scholarly journals Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA

2011 ◽  
Vol 39 (1) ◽  
pp. 321-326 ◽  
Author(s):  
Tobias Klatte ◽  
Laura Evans ◽  
Rebekah N. Whitehead ◽  
Jeffrey A. Cole
Keyword(s):  
Nitrate Reductase ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
Pcr Primers ◽  
Periplasmic Nitrate Reductase ◽  
Pcr Products ◽  
Successful Design ◽  
The One ◽  
Bacterial Genes ◽  
Biological Nitrogen

Generic primers are available for detecting bacterial genes required for almost every reaction of the biological nitrogen cycle, the one notable exception being napA (gene for the molybdoprotein of the periplasmic nitrate reductase) encoding periplasmic nitrate reductases. Using an iterative approach, we report the first successful design of three forward oligonucleotide primers and one reverse primer that, in three separate PCRs, can amplify napA DNA from all five groups of Proteobacteria. All 140 napA sequences currently listed in the NCBI (National Center for Biotechnology Information) database are predicted to be amplified by one or more of these primer pairs. We demonstrate that two pairs of these primers also amplify PCR products of the predicted sizes from DNA isolated from human faeces, confirming their ability to direct the amplification of napA fragments from mixed populations. Analysis of the resulting amplicons by high-throughput sequencing will enable a good estimate to be made of both the range and relative abundance of nitrate-reducing bacteria in any community, subject only to any unavoidable bias inherent in a PCR approach to molecular characterization of a highly diverse target.

High-throughput sequencing of kDNA amplicons for the analysis ofLeishmaniaminicircles and identification of Neotropical species

Parasitology ◽  
2017 ◽  
Vol 145 (5) ◽  
pp. 585-594 ◽  
Author(s):  
ARTHUR KOCHER ◽  
SOPHIE VALIÈRE ◽  
ANNE-LAURE BAÑULS ◽  
JÉRÔME MURIENNE
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Pcr Primers ◽  
Genomic Region ◽  
Kinetoplast Dna ◽  
Sequence Comparisons ◽  
Conserved Sequence ◽  
Species Complexes ◽  
Pcr Products ◽  
Minicircle Sequence

SUMMARYLeishmaniakinetoplast DNA contains thousands of small circular molecules referred to as kinetoplast DNA (kDNA) minicercles. kDNA minicircles are the preferred targets for sensitiveLeishmaniadetection, because they are present in high copy number and contain conserved sequence blocks in which polymerase chain reaction (PCR) primers can be designed. On the other hand, the heterogenic nature of minicircle networks has hampered the use of this peculiar genomic region for strain typing. The characterization ofLeishmaniaminicirculomes used to require isolation and cloning steps prior to sequencing. Here, we show that high-throughput sequencing of single minicircle PCR products allows bypassing these laborious laboratory tasks. The 120 bp long minicircle conserved region was amplified by PCR from 18Leishmaniastrains representative of the major species complexes found in the Neotropics. High-throughput sequencing of PCR products enabled recovering significant numbers of distinct minicircle sequences from each strain, reflecting minicircle class diversity. Minicircle sequence analysis revealed patterns that are congruent with current hypothesis ofLeishmaniarelationships. Then, we show that a barcoding-like approach based on minicircle sequence comparisons may allow reliable identifications ofLeishmaniaspp. This work opens up promising perspectives for the study of kDNA minicercles and a variety of applications inLeishmaniaresearch.

scholarly journals Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
Ryohei Nakao ◽  
Hideyuki Doi ◽  
Toshifumi Minamoto
Keyword(s):  
Fish Species ◽  
High Throughput Sequencing ◽  
Correlation Coefficients ◽  
Environmental Dna ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Taqman Probe ◽  
Individual Species ◽  
Natural Environments ◽  
Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

Exploring the plant environmental DNA diversity in soil from two sites on Deception Island (Antarctica, South Shetland Islands) using metabarcoding

2021 ◽  
pp. 1-10
Author(s):  
Micheline Carvalho-Silva ◽  
Luiz Henrique Rosa ◽  
Otávio H.B. Pinto ◽  
Thamar Holanda Da Silva ◽  
Diego Knop Henriques ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Crater Lake ◽  
Environmental Dna ◽  
Food Sources ◽  
South Shetland Islands ◽  
Deception Island ◽  
Shetland Islands ◽  
Operational Taxonomic Units ◽  
First Time ◽  
Impacted Site

Abstract The few Antarctic studies to date to have applied metabarcoding in Antarctica have primarily focused on microorganisms. In this study, for the first time, we apply high-throughput sequencing of environmental DNA to investigate the diversity of Embryophyta (Viridiplantae) DNA present in soil samples from two contrasting locations on Deception Island. The first was a relatively undisturbed site within an Antarctic Specially Protected Area at Crater Lake, and the second was a heavily human-impacted site in Whalers Bay. In samples obtained at Crater Lake, 84% of DNA reads represented fungi, 14% represented Chlorophyta and 2% represented Streptophyta, while at Whalers Bay, 79% of reads represented fungi, 20% represented Chlorophyta and < 1% represented Streptophyta, with ~1% of reads being unassigned. Among the Embryophyta we found 16 plant operational taxonomic units from three Divisions, including one Marchantiophyta, eight Bryophyta and seven Magnoliophyta. Sequences of six taxa were detected at both sampling sites, eight only at Whalers Bay and two only at Crater Lake. All of the Magnoliophyta sequences (flowering plants) represent species that are exotic to Antarctica, with most being plausibly linked to human food sources originating from local national research operator and tourism facilities.

scholarly journals Detection of erythromycin-resistant determinants by PCR.

1996 ◽  
Vol 40 (11) ◽  
pp. 2562-2566 ◽  
Author(s):  
J Sutcliffe ◽  
T Grebe ◽  
A Tait-Kamradt ◽  
L Wondrack
Keyword(s):  
Direct Sequencing ◽  
Pcr Primers ◽  
Efflux Pumps ◽  
Clinical Isolates ◽  
Gene Products ◽  
Mechanisms Of Resistance ◽  
Erythromycin Resistance ◽  
Surveillance Studies ◽  
Resistance Determinants ◽  
Pcr Products

Erythromycin resistance determinants include Erm methylases, efflux pumps, and inactivating enzymes. To distinguish the different mechanisms of resistance in clinical isolates, PCR primers were designed so that amplification of the partial gene products could be detected in multiplex PCRs. This methodology enables the direct sequencing of amplified PCR products that can be used to compare resistance determinants in clinical strains. Further, this methodology could be useful in surveillance studies of erythromycin-resistant determinants.

scholarly journals Simple and Visible Detection of Novel Astroviruses Causing Fatal Gout in Goslings Using One-Step Reverse Transcription Polymerase Spiral Reaction Method

2020 ◽  
Vol 7 ◽  
Author(s):  
Jun Ji ◽  
Qinxi Chen ◽  
Zhengli Yu ◽  
Xin Xu ◽  
Xinhao Mu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Cost Effective ◽  
Pcr Primers ◽  
Probit Regression ◽  
Conventional Pcr ◽  
Specific Detection ◽  
Isothermal Method ◽  
Naked Eye ◽  
One Step ◽  
The One

In this study, a one-step isothermal method combining polymerase spiral reaction (PSR) with reverse transcription (RT-PSR) was established for rapid and specific detection of novel astroviruses causing fatal gout in goslings (N-GoAstV). The one-step RT-PSR was accomplished at the optimal temperature of 62°C and time of 40 min and used primers simply designed as conventional PCR primers, and the results of detection were visible to the naked eye. The detection limit of PSR was above 34.7 copies/μL at a 95% probability level according to probit regression analysis. The assay specifically detected N-GoAstV, and no other reference viruses were detected. These results suggest that the newly established RT-PSR assay could, in one step, accomplish reverse-transcription, amplification, and result determination providing a visible, convenient, rapid, and cost-effective test that can be carried out onsite, in order to ensure timely quarantine of N-GoAstV-infected birds, leading to effective disease control.

scholarly journals High prevalence and diversity of beta-lactamase-encoding bacteria in cryosoils and ancient permafrost

2021 ◽  
Author(s):  
Sofia Rigou ◽  
Eugene Christo-Foroux ◽  
Sebastien Santini ◽  
Artemiy Goncharov ◽  
Jens Strauss ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Bacterial Genome ◽  
Environmental Dna ◽  
Single Copy ◽  
The Arctic ◽  
Human Society ◽  
Beta Lactamase ◽  
Extended Spectrum Beta Lactamase ◽  
Arctic Regions ◽  
Global Threat

Background: Antimicrobial resistance is one of the major challenges affecting public health. It is mostly due to the continuous emergence of extended-spectrum beta-lactamase from various environments followed by their rapid dissemination and selection in clinical settings. The warming of Earth' s climate is the other global threat facing human society, in particular with the Arctic regions experiencing a twice faster warming than the global average and permafrost affected by widespread thawing. A potentially dreadful combination of these two threats would be the release and dispersion of harmful microbes that have remained confined to largely uninhabited Arctic regions, or are stored dormant in permafrost. Methods: Environmental DNA was isolated from 12 soil samples from various Arctic and subarctic pristine regions in Siberia (Yakutia and Kamchatka), including 9 permafrost samples collected at various depths. The large datasets obtained from high throughput sequencing was assembled in contigs and their protein-gene contents predicted. We used exhaustive similarity searches to perform taxonomical assignments of bacterial, archaeal, and eukaryotic organisms, as well as DNA viruses. In addition, we specifically identified beta-lactamase genes and their prevalence per bacterial genome estimated through the detection of two universal single copy genes. Findings: A total of 9.217 1011 bp were exploited, leading to a total of 525,313 contigs at least 5kb in size. The DNA content of the various samples was found to be highly variable, not strictly correlated with the depth or radio-carbon-based deposit age, and most likely linked to the global density of microbes trapped in the corresponding permafrost layers. Bacteria account for more than 90% of the contigs in most samples, followed by Eukaryotes and Archaea (always lower than 10%). Viruses represented less than 2% of all contigs in all samples. The taxonomic profiles of surface cryosoils and deep permafrost samples exhibited a high diversity, including between permafrost samples originating from various depths in the same borehole. In all samples, bacterial contigs carrying different beta-lactamases from class A to D were identified. Interpretation: No clear common taxonomic feature could be found shared by surface cryosoils or ancient permafrost layers. However, most samples (9/12) exhibited a high frequency of beta-lactamase genes, with an estimated average close to 1 copy/bacterial genome. In addition to the well-documented reactivation of infectious ancient pathogens (bacteria, viruses, protozoa), we show now that global warming could contribute to the emergence of new antibiotic resistances through the mobilization by contemporary bacteria of ancient DNA released from thawing permafrost.

scholarly journals Intersimple Sequence Repeat Markers for Fingerprinting and Determining Genetic Relationships of Walnut (Juglans regia) Cultivars

2002 ◽  
Vol 127 (1) ◽  
pp. 75-81 ◽  
Author(s):  
Daniel Potter ◽  
Fangyou Gao ◽  
Giovanna Aiello ◽  
Charles Leslie ◽  
Gale McGranahan
Keyword(s):  
Genetic Relationships ◽  
Juglans Regia ◽  
Issr Markers ◽  
Genetic Distances ◽  
Sequence Repeat ◽  
Geographic Origins ◽  
Pcr Products ◽  
Extensive Exchange ◽  
The One ◽  
Issr Primers

The utility of intersimple sequence repeat (ISSR) markers for identification of English or Persian walnut (Juglans regia L.) cultivars was explored. Four cultivars were screened with 47 ISSR primers; eight of these primers, which generated reproducible and informative data, were selected for further study. Two individuals from each of 48 cultivars, including many currently important in the California walnut industry as well as accessions from Europe and Asia, were then examined with the eight ISSR primers. Polymerase chain reaction (PCR) products were separated on agarose gels and stained with ethidium bromide. Fifty-four bands were scored as present or absent in each cultivar; of these, 31 (57%) were polymorphic among the 48 cultivars. Combined data from the eight ISSR primers provided a unique fingerprint for each of the cultivars tested. Fifteen of the cultivars could be distinguished from all others with just one primer, 31 with a minimum of two primers, and two required three primers. Pairwise genetic distances between the cultivars were calculated and a dendrogram was generated using the neighbor-joining algorithm. Some of the groupings in the dendrogram corresponded to groups which, based on known pedigrees, are genealogically closely related. Others included accessions from diverse genetic and/or geographic origins. These results can be attributed to a combination of the limitations of the ISSR method for inferring genetic relationships, on the one hand, and the complex history of walnut cultivar development involving extensive exchange and breeding of germplasm from different geographic regions, on the other.

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