scholarly journals Quantitative Detection of the nosZ Gene, Encoding Nitrous Oxide Reductase, and Comparison of the Abundances of 16S rRNA, narG, nirK, and nosZ Genes in Soils

2006 ◽  
Vol 72 (8) ◽  
pp. 5181-5189 ◽  
Author(s):  
S. Henry ◽  
D. Bru ◽  
B. Stres ◽  
S. Hallet ◽  
L. Philippot
Keyword(s):  
Nitrous Oxide ◽  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Gene Copy ◽  
Nitrous Oxide Reductase ◽  
Gene Encoding ◽  
Copy Numbers ◽  
Dry Soil ◽  
Gene Copy Numbers

ABSTRACT Nitrous oxide (N2O) is an important greenhouse gas in the troposphere controlling ozone concentration in the stratosphere through nitric oxide production. In order to quantify bacteria capable of N2O reduction, we developed a SYBR green quantitative real-time PCR assay targeting the nosZ gene encoding the catalytic subunit of the nitrous oxide reductase. Two independent sets of nosZ primers flanking the nosZ fragment previously used in diversity studies were designed and tested (K. Kloos, A. Mergel, C. Rösch, and H. Bothe, Aust. J. Plant Physiol. 28:991-998, 2001). The utility of these real-time PCR assays was demonstrated by quantifying the nosZ gene present in six different soils. Detection limits were between 101 and 102 target molecules per reaction for all assays. Sequence analysis of 128 cloned quantitative PCR products confirmed the specificity of the designed primers. The abundance of nosZ genes ranged from 105 to 107 target copies g−1 of dry soil, whereas genes for 16S rRNA were found at 108 to 109 target copies g−1 of dry soil. The abundance of narG and nirK genes was within the upper and lower limits of the 16S rRNA and nosZ gene copy numbers. The two sets of nosZ primers gave similar gene copy numbers for all tested soils. The maximum abundance of nosZ and nirK relative to 16S rRNA was 5 to 6%, confirming the low proportion of denitrifiers to total bacteria in soils.

scholarly journals Photo-Induced Electron Transfer Real-Time PCR for Detection ofPlasmodium falciparum plasmepsin 2Gene Copy Number

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Samaly Santos Souza ◽  
Mariangela L'Episcopia ◽  
Carlo Severini ◽  
Venkatachalam Udhayakumar ◽  
Naomi W. Lucchi
Keyword(s):  
Electron Transfer ◽  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Gene Copy ◽  
Content Type ◽  
Copy Numbers ◽  
Ofplasmodium Falciparum ◽  
Photo Induced Electron Transfer ◽  
Gene Copy Numbers

ABSTRACTPiperaquine is an important partner drug used in artemisinin-based combination therapies (ACTs). An increase in theplasmepsin 2and3gene copy numbers has been associated with decreased susceptibility ofPlasmodium falciparumto piperaquine in Cambodia. Here, we developed a photo-induced electron transfer real-time PCR (PET-PCR) assay to quantify the copy number of theP. falciparumplasmepsin 2gene (PfPM2) that can be used in countries whereP. falciparumis endemic to enhance molecular surveillance.

scholarly journals Clinical Usefulness of 16S Ribosomal RNA Real-Time PCR for The Diagnosis of Scrub Typhus

2021 ◽  
Author(s):  
Dong-Min Kim ◽  
Na Ra Yun ◽  
Choon-Mee Kim ◽  
Da Young Kim ◽  
Jun-Won Seo
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Scrub Typhus ◽  
Clinical Usefulness ◽  
Asia Pacific ◽  
Gene Encoding ◽  
Copy Numbers ◽  
Severe Group ◽  
Pcr Targeting

Abstract Background: Scrub typhus is a major acute febrile disease in the Asia-Pacific region. The purpose of the present study is to investigate the clinical usefulness of real-time PCR (Q-PCR) of 16S rRNA for the diagnosis of scrub typhus.Methods: We examined blood specimens from 148 adult patients who were confirmed to have scrub typhus from September 2008 to December 2009. Among the 148 scrub typhus patients, 36 patients were treated with antibiotics before admission. To evaluate the clinical usefulness of 16S rRNA Q-PCR, we compared its diagnostic accuracy to the accuracy of the following methods: nested PCR (N-PCR) targeting the gene encoding the 56-kDa protein, Q-PCR targeting the gene encoding the 47-kDa protein, and conventional PCR (C-PCR) targeting the 16S rRNA gene.Results: According to 16S rRNA Q-PCR and 47-kDa Q-PCR, the mild group had copy numbers of 234.4 and 130.5, whereas the severe group had copy numbers of 584.4 and 244.7, respectively. In both tests, the mean copy numbers were significantly greater in the severe group (P<0.01 and P=0.01). 16S rRNA Q-PCR detected Orientia tsutsugamushi infections with a sensitivity of 91.9%, and 56-kDa N-PCR, 47-kDa Q-PCR, and 16S rRNA C-PCR exhibited lower sensitivities of 81.1%, 74.3%, and 87.8%, respectively, for all 148 patients. In addition, 16S rRNA Q-PCR exhibited a sensitivity of 99.1% in the 112 patients who were not treated with antibiotics before admission.Conclusion: 16S rRNA Q-PCR is clinically useful for the rapid diagnosis of scrub typhus and is more accurate than the 56-kDa N-PCR, 47-kDa Q-PCR, and 16S C-PCR methods.

scholarly journals Clinical usefulness of 16S ribosomal RNA real-time PCR for the diagnosis of scrub typhus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Na Ra Yun ◽  
Choon-Mee Kim ◽  
Da Young Kim ◽  
Jun-Won Seo ◽  
Dong-Min Kim
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Scrub Typhus ◽  
Clinical Usefulness ◽  
Asia Pacific ◽  
Gene Encoding ◽  
Copy Numbers ◽  
Severe Group ◽  
Pcr Targeting

AbstractScrub typhus is a major acute febrile disease in the Asia–Pacific region. The purpose of the present study is to investigate the clinical usefulness of real-time PCR (Q-PCR) of 16S rRNA for the diagnosis of scrub typhus. We examined blood specimens from 148 adult patients who were confirmed to have scrub typhus from September 2008 to December 2009. Among the 148 scrub typhus patients, 36 patients were treated with antibiotics before admission. To evaluate the clinical usefulness of 16S rRNA Q-PCR, we compared its diagnostic accuracy to the accuracy of the following methods: nested PCR (N-PCR) targeting the gene encoding the 56-kDa protein, Q-PCR targeting the gene encoding the 47-kDa protein, and conventional PCR (C-PCR), targeting the 16S rRNA gene. According to 16S rRNA Q-PCR and 47-kDa Q-PCR, the mild group had copy numbers of 234.4 ± 261.9 and 130.5 ± 128.3, whereas the severe group had copy numbers of 584.4 ± 911.4 and 244.7 ± 210.9, respectively. In both tests, the mean copy numbers were significantly greater in the severe group (P = 0.037 and P = 0.035). 16S rRNA Q-PCR detected Orientia tsutsugamushi infections with a sensitivity of 91.9% (95% CI 86.3–95.7), and 56-kDa N-PCR, 47-kDa Q-PCR, and 16S rRNA C-PCR exhibited lower sensitivities of 81.1% (95% CI 73.8–87.0), 74.3% (95% CI 66.5–81.1), and 87.8% (95% CI 81.5–92.6), respectively, for all 148 patients. In addition, 16S rRNA Q-PCR exhibited a sensitivity of 99.1% (95% CI 95.1–100.0) in the 112 patients who were not treated with antibiotics before admission. 16S rRNA Q-PCR is clinically useful for the rapid diagnosis of scrub typhus and is more accurate than the 56-kDa N-PCR, 47-kDa Q-PCR, and 16S C-PCR methods.

scholarly journals Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes

2003 ◽  
Vol 69 (12) ◽  
pp. 7289-7297 ◽  
Author(s):  
Jaana Vaitomaa ◽  
Anne Rantala ◽  
Katrianna Halinen ◽  
Leo Rouhiainen ◽  
Petra Tallberg ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Methods ◽  
Gene Copy ◽  
Quantitative Real Time Pcr ◽  
Microcystin Synthetase ◽  
Copy Numbers ◽  
Restoration Strategies ◽  
Cell Densities ◽  
Gene Copy Numbers

ABSTRACT Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanjärvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanjärvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and Anabaena. The main microcystin producer in Lake Tuusulanjärvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of Anabaena. Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.

Possible gene dosage effect of glutathione-S-transferases on atopic asthma: Using real-time PCR for quantification of GSTM1 and GSTT1 gene copy numbers

2004 ◽  
Vol 24 (3) ◽  
pp. 208-214 ◽  
Author(s):  
Charlotte Brasch-Andersen ◽  
Lene Christiansen ◽  
Qihua Tan ◽  
Annette Haagerup ◽  
J�rgen Vestbo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gene Dosage ◽  
Atopic Asthma ◽  
Gene Copy ◽  
Gene Dosage Effect ◽  
Gstt1 Gene ◽  
Copy Numbers ◽  
Glutathione S Transferases ◽  
Gene Copy Numbers

Real-time PCR-based determination of gene copy numbers inPichia pastoris

2010 ◽  
Vol 5 (4) ◽  
pp. 413-420 ◽  
Author(s):  
Sandra Abad ◽  
Kerstin Kitz ◽  
Astrid Hörmann ◽  
Ulrike Schreiner ◽  
Franz S. Hartner ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gene Copy ◽  
Copy Numbers ◽  
Gene Copy Numbers

scholarly journals High-resolution microbiome analysis enabled by linking of 16S rRNA gene sequences with adjacent genomic contexts

2021 ◽  
Vol 7 (9) ◽  
Author(s):  
Žana Kapustina ◽  
Justina Medžiūnė ◽  
Gediminas Alzbutas ◽  
Irmantas Rokaitis ◽  
Karolis Matjošaitis ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Sequencing ◽  
Gene Copy ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Copy Numbers ◽  
Rrna Sequencing ◽  
Metagenome Sequencing ◽  
Gene Copy Numbers

Sequence-based characterization of bacterial communities has long been a hostage of limitations of both 16S rRNA gene and whole metagenome sequencing. Neither approach is universally applicable, and the main efforts to resolve constraints have been devoted to improvement of computational prediction tools. Here, we present semi-targeted 16S rRNA sequencing (st16S-seq), a method designed for sequencing V1–V2 regions of the 16S rRNA gene along with the genomic locus upstream of the gene. By in silico analysis of 13 570 bacterial genome assemblies, we show that genome-linked 16S rRNA sequencing is superior to individual hypervariable regions or full-length gene sequences in terms of classification accuracy and identification of gene copy numbers. Using mock communities and soil samples we experimentally validate st16S-seq and benchmark it against the established microbial classification techniques. We show that st16S-seq delivers accurate estimation of 16S rRNA gene copy numbers, enables taxonomic resolution at the species level and closely approximates community structures obtainable by whole metagenome sequencing.

scholarly journals Nitric Oxide Reductase-Targeted Real-Time PCR Quantification of Denitrifier Populations in Soil

2007 ◽  
Vol 73 (13) ◽  
pp. 4250-4258 ◽  
Author(s):  
C. E. Dandie ◽  
M. N. Miller ◽  
D. L. Burton ◽  
B. J. Zebarth ◽  
J. T. Trevors ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Total Population ◽  
Rrna Gene ◽  
Nitric Oxide Reductase ◽  
Soil Microcosms ◽  
Copy Numbers

ABSTRACT The quantification of denitrifying bacteria is a component in the further understanding of denitrification processes in the environment. Real-time PCR primers were designed to target two segments of the denitrifier population (cnorB P [Pseudomonas mandelii and closely related strains] and cnorB B [Bosea, Bradyrhizobium, and Ensifer spp.]) in agricultural soils based on functional cnorB (nitric oxide reductase) gene sequences. Total population numbers were measured using 16S rRNA gene real-time PCR. Two soil microcosm experiments were conducted. Experiment 1 examined the response of the indigenous soil microbial population to the addition of 500 mg/kg glucose-C daily over 7 days in soil microcosms. Changes in the total population were correlated (r = 0.83) between 16S rRNA gene copy numbers and microbial biomass carbon estimates. Members of the cnorB P population of denitrifiers showed typical r-strategy by being able to increase their proportion in the total population from starting levels of <0.1% to around 2.4% after a daily addition of 500 mg/kg glucose-C. The cnorB B guild was not able to increase its relative percentage of the total population in response to the addition of glucose-C, instead increasing copy numbers only in proportion with the total population measured by 16S rRNA genes. Experiment 2 measured population dynamics in soil after the addition of various amounts of glucose-C (0 to 500 mg/kg) and incubation under denitrifying conditions. cnorB P populations increased proportionally with the amount of glucose-C added (from 0 to 500 mg/kg). In soil microcosms, denitrification rates, respiration, and cnorB P population densities increased significantly with increasing rates of glucose addition. cnorB B guild densities did not increase significantly under denitrifying conditions in response to increasing C additions.

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