Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes

ABSTRACT For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).

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Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA

Applied and Environmental Microbiology ◽

10.1128/aem.64.2.795-799.1998 ◽

1998 ◽

Vol 64 (2) ◽

pp. 795-799 ◽

Cited By ~ 1058

Author(s):

Julian R. Marchesi ◽

Takuichi Sato ◽

Andrew J. Weightman ◽

Tracey A. Martin ◽

John C. Fry ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Gene Amplification ◽

Environmental Samples ◽

Bacterial Species ◽

Pcr Primers ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Specific Pcr

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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Species-Specific PCR for Identification of Common Contaminant Mollicutes in Cell Culture

Applied and Environmental Microbiology ◽

10.1128/aem.67.7.3195-3200.2001 ◽

2001 ◽

Vol 67 (7) ◽

pp. 3195-3200 ◽

Cited By ~ 46

Author(s):

Fanrong Kong ◽

Gregory James ◽

Susanna Gordon ◽

Anna Zelynski ◽

Gwendolyn L. Gilbert

Keyword(s):

16S Rrna ◽

Cell Cultures ◽

Bacterial Species ◽

Intergenic Spacer ◽

16S Rrna Genes ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Specific Pcr ◽

Species Specific

ABSTRACT Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5′ end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5′ end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.

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A nested PCR-based method for detection ofStreptococcus agalactiae16S rRNA in milk and its application

Chinese Journal of Agricultural Biotechnology ◽

10.1079/cjb200694 ◽

2006 ◽

Vol 3 (2) ◽

pp. 115-118

Author(s):

Jia Yu-Ping ◽

Zhou Dong-Shun ◽

Zhao Hong-Kun ◽

Wan Ren-Zhong ◽

Liu Wen-Qiang ◽

...

Keyword(s):

16S Rrna ◽

Simple Procedure ◽

Bovine Mastitis ◽

Dairy Herd ◽

Raw Milk ◽

16S Rrna Genes ◽

Rrna Genes ◽

Nested Polymerase Chain Reaction ◽

Specific Pcr ◽

And Control

AbstractBovine mastitis caused byStreptococcus agalactiaeis mainly subclinical and therefore can be diagnosed only in the laboratory. A nested polymerase chain reaction (PCR)-based method for specific, sensitive and rapid detection ofS. agalactiaein raw milk was developed. The general streptococci primers, which anneal to conserved areas within the 16S rRNA subunit gene, were used as positive controls. The specificity ofS. agalactiaeprimers is based on various areas within conserved areas of the 16S rRNA genes ofS. agalactiae. Results have indicated that the method enables the detection of 1 CFU/ml ofS. agalactiaein raw milk after enrichment, followed by DNA extraction using a rapid and simple procedure developed for this purpose, and specific PCR reaction. The method developed can be used efficiently in the early infectious status investigation ofS. agalactiaein the dairy herd and in prevention and control ofS. agalactiaespread in a herd.

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Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

Applied and Environmental Microbiology ◽

10.1128/aem.65.2.422-430.1999 ◽

1999 ◽

Vol 65 (2) ◽

pp. 422-430 ◽

Cited By ~ 170

Author(s):

Ulrich Nübel ◽

Ferran Garcia-Pichel ◽

Michael Kühl ◽

Gerard Muyzer

Keyword(s):

16S Rrna ◽

Microbial Mats ◽

Denaturing Gradient Gel Electrophoresis ◽

Morphological Diversity ◽

Numerical Approach ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Specific Pcr ◽

Phototrophic Microorganisms

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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‘Candidatus Mycoplasma haemoalbiventris’ and tick-borne pathogens screening in white-eared opossums (Didelphis albiventris) from Curitiba and Foz do Iguaçu Cities, Paraná State, southern Brazil

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612021072 ◽

2021 ◽

Vol 30 (4) ◽

Author(s):

Renata Prestes Antonangelo de Oliveira ◽

Flávia Carolina Meira Collere ◽

Larissa Dantas Roeder Ferrari ◽

Vanessa dos Santos Coradi ◽

Nathália de Albuquerque Soares ◽

...

Keyword(s):

16S Rrna ◽

Clinical Signs ◽

16S Rrna Genes ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Southern Brazil ◽

Specific Pcr ◽

Gapdh Gene ◽

Didelphis Albiventris

Abstract Hemoplasmas are epierythrocytic bacteria that infect mammals. ‘Candidatus Mycoplasma haemoalbiventris’ was detected in white-eared opossums (Didelphis albiventris) from southern and central-western Brazil. The present study aimed at: i) screening opossums for tick-borne (TBP) pathogens (Piroplasmida and Anaplasmataceae) and ii) detecting and characterizing hemoplasma species infecting opossums from Curitiba and Foz do Iguaçu cities in the Paraná State, southern Brazil. Thirty blood samples from white-eared opossums were evaluated by PCR assays. Animals were not infested by ectoparasites. The mammalian endogenous gapdh gene was consistently amplified in all samples. All opossums tested negative for Theileria/Babesia spp. and Ehrlichia/Anaplasma spp. by PCR based on 18S rRNA and 16S rRNA genes, respectively. A genus-specific PCR assay based on the 16S rRNA gene of hemoplasmas showed that three/13 (23.08%; CI 95%: 8.18-50.26%) opossums from Foz do Iguaçu were positive for hemotropic Mycoplasma sp. All opossums from Curitiba tested negative for hemoplasmas. Sequencing of both the 16S and 23S rRNA genes revealed that the animals were infected by ‘Ca. M. haemoalbiventris’. Although ‘Ca. M. haemoalbiventris’ is prevalent in opossums in Brazil, clinical signs associated with its infection and its putative vectors remain unknown.

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Evaluations of Different Hypervariable Regions of Archaeal 16S rRNA Genes in Profiling of Methanogens by Archaea-Specific PCR and Denaturing Gradient Gel Electrophoresis

Applied and Environmental Microbiology ◽

10.1128/aem.00684-07 ◽

2007 ◽

Vol 74 (3) ◽

pp. 889-893 ◽

Cited By ~ 118

Author(s):

Zhongtang Yu ◽

Rubén García-González ◽

Floyd L. Schanbacher ◽

Mark Morrison

Keyword(s):

16S Rrna ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Specific Pcr ◽

Pcr Products ◽

Gradient Gel Electrophoresis ◽

V Region

ABSTRACT Different hypervariable (V) regions of the archaeal 16S rRNA gene (rrs) were compared systematically to establish a preferred V region(s) for use in Archaea-specific PCR-denaturing gradient gel electrophoresis (DGGE). The PCR products of the V3 region produced the most informative DGGE profiles and permitted identification of common methanogens from rumen samples from sheep. This study also showed that different methanogens might be detected when different V regions are targeted by PCR-DGGE. Dietary fat appeared to transiently stimulate Methanosphaera stadtmanae but inhibit Methanobrevibacter sp. strain AbM4 in rumen samples.

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Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene

Journal of the Asiatic Society of Bangladesh Science ◽

10.3329/jasbs.v41i1.46190 ◽

2015 ◽

Vol 41 (1) ◽

pp. 51-58

Author(s):

Mohammad Shamimul Alam ◽

Hawa Jahan ◽

Rowshan Ara Begum ◽

Reza M Shahjahan

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Fish Larva ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Valuable Insight ◽

Multiple Sequence ◽

Pcr Rflp ◽

Alternative Means

Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

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Effects of Supplement of Marichromatium gracile YL28 on Water Quality and Microbial Structures in Shrimp Mariculture Ecosystems

Genes ◽

10.3390/genes12010040 ◽

2020 ◽

Vol 12 (1) ◽

pp. 40

Author(s):

Liang Cui ◽

Bitong Zhu ◽

Xiaobo Zhang ◽

Zhuhua Chan ◽

Chungui Zhao ◽

...

Keyword(s):

Water Quality ◽

16S Rrna ◽

Relative Abundance ◽

Animal Health ◽

Denitrifying Bacteria ◽

Nitrite Reduction ◽

16S Rrna Genes ◽

Rrna Genes ◽

Sequencing Analysis ◽

Shrimp Culture

The elevated NH3-N and NO2-N pollution problems in mariculture have raised concerns because they pose threats to animal health and coastal and offshore environments. Supplement of Marichromatium gracile YL28 (YL28) into polluted shrimp rearing water and sediment significantly decreased ammonia and nitrite concentrations, showing that YL28 functioned as a novel safe marine probiotic in the shrimp culture industry. The diversity of aquatic bacteria in the shrimp mariculture ecosystems was studied by sequencing the V4 region of 16S rRNA genes, with respect to additions of YL28 at the low and high concentrations. It was revealed by 16S rRNA sequencing analysis that Proteobacteria, Planctomycete and Bacteroidetes dominated the community (>80% of operational taxonomic units (OTUs)). Up to 41.6% of the predominant bacterial members were placed in the classes Gammaproteobacteria (14%), Deltaproteobacteria (14%), Planctomycetacia (8%) and Alphaproteobacteria (5.6%) while 40% of OTUs belonged to unclassified ones or others, indicating that the considerable bacterial populations were novel in our shrimp mariculture. Bacterial communities were similar between YL28 supplements and control groups (without addition of YL28) revealed by the β-diversity using PCoA, demonstrating that the additions of YL28 did not disturb the microbiota in shrimp mariculture ecosystems. Instead, the addition of YL28 increased the relative abundance of ammonia-oxidizing and denitrifying bacteria. The quantitative PCR analysis further showed that key genes including nifH and amoA involved in nitrification and nitrate or nitrite reduction significantly increased with YL28 supplementation (p < 0.05). The supplement of YL28 decreased the relative abundance of potential pathogen Vibrio. Together, our studies showed that supplement of YL28 improved the water quality by increasing the relative abundance of ammonia-oxidizing and denitrifying bacteria while the microbial community structure persisted in shrimp mariculture ecosystems.

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Ultra-accurate microbial amplicon sequencing with synthetic long reads

Microbiome ◽

10.1186/s40168-021-01072-3 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Benjamin J. Callahan ◽

Dmitry Grinevich ◽

Siddhartha Thakur ◽

Michael A. Balamotis ◽

Tuval Ben Yehezkel

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Amplicon Sequencing ◽

Species Level ◽

Full Length ◽

16S Rrna Genes ◽

Rrna Genes ◽

Strain Identification ◽

Long Reads ◽

Long Read

Abstract Background Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Methods Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. Results LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. Conclusions The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.

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