Pt_12 (December 1, 2012): 2910–15. http://dx.doi.org/10.1099/ijs.0.038612-0.

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Plants of Convolvulus arvensis exhibiting symptoms of undersized leaves, shoot proliferation and yellowing, collectively defined as bindweed yellows, were sampled in different regions of Europe and assessed for phytoplasma infection by PCR amplification using phytoplasma universal rRNA operon primer pairs. Positive results were obtained for all diseased plants. RFLP analysis of amplicons comprising the16S rRNA gene alone or the16S rRNA gene and 16-23S intergenic spacer region indicated that the detected phytoplasmas were distinguishable from all other previously described rRNA gene sequences. Analysis of 16S rRNA gene sequences derived from seven selected phytoplasma strains (BY-S57/11, BY-S62/11, BY-I1015, BY-I1016, BY-BH1, BY-BH2 and BY-G) showed that they were nearly identical (99.9–100 % gene sequence similarity) but shared less than 97.5 % similarity with comparable sequences of other phytoplasmas. Thus, BY phytoplasmas represent a new taxon whose closest relatives are stolbur phytoplasma strains and ‘ Candidatus Phytoplasma fragariae ’ with which they share 97.2 % and 97.1 % 16S rRNA gene sequence similarity, respectively. Phylogenetic analysis of 16S rRNA gene sequences confirmed that bindweed yellows phytoplasma strains collectively represent a distinct lineage within the phytoplasma clade and share a common ancestor with previously published or proposed ‘Candidatus Phytoplasma’ taxa within a major branch including aster yellows and stolbur phytoplasmas. On the basis of unique 16S rRNA gene sequences and biological properties that include a single host plant species and a geographical distribution limited to parts of Europe, the bindweed yellows (BY) phytoplasmas represent a coherent but discrete taxon, ‘Candidatus Phytoplasma convolvuli’, with strain BY-S57/11 (GenBank accession no. JN833705) as the reference strain.

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Reigel, Alicia M., Sarah M. Owens, and Michael E. Hellberg. "Reducing host DNA contamination in 16S rRNA gene surveys of anthozoan microbiomes using PNA clamps." Coral Reefs 39, no. 6 (October 6, 2020): 1817–27. http://dx.doi.org/10.1007/s00338-020-02006-5.

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AbstractEfforts to study the microbial communities associated with corals can be limited by inefficiencies in the sequencing process due to high levels of host amplification by universal bacterial 16S rRNA gene primers. Here, we develop an inexpensive peptide nucleic acid (PNA) clamp that binds to a target sequence of host DNA during PCR and blocks amplification. We then test the ability of this PNA clamp to mitigate host contamination and increase overall microbial sequence coverage on samples from three coral species: the gorgonians Eunicea flexuosa and Gorgonia ventalina, and the scleractinian Porites panamensis. The 20-bp PNA clamp was designed using DNA from E. flexuosa. Adding the PNA clamp during PCR increased the percentage of microbial reads in E. flexuosa samples more than 11-fold. Microbial community diversity was similar without- and with-PNA clamps, as were the relative frequencies of the ten most abundant ASVs (amplicon sequence variants), indicating that the clamps successfully blocked host DNA amplification while simultaneously increasing microbial DNA amplification proportionally across the most abundant taxa. The reduction of E. flexuosa DNA correlated with an increase in the abundance of rarer taxa. The clamp also increased the average percentage of microbial reads in another gorgonian, G. ventalina, by 8.6-fold without altering the microbial community beta diversity, and in a distantly related scleractinian coral, P. panamensis, by nearly double. The reduction of host contamination correlated with the number of nucleotide mismatches between the host amplicon and the PNA clamp. The PNA clamp costs as little as $0.48 per sample, making it an efficient and cost-effective solution to increase microbial sequence coverage for high-throughput sequencing of coral microbial communities.

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Nehmï¿½, Benjamin, Yan Gilbert, Valï¿½rie Lï¿½tourneau, Robert J. Forster, Marc Veillette, Richard Villemur, and Caroline Duchaine. "Culture-Independent Characterization of Archaeal Biodiversity in Swine Confinement Building Bioaerosols." Applied and Environmental Microbiology 75, no. 17 (June 26, 2009): 5445–50. http://dx.doi.org/10.1128/aem.00726-09.

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ABSTRACT It was previously demonstrated that microbial communities of pig manure were composed of both bacteria and archaea. Recent studies have shown that bacteria are aerosolized from pig manure, but none have ever focused on the airborne archaeal burden. We sought here to develop and apply molecular ecology approaches to thoroughly characterize airborne archaea from swine confinement buildings (SCBs). Eight swine operations were visited, twice in winter and once during summer. Institute of Occupational Medicine cassettes loaded with 25-mm gelatin filters were used to capture the inhalable microbial biomass. The total genomic DNA was extracted and used as a template for PCR amplification of the archaeal 16S rRNA gene. High concentrations of archaea were found in SCB bioaerosols, being as high as 108 16S rRNA gene copies per cubic meter of air. Construction and sequencing of 16S rRNA gene libraries revealed that all sequences were closely related to methanogenic archaea, such as Methanosphaera stadtmanae (94.7% of the archaeal biodiversity). Archaeal community profiles were compared by 16S rRNA gene denaturing gradient gel electrophoresis. This analysis showed similar fingerprints in each SCB and confirmed the predominance of methanogenic archaea in the bioaerosols. This study sheds new light on the nature of bioaerosols in SCBs and suggests that archaea are also aerosolized from pig manure.

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Loy, Alexander, Claudia Schulz, Sebastian Lücker, Andreas Schöpfer-Wendels, Kilian Stoecker, Christian Baranyi, Angelika Lehner, and Michael Wagner. "16S rRNA Gene-Based Oligonucleotide Microarray for Environmental Monitoring of the Betaproteobacterial Order “Rhodocyclales”." Applied and Environmental Microbiology 71, no. 3 (March 2005): 1373–86. http://dx.doi.org/10.1128/aem.71.3.1373-1386.2005.

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ABSTRACT For simultaneous identification of members of the betaproteobacterial order “Rhodocyclales” in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the “Rhodocyclales.” The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent “Rhodocyclales” diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed “Rhodocyclales”-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of “Rhodocyclales” populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.

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Jahan, Hawa, Maria Akter, Rowshan Ara Begum, and Reza Md Shahjahan. "Identification and comparison of three carp fishes based on mitochondrial 16S rRNA gene." Dhaka University Journal of Biological Sciences 26, no. 2 (July 10, 2017): 167–74. http://dx.doi.org/10.3329/dujbs.v26i2.46396.

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Identification of Labeo rohita, L. bata and L. gonius is sometimes problematic when usual morphological features are lost and it is difficult to differentiate them with traditional morphological features at their diverse developmental stages. PCR-sequencing provides an authentic alternative means of identification of individuals at species level. Three local carp fishes were collected and 16S rRNA genes were sequenced by sanger sequencing method after PCR amplification using universal primers. Obtained sequences were found accurate with blast search result which showed maximum range of similarity with the existing respective gene fragments present in GenBank database. Sequences were compared and multiple sequence alignment has revealed some polymorphic sites which can be used to differentiate these three species from one another. This study may provide valuable understanding to study their population in future. Dhaka Univ. J. Biol. Sci. 26(2): 167-174, 2017 (July)

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Fredricks, David N., and David A. Relman. "Improved Amplification of Microbial DNA from Blood Cultures by Removal of the PCR Inhibitor Sodium Polyanetholesulfonate." Journal of Clinical Microbiology 36, no. 10 (1998): 2810–16. http://dx.doi.org/10.1128/jcm.36.10.2810-2816.1998.

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Molecular methods are increasingly used to identify microbes in clinical samples. A common technical problem with PCR is failed amplification due to the presence of PCR inhibitors. Initial attempts at amplification of the bacterial 16S rRNA gene from inoculated blood culture media failed for this reason. The inhibitor persisted, despite numerous attempts to purify the DNA, and was identified as sodium polyanetholesulfonate (SPS), a common additive to blood culture media. Like DNA, SPS is a high-molecular-weight polyanion that is soluble in water but insoluble in alcohol. Accordingly, SPS tends to copurify with DNA. An extraction method was designed for purification of DNA from blood culture media and removal of SPS. Blood culture media containing human blood and spiked with Escherichia coli was subjected to an organic extraction procedure with benzyl alcohol, and removal of SPS was documented spectrophotometrically. Successful amplification of the extracted E. coli 16S rRNA gene was achieved by adding 5 μl of undiluted processed sample DNA to a 50-μl PCR mixture. When using other purification methods, the inhibitory effect of SPS could be overcome only by dilution of these samples. By our extraction technique, even uninoculated blood culture media were found to contain bacterial DNA when they were subjected to broad-range 16S rRNA gene consensus PCR. We conclude that the blood culture additive SPS is a potent inhibitor of PCR, is resistant to removal by traditional DNA purification methods, but can be removed by a benzyl alcohol extraction protocol that results in improved PCR performance.

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Dunbar, John, Shannon Takala, Susan M. Barns, Jody A. Davis, and Cheryl R. Kuske. "Levels of Bacterial Community Diversity in Four Arid Soils Compared by Cultivation and 16S rRNA Gene Cloning." Applied and Environmental Microbiology 65, no. 4 (April 1, 1999): 1662–69. http://dx.doi.org/10.1128/aem.65.4.1662-1669.1999.

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ABSTRACT Techniques based on amplification of 16S rRNA genes for comparing bacterial communities are now widely used in microbial ecology, but calibration of these techniques with traditional tools, such as cultivation, has been conspicuously absent. In this study, we compared levels of bacterial community diversity in two pinyon rhizosphere soil samples and two between-tree (interspace) soil samples by analyzing 179 cultivated bacterial isolates and 801 16S rRNA genes amplified from extracted soil DNA. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes RsaI and BstUI. The average level of 16S rRNA gene sequence similarity of members of a phylotype was 86.6% based on an analysis of partial sequences. A total of 498 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, while 34 phylotypes occurred among the cultivated isolates. Analysis of sequences from a subset of the phylotypes showed that at least seven bacterial divisions were represented in the clone libraries, whereas the isolates represented only three. The phylotype richness, frequency distribution (evenness), and composition of the four culture collections and the four clone libraries were investigated by using a variety of diversity indices. Although cultivation and 16S rRNA cloning analyses gave contradictory descriptions of the relative phylotype richness for one of the four environments, the two methods identified qualitatively consistent relationships when levels of evenness were compared. The levels of phylotype similarity between communities were uniformly low (15 to 31%). Both methods consistently indicated that one environment was distinct from the other three. Our data illustrate that while 16S rDNA cloning and cultivation generally describe similar relationships between soil microbial communities, significant discrepancies can occur.

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Slany, Michal, Martina Vanerkova, Eva Nemcova, Barbora Zaloudikova, Filip Ruzicka, and Tomas Freiberger. "Differentiation of Staphylococcus spp. by high-resolution melting analysis." Canadian Journal of Microbiology 56, no. 12 (December 2010): 1040–49. http://dx.doi.org/10.1139/w10-091.

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High-resolution melting analysis (HRMA) is a fast (post-PCR) high-throughput method to scan for sequence variations in a target gene. The aim of this study was to test the potential of HRMA to distinguish particular bacterial species of the Staphylococcus genus even when using a broad-range PCR within the 16S rRNA gene where sequence differences are minimal. Genomic DNA samples isolated from 12 reference staphylococcal strains ( Staphylococcus aureus , Staphylococcus capitis , Staphylococcus caprae , Staphylococcus epidermidis , Staphylococcus haemolyticus , Staphylococcus hominis , Staphylococcus intermedius , Staphylococcus saprophyticus , Staphylococcus sciuri , Staphylococcus simulans , Staphylococcus warneri , and Staphylococcus xylosus ) were subjected to a real-time PCR amplification of the 16S rRNA gene in the presence of fluorescent dye EvaGreen™, followed by HRMA. Melting profiles were used as molecular fingerprints for bacterial species differentiation. HRMA of S. saprophyticus and S. xylosus resulted in undistinguishable profiles because of their identical sequences in the analyzed 16S rRNA region. The remaining reference strains were fully differentiated either directly or via high-resolution plots obtained by heteroduplex formation between coamplified PCR products of the tested staphylococcal strain and phylogenetically unrelated strain.

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Martini, M., I. M. Lee, K. D. Bottner, Y. Zhao, S. Botti, A. Bertaccini, N. A. Harrison, et al. "Ribosomal protein gene-based phylogeny for finer differentiation and classification of phytoplasmas." International Journal of Systematic and Evolutionary Microbiology 57, no. 9 (September 1, 2007): 2037–51. http://dx.doi.org/10.1099/ijs.0.65013-0.

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Extensive phylogenetic analyses were performed based on sequences of the 16S rRNA gene and two ribosomal protein (rp) genes, rplV (rpl22) and rpsC (rps3), from 46 phytoplasma strains representing 12 phytoplasma 16Sr groups, 16 other mollicutes and 28 Gram-positive walled bacteria. The phylogenetic tree inferred from rp genes had a similar overall topology to that inferred from the 16S rRNA gene. However, the rp gene-based tree gave a more defined phylogenetic interrelationship among mollicutes and Gram-positive walled bacteria. Both phylogenies indicated that mollicutes formed a monophyletic group. Phytoplasmas clustered with Acholeplasma species and formed one clade paraphyletic with a clade consisting of the remaining mollicutes. The closest relatives of mollicutes were low-G+C-content Gram-positive bacteria. Comparative phylogenetic analyses using the 16S rRNA gene and rp genes were performed to evaluate their efficacy in resolving distinct phytoplasma strains. A phylogenetic tree was constructed based on analysis of rp gene sequences from 87 phytoplasma strains belonging to 12 16Sr phytoplasma groups. The phylogenetic relationships among phytoplasmas were generally in agreement with those obtained on the basis of the 16S rRNA gene in the present and previous works. However, the rp gene-based phylogeny allowed for finer resolution of distinct lineages within the phytoplasma 16Sr groups. RFLP analysis of rp gene sequences permitted finer differentiation of phytoplasma strains in a given 16Sr group. In this study, we also designed several semi-universal and 16Sr group-specific rp gene-based primers that allow for the amplification of 11 16Sr group phytoplasmas.

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Chouari, Rakia, Denis Le Paslier, Catherine Dauga, Patrick Daegelen, Jean Weissenbach, and Abdelghani Sghir. "Novel Major Bacterial Candidate Division within a Municipal Anaerobic Sludge Digester." Applied and Environmental Microbiology 71, no. 4 (April 2005): 2145–53. http://dx.doi.org/10.1128/aem.71.4.2145-2153.2005.

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ABSTRACT In a previous study, we analyzed the molecular diversity of Planctomycetales by PCR amplification and sequencing of 16S rRNA clone libraries generated from a municipal wastewater plant, using planctomycete-specific and universal primer sets (R. Chouari, D. Le Paslier, P. Daegelen, P. Ginestet, J. Weissenbach, and A. Sghir, Appl. Environ. Microbiol. 69:7354-7363, 2003). Only a small fraction (4%) of the 16S rRNA gene sequences of the digester clone library corresponded to the Planctomycetales division. Importantly, 85.9% of the digester clone sequences are grouped into two different clusters named WWE1 (81.4% of the sequences) and WWE2 (4.5%) and are distantly affiliated with unidentified bacterial sequences retrieved from a methanogenic reactor community and from a termite gut, respectively. In phylogenetic analysis using 16S rRNA gene sequence representatives of the main phylogenetic bacterial divisions, the two clusters are monophyletic, branch apart from each other, and are distantly related to Planctomycetales and other bacterial divisions. A novel candidate division is proposed for WWE1, while the WWE2 cluster strongly affiliates with the recently proposed Lentisphearae phylum. We designed and validated a 16S rRNA probe targeting WWE1 16S rRNA sequences by both fluorescent in situ hybridization (FISH) and dot blot hybridization (DBH). Results of FISH analysis show that WWE1 representative microorganisms are rods or filamentous shaped, while DBH shows that WWE1 accounts for 12% of the total bacterial rRNA within the anaerobic digester. The remaining 16S rRNA gene sequences are affiliated with Verrucomicrobia or recently described candidate divisions with no known pure culture representatives, such as OD1, BRC1, or NBL-UPA2, making up less than 3.5% of the clone library, respectively. This inventory expands the known diversity of the latter bacterial division-level lineages.

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Wantania, Letha L., Stenly Wullur, Elvi L. Ginting, Desy M. H. Mantiri, Suzanne L. Undap, Deiske A. Sumilat, and Grevo S. Gerung. "Isolation and amplification of 16S rRNA gen of Associated Microbial isolates in Red Algae Kappaphycus alvarezii from Belang, Southeast Minahasa Regency, North Sulawesi." JURNAL ILMIAH PLATAX 7, no. 1 (January 30, 2019): 220. http://dx.doi.org/10.35800/jip.7.1.2019.22808.

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This study aims to obtain isolates and amplify the associative bacterial 16SrRNA gene in K. alvarezii algae. The K. alvarezii algae was collected from seaweed cultivation area in Belang, Southeast Minahasa, North Sulawesi. Associative bacteria were sampled from K. alvarezii algae, grown in Nutrient Agar and separated based on their morphological characteristics. Each isolates were extracted their DNA genome and.the16S rRNA gene of each isolate was amplified using PCR. Eight associative bacterial from K. alvarezii algae were successfully isolated based on morphological characteristics which were dominated by round shape, smooth edges, convex elevation, and white color of the isolates. The results of genomic DNA extraction from each of these isolates were successfully used as templates to amplify the 16s rRNA gene.Key word: Bacteria, Kappaphycus alvarezii, Taxsonomy, 16S rRNAABSTRAKPenelitian ini bertujuan untuk mendapatkan isolat dan mengamplifikasi gen 16SrRNA bakteri asosiatif pada alga K. alvarezii. Metode penelitian diawali dengan pengambilan sampel alga K. alvarezii di lokasi pembudidayaan rumput laut di desa Belang, Minahasa Tenggara, Sulawesi Utara. Selanjutnya, bakteri asosiatif pada alga K. alvarezii tersebut ditumbuhkan dalam media Nutrient Agar, isolat yang tumbuh kemudian dipisahkan berdasarkan karakteristik morfologinya. dan gen 16S rRNA masing-masing isolat diamplifikasi menggunakn PCR. Delapan isolat bakteri asosiatif pada alga K. alvarezii berhasil diisolasi dengan karakteristik morfologi berbeda yang didominasi dengan bentuk round, tepian smooth, elevasi convex, dan warna putih. Hasil ekstraksi DNA genom dari masing-masing isolat tersebut berhasil digunakan sebagai template untuk mengamplifikasi gen 16s rRNA.Kata Kunci: Bakteri, Kappaphycus alvarezii, Taksonomi, 16S rRNA

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Sumner, John W., Gregory A. Storch, Richard S. Buller, Allison M. Liddell, Steven L. Stockham, Yasuko Rikihisa, Sharon Messenger, and Christopher D. Paddock. "PCR Amplification and Phylogenetic Analysis ofgroESL Operon Sequences from Ehrlichia ewingiiand Ehrlichia muris." Journal of Clinical Microbiology 38, no. 7 (2000): 2746–49. http://dx.doi.org/10.1128/jcm.38.7.2746-2749.2000.

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Broad-range PCR primers were used to amplify part of thegroESL operon of the canine pathogen Ehrlichia ewingii, recently recognized as a human pathogen, and the murine pathogen Ehrlichia muris. Phylogenetic analysis supported the relationships among Ehrlichia species previously determined by comparison of 16S rRNA gene sequences. These sequences provide additional PCR targets for species for which few gene sequences have been determined.

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Slobodkina, Galina B., Nikolai A. Chernyh, Alexander I. Slobodkin, Irina V. Subbotina, Elizaveta A. Bonch-Osmolovskaya, and Alexander V. Lebedinsky. "PCR-Based Identification of Hyperthermophilic Archaea of the Family Thermococcaceae." Applied and Environmental Microbiology 70, no. 9 (September 2004): 5701–3. http://dx.doi.org/10.1128/aem.70.9.5701-5703.2004.

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ABSTRACT A method for rapid detection and identification of hyperthermophilic archaea of the family Thermococcaceae based on PCR amplification of 16S rRNA gene fragments with primers TcPc 173F (5′-TCCCCCATAGGYCTGRGGTACTGGAAGGTC-3′) and TcPc 589R (5′-GCCGTGRGATTTCGCCAGGGACTTACGGGC-3′) was developed and used for identification of new isolates.

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Alraddadi, B., S. Al-Azri, and KR Forward. "Influence of 16S Ribosomal RNA Gene Polymerase Chain Reaction and Sequencing on Antibiotic Management of Bone and Joint Infections." Canadian Journal of Infectious Diseases and Medical Microbiology 24, no. 2 (2013): 85–88. http://dx.doi.org/10.1155/2013/747145.

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INTRODUCTION: Amplification of the 16S ribosomal RNA gene by polymerase chain reaction (PCR) followed by analysis of generated sequences can be an important adjunct to conventional cultures.OBJECTIVE: To determine how the results of this approach influence physicians’ decisions regarding the management of bone and joint infections.METHOD: Clinical and laboratory findings of patients seen at the Queen Elizabeth II Health Sciences Centre (Halifax, Nova Scotia) between December 2005 and September 2009 were reviewed. Patients who had negative cultures but likely or possible bone and joint infections were further evaluated using 16S rRNA PCR. The impact of the 16S rRNA PCR result on antibiotic management was evaluated and it was assessed whether untreated patients with negative 16S rRNA PCR subsequently presented with infections, suggesting a false-negative result.RESULT: A total of 36 patients (mean age 62 years) were reviewed. Thirty-two patients were evaluated by infectious disease consultants; of these, 20 were considered likely to have infections. Seventeen patients were admitted with suspected prosthetic joint infections. Twenty-nine patients received antimicrobial treatment before the sample for the 16S rRNA PCR assay was obtained. Of the 36 patients, 26 (72.2%) were treated appropriately with modifications to their antibiotic regimen in response to the 16S rRNA PCR assay results. Antimicrobials were discontinued for 19 patients based on negative PCR assay and, in seven patients, antibiotics were changed based on a positive result. There were no relapses among patients with negative PCR assay in whom antibiotics were discontinued.CONCLUSION: 16S ribosomal RNA gene PCR and sequencing is a valuable tool in the guidance of antimicrobial therapy for bone and joint infections.

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Junier, Pilar, Ok-Sun Kim, Ora Hadas, Johannes F. Imhoff, and Karl-Paul Witzel. "Evaluation of PCR Primer Selectivity and Phylogenetic Specificity by Using Amplification of 16S rRNA Genes from Betaproteobacterial Ammonia-Oxidizing Bacteria in Environmental Samples." Applied and Environmental Microbiology 74, no. 16 (June 20, 2008): 5231–36. http://dx.doi.org/10.1128/aem.00288-08.

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ABSTRACT The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (βAOB) was evaluated. βAOB represent a group of phylogenetically related organisms for which the 16S rRNA gene approach is especially suitable. We used experimental comparisons of primer performance with water samples, together with an in silico analysis of published sequences and a literature review of clone libraries made with four specific PCR primers for the βAOB 16S rRNA gene. With four aquatic samples, the primers NitA/NitB produced the highest frequency of ammonia-oxidizing-bacterium-like sequences compared to clone libraries with products amplified with the primer combinations βAMOf/βAMOr, βAMOf/Nso1255g, and NitA/Nso1225g. Both the experimental examination of ammonia-oxidizing-bacterium-specific 16S rRNA gene primers and the literature search showed that neither specificity nor sensitivity of primer combinations can be evaluated reliably only by sequence comparison. Apparently, the combination of sequence comparison and experimental data is the best approach to detect possible biases of PCR primers. Although this study focused on βAOB, the results presented here more generally exemplify the importance of primer selection and potential primer bias when analyzing microbial communities in environmental samples.

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Tannock, G. W., A. Tilsala-Timisjarvi, S. Rodtong, J. Ng, K. Munro, and T. Alatossava. "Identification of Lactobacillus Isolates from the Gastrointestinal Tract, Silage, and Yoghurt by 16S-23S rRNA Gene Intergenic Spacer Region Sequence Comparisons." Applied and Environmental Microbiology 65, no. 9 (1999): 4264–67. http://dx.doi.org/10.1128/aem.65.9.4264-4267.1999.

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Lactobacillus isolates were identified by PCR amplification and sequencing of the region between the 16S and 23S rRNA genes (spacer region). The sequences obtained from the isolates were compared to those of reference strains held in GenBank. A similarity of 97.5% or greater was considered to provide identification. To check the reliability of the method, the V2-V3 region of the 16S rRNA gene was amplified and sequenced in the case of isolates whose spacer region sequences were less than 99% similar to that of a reference strain. Confirmation of identity was obtained in all instances. Spacer region sequencing provided rapid and accurate identification ofLactobacillus isolates obtained from gastrointestinal, yoghurt, and silage samples. It had an advantage over 16S V2-V3 sequence comparisons because it distinguished between isolates ofLactobacillus casei and Lactobacillus rhamnosus.

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Mirza, M. S., J. L. W. Rademaker, J. D. Janse, and A. D. L. Akkermans. "Specific 16S ribosomal RNA targeted oligonucleotide probe against Clavibacter michiganensis subsp. sepedonicus." Canadian Journal of Microbiology 39, no. 11 (November 1, 1993): 1029–34. http://dx.doi.org/10.1139/m93-156.

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In this article we report on the polymerase chain reaction amplification of a partial 16S rRNA gene from the plant pathogenic bacterium Clavibacter michiganensis subsp. sepedonicus. A partial sequence (about 400 base pairs) of the gene was determined that covered two variable regions important for oligonucleotide probe development. A specific 24mer oligonucleotide probe targeted against the V6 region of 16S rRNA was designed. Specificity of the probe was determined using dot blot hybridization. Under stringent conditions (60 °C), the probe hybridized with all 16 Cl. michiganensis subsp. sepedonicus strains tested. Hybridization did not occur with 32 plant pathogenic and saprophytic bacteria used as controls under the same conditions. Under less stringent conditions (55 °C) the related Clavibacter michiganensis subsp. insidiosus, Clavibacter michiganensis subsp. nebraskensis, and Clavibacter michiganensis subsp. tesselarius also showed hybridization. At even lower stringency (40 °C), all Cl. michiganensis subspecies tested including Clavibacter michiganensis subsp. michiganensis showed hybridization signal, suggesting that under these conditions the probe may be used as a species-specific probe for Cl. michiganensis.Key words: Clavibacter michiganensis subsp. sepedonicus, PCR, 16S rRNA, oligonucleotide probe.

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Journal articles on the topic '16S rRNA gene amplification'

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