scholarly journals Hierarchical Oligonucleotide Primer Extension as a Time- and Cost-Effective Approach for Quantitative Determination of Bifidobacterium spp. in Infant Feces

2009 ◽  
Vol 75 (8) ◽  
pp. 2573-2576 ◽  
Author(s):  
Pei-Ying Hong ◽  
Gaik Chin Yap ◽  
Bee Wah Lee ◽  
Kaw Yan Chua ◽  
Wen-Tso Liu
Keyword(s):  
Quantitative Determination ◽  
Bifidobacterium Longum ◽  
Cost Effective ◽  
Primer Extension ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Oligonucleotide Primer ◽  
Effective Manner ◽  
Relative Abundances

ABSTRACT The Bifidobacterium spp. present in 10 infant fecal samples (4 from infants with eczema and 6 from healthy infants) were quantified with both hierarchical oligonucleotide primer extension (HOPE) and fluorescence in situ hybridization-flow cytometry. The relative abundances of Bifidobacterium longum and B. catenulatum with respect to the total bifidobacteria had a poor correlation (ρ, <0.600; P value, >0.208), presumably due to differences in primer specificity and the level of hybridization stringency of both methods. In contrast, the relative abundances of organisms of the genus Bifidobacterium against the total amplified 16S rRNA genes and those of B. adolescentis, B. bifidum, and B. breve against the genus Bifidobacterium exhibited a good statistical correlation (ρ, >0.783; P value, <0.066). This good comparability supports HOPE as a method to achieve high-throughput quantitative determination of bacterial targets in a time- and cost-effective manner.

scholarly journals Use of a Hierarchical Oligonucleotide Primer Extension Approach for Multiplexed Relative Abundance Analysis of Methanogens in Anaerobic Digestion Systems

2013 ◽  
Vol 79 (24) ◽  
pp. 7598-7609 ◽  
Author(s):  
Jer-Horng Wu ◽  
Hui-Ping Chuang ◽  
Mao-Hsuan Hsu ◽  
Wei-Yu Chen
Keyword(s):  
Anaerobic Digestion ◽  
16S Rrna ◽  
Industrial Wastewater ◽  
Anaerobic Treatment ◽  
Primer Extension ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Oligonucleotide Primer ◽  
Content Type ◽  
Relative Abundances

ABSTRACTIn this study, we established a rapid multiplex method to detect the relative abundances of amplified 16S rRNA genes from known cultivatable methanogens at hierarchical specificities in anaerobic digestion systems treating industrial wastewater and sewage sludge. The method was based on the hierarchical oligonucleotide primer extension (HOPE) technique and combined with a set of 27 primers designed to target the total archaeal populations and methanogens from 22 genera within 4 taxonomic orders. After optimization for their specificities and detection sensitivity under the conditions of multiple single-nucleotide primer extension reactions, the HOPE approach was applied to analyze the methanogens in 19 consortium samples from 7 anaerobic treatment systems (i.e., 513 reactions). Among the samples, the methanogen populations detected with order-level primers accounted for >77.2% of the PCR-amplified 16S rRNA genes detected using anArchaea-specific primer. The archaeal communities typically consisted of 2 to 7 known methanogen genera within theMethanobacteriales,Methanomicrobiales, andMethanosarcinalesand displayed population dynamic and spatial distributions in anaerobic reactor operations. Principal component analysis of the HOPE data further showed that the methanogen communities could be clustered into 3 distinctive groups, in accordance with the distribution of theMethanosaeta,Methanolinea, andMethanomethylovorans, respectively. This finding suggested that in addition to acetotrophic and hydrogenotrophic methanogens, the methylotrophic methanogens might play a key role in the anaerobic treatment of industrial wastewater. Overall, the results demonstrated that the HOPE approach is a specific, rapid, and multiplexing platform to determine the relative abundances of targeted methanogens in PCR-amplified 16S rRNA gene products.

scholarly journals Relative Abundance of Bacteroides spp. in Stools and Wastewaters as Determined by Hierarchical Oligonucleotide Primer Extension

2008 ◽  
Vol 74 (9) ◽  
pp. 2882-2893 ◽  
Author(s):  
Pei-Ying Hong ◽  
Jer-Horng Wu ◽  
Wen-Tso Liu
Keyword(s):  
16S Rrna ◽  
Fecal Microbiota ◽  
Primer Extension ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Oligonucleotide Primer ◽  
Healthy Human ◽  
Dna Templates ◽  
Human Adults ◽  
Relative Abundances

ABSTRACT A molecular method, termed hierarchical oligonucleotide primer extension (HOPE), was used to determine the relative abundances of predominant Bacteroides spp. present in fecal microbiota and wastewaters. For this analysis, genomic DNA in feces of healthy human adults, bovines, and swine and in wastewaters was extracted and total bacterial 16S rRNA genes were PCR amplified and used as the DNA templates for HOPE. Nineteen oligonucleotide primers were designed to detect 14 Bacteroides spp. at different hierarchical levels (domain, order, cluster, and species) and were arranged into and used in six multiplex HOPE reaction mixtures. Results showed that species like B. vulgatus, B. thetaiotaomicron, B. caccae, B. uniformis, B. fragilis, B. eggerthii, and B. massiliensis could be individually detected in human feces at abundances corresponding to as little as 0.1% of PCR-amplified 16S rRNA genes. Minor species like B. pyogenes, B. salyersiae, and B. nordii were detected only collectively using a primer that targeted the B. fragilis subgroup (corresponding to ∼0.2% of PCR-amplified 16S rRNA genes). Furthermore, Bac303-related targets (i.e., most Bacteroidales) were observed to account for 28 to 44% of PCR-amplified 16S rRNA genes from human fecal microbiota, and their abundances were higher than those detected in the bovine and swine fecal microbiota and in wastewaters by factors of five and two, respectively. These results were comparable to those obtained by quantitative PCR and to those reported previously from studies using whole-cell fluorescence hybridization and 16S rRNA clone library methods, supporting the conclusion that HOPE can be a sensitive, specific, and rapid method to determine the relative abundances of Bacteroides spp. predominant in fecal samples.

scholarly journals Simultaneous Stripping of Ammonia from Leachate: Experimental Insights and Key Microbial Players

Water ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2494
Author(s):  
Łukasz Jurczyk ◽  
Justyna Koc-Jurczyk ◽  
Adam Masłoń
Keyword(s):  
Removal Efficiency ◽  
Reaction Rate ◽  
Batch Reactor ◽  
Cost Effective ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Community Shift ◽  
Biological Removal ◽  
Novel Approach ◽  
Aeration System

Air stripping is commonly used to remove the ammonia in multistage treatment systems for municipal landfill leachate (LFL). This paper proposes a novel approach combining the process of stripping with biological removal of ammonia, based on simultaneous nitrification and denitrification (SND) in a single hybrid sequencing batch reactor (HSBR). To avoid the accumulation of free ammonia (N-FAN), the shallow aeration system was used for the treatment of raw LFL with N-TAN level of 1520 mg/L and pH 9.24. The mean N-FAN removal efficiency of 69% with the reaction rate of 55 mg L−1 h−1 and mean ammonium (N-NH4+) removal efficiency of 84% with the reaction rate of 44 mg L−1 h−1 were achieved within a month in such an HSBR (R1). The comparative HSBR (R2), with conventional aeration system maintaining the same concentration of dissolved oxygen (DO ≤ 1 mg/L), was removing only trace amounts of N-FAN and 48% of N-NH4+. The quantitative analysis of 16S rRNA genes indicated that the number of total bacteria, Actinobacteria, Bacteroidetes, Firmicutes, and Beta- and Gammaproteobacteria increased during the operation of both HSBRs, but was always higher in R1. Moreover, the bacterial community shift was observed since the beginning of the experiment; the relative abundance of Firmicutes, and Beta- and Gammaproteobacteria increased by 5.01, 3.25 and 9.67% respectively, whilst the abundance of Bacteroidetes and Actinobacteria decreased by 15.59 and 0.95%. All of the surveyed bacteria groups, except Gammaproteobacteria, correlated significantly negatively (p < 0.001) with the concentrations of N-NH4+ in the outflows from R1. The results allow us to suppose that simultaneous stripping and SND in a single reactor could be a promising, cost-effective and easy-to-operate solution for LFL treatment.

scholarly journals Spectrophotometric determination of sotalol in tablets

2018 ◽  
pp. 49-55
Author(s):  
Y. M. Zhuk ◽  
S. O. Vasyuk
Keyword(s):  
Quantitative Determination ◽  
Room Temperature ◽  
Cost Effective ◽  
Pharmaceutical Formulations ◽  
Molar Absorptivity ◽  
Pharmaceutical Drugs ◽  
Analysis Method ◽  
Highly Sensitive ◽  
Colored Product

In this investigation a visible spectrophotometric method for the determination of sotalol based on the absorbance of colored product of the reaction between sotalol hydrochloride and bromcresol purple in acetone medium at 399 nm measurement was developed. The optimal conditions for the quantitative determination of sotalol hydrochloride in the content of pharmaceutical drugs were established. The stoichiometric relationship coefficients between sotalol hydrochloride and bromcresol purple were determined. The validation of the worked out procedure on such validated characteristics as linearity, precision, accuracy and robustness was carried out. The aim. To develop a highly sensitive, easy to use, cost-effective and valid method for quantitative determination of sotalol hydrochloride in dosage forms. The analysis method. Visible spectrophotometry. The analytical parameters such as molar absorptivity, Beer’s law limits and Sandell’s sensitivity values were calculated. The developed methods give the result with repeatability sufficient for dependable determination the investigated substance in pharmaceutical formulations. Accuracy established by analyte addition technique. Determined factors that influence on the absorbance value: reagent quantity and timing stability. Sample solutions stable during 30 min. Addition to sample solution ± 10% bromcresol purple solution is not change the absorbance value. Established that reaction between sotalol hydrochloride and bromcresol purple proceeds in acetone medium at room temperature. Molar absorption coefficient is 2,62∙103.

scholarly journals UV Spectrophotometric Method Development and Validation of Fimasartan Drug and Its Tablet Formulation

1970 ◽  
Vol 7 (5) ◽  
pp. 25-29
Author(s):  
Kaushik S Agrawal ◽  
Lokesh R Gandhi ◽  
Nitin S. Bhajipale S Bhajipale3
Keyword(s):  
Standard Deviation ◽  
Quantitative Determination ◽  
Relative Standard Deviation ◽  
Spectrophotometric Method ◽  
Method Development ◽  
Cost Effective ◽  
Tablet Formulation ◽  
Relative Standard ◽  
Bulk Drug

A novel, safe and sensitive method of Spectrophotometric estimation in UV - region has been developed for the assay of Fimasartan in its tablet formulation. The present study was undertaken to develop and validate a simple, accurate, precise, reproducible and cost effective UV spectrophotometric method for the estimation of Fimasartan bulk and pharmaceutical formulation. The method have been developed and validated for the assay of Fimasartan using Methanol as diluent. Absorption maximum (λmax) of the drug was found to be 240nm. The quantitative determination of the drug was carried out at 240nm. The method was shown linear in the mentioned concentrations having correlation coefficient R2 of 0.999. The recovery values for Fimasartan ranged from 98.74% to 99.23%.The Percent Relative Standard Deviation of interday and intraday was 0.85% and 0.75% respectively. All the parameters of the analysis were chosen according to the International Conference on Harmonisation guideline and validated statistically using Relative Standard Deviation and Percent Relative Standard Deviation. Hence, proposed method was precise, accurate and cost effective. This method could be applicable for quantitative determination of the bulk drug as well as dosage formulation.   KEY WORDS: 

scholarly journals ДОСЛІДЖЕННЯ ВАЛІДАЦІЙНИХ ХАРАКТЕРИСТИК МЕТОДИКИ КІЛЬКІСНОГО ВИЗНАЧЕННЯ ПАРАЦЕТАМОЛУ ТА ЙОГО ОСНОВНОЇ ДОМІШКИ В РЕКТАЛЬНИХ СУПОЗИТОРІЯХ

2021 ◽  
Vol 146 (3) ◽  
pp. 139-154
Author(s):  
О. О. Салій ◽  
Г. І. Кузьміна ◽  
Л. А. Фуклева ◽  
В. В. Манацюк
Keyword(s):  
Liquid Chromatography ◽  
Quantitative Determination ◽  
High Performance ◽  
Signal To Noise Ratio ◽  
Statistical Processing ◽  
Cost Effective ◽  
Hplc Method ◽  
Uv Detector ◽  
Main Impurity

Еxploration of validation characteristicsof the method for the quantitative determination of paracetamol and its main impurity 4-aminophenol in rectal suppositories by spectrophotometry (UV) and high performance liquid chromatography (HPLC). Spectrophotometric measurements were carried out on a UV VIS Lambda 35 spectrophotometer (Perkin Elmer, USA) in cuvettes l = 1 cm. We used a Waters 2695 liquid chromatography with a Waters 2489 UV-detector, as well as a Nucleosil C18 chromatographic column with a size of 250 × 4.6 mm, filled with octadecylsilyl sorbent with a particle size of 10 microns. We used reagents: purified water, which was obtained from the Milli Q plant, manufactured by Millipore Corporation (Germany), sodium hydroxide Sigma-Aldrich, cat. № 06203, sodium 1-butanesulfonate Sigma-Aldrich, cat. № 19022-10G-F; ethanol 96%, methanol Sigma-Aldrich (purity 99.9%), formic acid Sigma-Aldrich, cat. № 33015. Validation characteristics were confirmed as specificity, correctness, precision. The total predicted uncertainty of the analytical method for quantitative determination and the limit of quantitative determination of the main impurity of paracetamol, at which the signal-to-noise ratio is fulfilled, is 10% of the initial concentration of the reference solution (0.5 μg / ml). Confirmed linearity for quantitative determination of paracetamol content in the range of 80 to 120% of the nominal value. Statistical processing of experimental data was carried out; the correlation coefficient of the linear dependence (r) between the entered and found values for the quantitative determination of paracetamol is > 0.990, which indicates the correctness of the method. Methods for the quantitative determination of paracetamol and its main impurity in suppositories have been developed and validated. The method for quantitative determination of paracetamol in suppositories is significantly simpler for routine control of a batch of drugs and is cost-effective compared to the HPLC method. The obtained experimental results indicate that according to the studied validation characteristics, the technique is correct and can be reproduced in other laboratories.

scholarly journals A Simple and Cost-Effective TLC-Densitometric Method for the Quantitative Determination of Acetylsalicylic Acid and Ascorbic Acid in Combined Effervescent Tablets

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3115 ◽  
Author(s):  
Alina Pyka-Pająk ◽  
Małgorzata Dołowy ◽  
Wioletta Parys ◽  
Katarzyna Bober ◽  
Grażyna Janikowska
Keyword(s):  
Ascorbic Acid ◽  
Quantitative Determination ◽  
Acetylsalicylic Acid ◽  
High Performance ◽  
Limit Of Detection ◽  
Volume Ratio ◽  
Cost Effective ◽  
Pharmaceutical Formulations ◽  
Limit Of Quantification

A new, simple, and cost-effective TLC-densitometric method has been established for the simultaneous quantitative determination of acetylsalicylic acid and ascorbic acid in combined effervescent tablets. Separation was performed on aluminum silica gel 60F254 plates using chloroform-ethanol-glacial acid at a volume ratio of 5:4:0.03 as the mobile phase. UV densitometry was performed in absorbance mode at 200 nm and 268 nm for acetylsalicylic acid and ascorbic acid, respectively. The presented method was validated as per ICH guidelines by specificity, linearity, accuracy, precision, limit of detection, limit of quantification, and robustness. Method validations indicate a good sensitivity with a low value of LOD and LOQ of both examined active substances. The linearity range was found to be 1.50–9.00 μg/spot and 1.50–13.50 μg/spot for acetylsalicylic and ascorbic acid, respectively. A coefficient of variation that was less than 3% confirms the satisfactory accuracy and precision of the proposed method. The results of the assay of combined tablet formulation equal 97.1% and 101.6% in relation to the label claim that acetylsalicylic acid and ascorbic acid fulfill pharmacopoeial requirements. The developed TLC-densitometric method can be suitable for the routine simultaneous analysis of acetylsalicylic acid and ascorbic acid in combined pharmaceutical formulations. The proposed TLC-densitometry may be an alternative method to the modern high-performance liquid chromatography in the quality control of above-mentioned substances, and it can be applied when HPLC or GC is not affordable in the laboratory.

scholarly journals Differences in the Diversity and Structure of the Gut Microbiome in Different Life Stages of the American Cockroach (Periplaneta americana)

2020 ◽  
Author(s):  
Zhiyu Chen ◽  
Bin Yang ◽  
Peiyu Ou ◽  
Xiaobao Jin
Keyword(s):  
Gut Microbiome ◽  
Metabolic Pathways ◽  
Periplaneta Americana ◽  
Life Stage ◽  
American Cockroach ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Functional Genes ◽  
Life Stages ◽  
Relative Abundances

Abstract Gut microbes play critical roles in host nutrition, physiology, and behavior. Periplaneta americana is a famous urban pest which is widely distributed in the tropics and subtropics, but very few information is available on the gut microbiome of Periplaneta americana, particularly in its different life stages. Here, we characterized the diversity and structure of gut microbiome in eggs, nymph and adult life stages of Periplaneta americana using high-throughput 16S rRNA genes sequencing. Both the results of Alpha- and Beta-diversity analysis showed the diversity and structure of gut microbiome were significant different among the eggs, nymph and adult stages. The result of species distribution showed the predominant phyla in three life stages were Bacteroidetes , Firmicutes and Proteobacteria , but the relative abundances of these bacteria were significant different among each life stage. 1,169 operational taxonomic units were shared by three stages, which indicating the gut microbiome may be inherited to offspring from parents of Periplaneta americana. According to the prediction of functional genes in metabolic pathways, most of them were distributed in the metabolic pathways of basic physiology such as nutrition, growth, development and immunity, etc. The relative abundances of functional genes in metabolic pathways were significant different among life stages of Periplaneta americana, indicating the gut microbiome might play an important role in the physiology across its different life stages. This study revealed the diversity and structure of gut microbiome in different life stages of Periplaneta americana, which may contribute to us to understand it’s physiology and behaviors.

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