scholarly journals Rapid Filtration Separation-Based Sample Preparation Method for Bacillus Spores in Powdery and Environmental Matrices

2011 ◽  
Vol 78 (5) ◽  
pp. 1505-1512 ◽  
Author(s):  
Sandra Isabel ◽  
Maurice Boissinot ◽  
Isabelle Charlebois ◽  
Chantal M. Fauvel ◽  
Lu-E Shi ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dna Extraction ◽  
Environmental Samples ◽  
Extraction Procedure ◽  
Pcr Detection ◽  
Filter Method ◽  
Nucleic Acid Detection ◽  
Quality Level ◽  
Content Type ◽  
Bacillus Spores

ABSTRACTAuthorities frequently need to analyze suspicious powders and other samples for biothreat agents in order to assess environmental safety. Numerous nucleic acid detection technologies have been developed to detect and identify biowarfare agents in a timely fashion. The extraction of microbial nucleic acids from a wide variety of powdery and environmental samples to obtain a quality level adequate for these technologies still remains a technical challenge. We aimed to develop a rapid and versatile method of separating bacteria from these samples and then extracting their microbial DNA.Bacillus atrophaeussubsp.globigiiwas used as a simulant ofBacillus anthracis. We studied the effects of a broad variety of powdery and environmental samples on PCR detection and the steps required to alleviate their interference. With a benchmark DNA extraction procedure, 17 of the 23 samples investigated interfered with bacterial lysis and/or PCR-based detection. Therefore, we developed thedual-filter method forappliedrecovery of microbial particles fromenvironmental and powdery samples (DARE). The DARE procedure allows the separation of bacteria from contaminating matrices that interfere with PCR detection. This procedure required only 2 min, while the DNA extraction process lasted 7 min, for a total of <10 min. This sample preparation procedure allowed the recovery of cleaned bacterial spores and relieved detection interference caused by a wide variety of samples. Our procedure was easily completed in a laboratory facility and is amenable to field application and automation.

scholarly journals Taxonomic identification of airborne pollen from complex environmental samples by DNA metabarcoding: a methodological study for optimizing protocols

2017 ◽  
Author(s):  
Kleopatra Leontidou ◽  
Cristiano Vernesi ◽  
Johannes De Groeve ◽  
Fabiana Cristofolini ◽  
Despoina Vokou ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Chloroplast Dna ◽  
Dna Extraction ◽  
Environmental Samples ◽  
Airborne Pollen ◽  
Reference Database ◽  
Taxonomic Assignment ◽  
Spore Trap ◽  
Custom Made ◽  
Dna Metabarcoding

AbstractMetabarcoding is a promising DNA-based method for identifying airborne pollen from environmental samples with advantages over microscopic methods. This method requires several preparatory steps of the samples, with the extraction protocol being of fundamental importance to obtain an optimal DNA yield. Currently, there is no consensus in sample preparation and DNA extraction, especially for gravimetric pollen samplers. Therefore, the aim of this study was to develop protocols to process environmental samples for pollen DNA extraction and further metabarcoding analysis, and to assess the efficacy of these protocols for the taxonomic assignment of airborne pollen, collected by gravimetric (Tauber trap) and volumetric samplers (Burkard spore trap). Protocols were tested across an increasing complexity of samples, from single-species pure pollen to environmental samples. A short fragment (about 150 base pair) of chloroplast DNA was amplified by universal primers for plants (trnL). After PCR amplification, amplicons were Sanger-sequenced and taxonomic assignment was accomplished by comparison to a custom-made reference database including chloroplast DNA sequences of 46 plant families, including most of the anemophilous taxa occurring in the study area (Trentino, Italy, Eastern Italian Alps). Using as a benchmark the classical morphological pollen analysis, it emerged that DNA metabarcoding is applicable efficiently across a complexity of samples, provided that sample preparation, DNA extraction and amplification protocols are specifically optimized.

scholarly journals Comparison of commercial DNA kits and traditional DNA extraction procedure in PCR detection of pork in dry/fermented sausages

Poljoprivreda ◽  
2015 ◽  
Vol 21 (1 Supplement) ◽  
pp. 199-202 ◽  
Author(s):  
Ivona Djurkin Kusec ◽  
◽  
Zarko Radisic ◽  
Miodrag Komlenic ◽  
Goran Kusec
Keyword(s):  
Dna Extraction ◽  
Extraction Procedure ◽  
Pcr Detection ◽  
Fermented Sausages ◽  
Dry Fermented Sausages

Pollutant Identification by Selected Area Electron Diffraction

1974 ◽  
Vol 32 ◽  
pp. 532-533
Author(s):  
R. E. Ferrell ◽  
G. G. Paulson ◽  
C. W. Walker
Keyword(s):  
Thermal Treatment ◽  
Electron Diffraction ◽  
Sample Preparation ◽  
Crystal Structures ◽  
Water Samples ◽  
Environmental Samples ◽  
Short Review ◽  
Selected Area Electron Diffraction ◽  
Multiple Component

Selected area electron diffraction (SAD) has been used successfully to determine crystal structures, identify traces of minerals in rocks, and characterize the phases formed during thermal treatment of micron-sized particles. There is an increased interest in the method because it has the potential capability of identifying micron-sized pollutants in air and water samples. This paper is a short review of the theory behind SAD and a discussion of the sample preparation employed for the analysis of multiple component environmental samples.

Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

2017 ◽  
Vol 6 (04) ◽  
pp. 5347 ◽  
Author(s):  
Omar B. Ahmed* ◽  
Anas S. Dablool
Keyword(s):  
Dna Extraction ◽  
Control Method ◽  
Extraction Procedure ◽  
Cost Effective ◽  
High Yield ◽  
Gram Negative Bacteria ◽  
Bacterial Dna ◽  
Gram Negative ◽  
E Coli

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

scholarly journals Amplicon-sequencing of raw milk microbiota: impact of DNA extraction and library-PCR

2021 ◽  
Author(s):  
Annemarie Siebert ◽  
Katharina Hofmann ◽  
Lena Staib ◽  
Etienne V. Doll ◽  
Siegfried Scherer ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dna Extraction ◽  
Raw Milk ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Microbiome Analysis ◽  
Eukaryotic Dna ◽  
Selective Lysis ◽  
The Impact

Abstract The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy® PowerFood® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers. Key points • Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota. • Reduction of eukaryotic DNA by enzymatic digestion. • Shift of detected microbiome caused by high cycle numbers in library-PCR.

Study of a discrete grey forecasting model based on the quality cost characteristic curve

2017 ◽  
Vol 7 (3) ◽  
pp. 376-384 ◽  
Author(s):  
Wenjie Dong ◽  
Sifeng Liu ◽  
Zhigeng Fang ◽  
Xiaoyu Yang ◽  
Qian Hu ◽  
...  
Keyword(s):  
Prediction Accuracy ◽  
Characteristic Curve ◽  
Grey System Theory ◽  
Forecasting Model ◽  
Quality Level ◽  
Cost Models ◽  
Quality Cost ◽  
Content Type ◽  
Grey Forecasting ◽  
Grey Forecasting Model

Purpose The purpose of this paper is to clarify several commonly used quality cost models based on Juran’s characteristic curve. Through mathematical deduction, the lowest point of quality cost and the lowest level of quality level (often depicted by qualification rate) can be obtained. This paper also aims to introduce a new prediction model, namely discrete grey model (DGM), to forecast the changing trend of quality cost. Design/methodology/approach This paper comes to the conclusion by means of mathematical deduction. To make it more clear, the authors get the lowest quality level and the lowest quality cost by taking the derivative of the equation of quality cost and quality level. By introducing the weakening buffer operator, the authors can significantly improve the prediction accuracy of DGM. Findings This paper demonstrates that DGM can be used to forecast quality cost based on Juran’s cost characteristic curve, especially when the authors do not have much information or the sample capacity is rather small. When operated by practical weakening buffer operator, the randomness of time series can be obviously weakened and the prediction accuracy can be significantly improved. Practical implications This paper uses a real case from a literature to verify the validity of discrete grey forecasting model, getting the conclusion that there is a certain degree of feasibility and rationality of DGM to forecast the variation tendency of quality cost. Originality/value This paper perfects the theory of quality cost based on Juran’s characteristic curve and expands the scope of application of grey system theory.

scholarly journals Rapidec Carba NP Test for Rapid Detection of Carbapenemase Producers

2015 ◽  
Vol 53 (9) ◽  
pp. 3003-3008 ◽  
Author(s):  
Laurent Poirel ◽  
Patrice Nordmann
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sample Preparation ◽  
Acinetobacter Baumannii ◽  
Sensitivity And Specificity ◽  
Rapid Detection ◽  
Content Type

Performances of the Rapidec Carba NP test (bioMérieux) were evaluated for detection of all types of carbapenemases inEnterobacteriaceae,Acinetobacter baumannii, andPseudomonas aeruginosa. In less than 2 h after sample preparation, it showed a sensitivity and specificity of 96%. This ready-to-use test is well adapted to the daily need for detection of carbapenemase producers in any laboratory worldwide.

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