scholarly journals Quantification of Toxic Cyanobacteria in Water by Use of Competitive PCR Followed by Sequence-Specific Labeling of Oligonucleotide Probes

1998 ◽  
Vol 64 (7) ◽  
pp. 2639-2643 ◽  
Author(s):  
Knut Rudi ◽  
Olav M. Skulberg ◽  
Frank Larsen ◽  
Kjetill S. Jakobsen
Keyword(s):  
Sample Preparation ◽  
Solid Phase ◽  
Bacterial Species ◽  
Simultaneous Detection ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Oligonucleotide Probes ◽  
Competitive Pcr ◽  
Wide Range ◽  
Preparation Strategy

ABSTRACT A complete nucleic-acid-based assay which consists of sample preparation, DNA amplification, and chromogenic detection was developed for quantifying potential toxin-producing cyanobacteria of interest to the public. The sample preparation strategy involves the same solid phase for cell concentration and DNA purification. For the detection step, we used a combination of competitive PCR amplification, sequence-specific labeling of oligonucleotide probes, hybridization of the labeled oligonucleotides to immobilized complements and, finally, chromogenic detection. The complete assay was tested with water containing toxin-producing cyanobacteria belonging to the genusMicrocystis. A detection limit of 100 cells/ml and a quantitative range of more than 3 orders of magnitude were obtained. This approach can easily be adapted to a wide range of bacterial species and has the potential for simultaneous detection and quantitation of several different target organisms by a single assay.

scholarly journals Modern Methods of Sample Preparation for the Analysis of Oxylipins in Biological Samples

Molecules ◽  
2019 ◽  
Vol 24 (8) ◽  
pp. 1639 ◽  
Author(s):  
Liakh ◽  
Pakiet ◽  
Sledzinski ◽  
Mika
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Biological Samples ◽  
Solid Phase ◽  
Biological Tissues ◽  
Gas Chromatography Mass Spectrometry ◽  
Liquid Liquid Extraction ◽  
Wide Range ◽  
Popular Subject ◽  
Technological Platforms

Oxylipins are potent lipid mediators derived from polyunsaturated fatty acids, which play important roles in various biological processes. Being important regulators and/or markers of a wide range of normal and pathological processes, oxylipins are becoming a popular subject of research; however, the low stability and often very low concentration of oxylipins in samples are a significant challenge for authors and continuous improvement is required in both the extraction and analysis techniques. In recent years, the study of oxylipins has been directly related to the development of new technological platforms based on mass spectrometry (LC–MS/MS and gas chromatography–mass spectrometry (GC–MS)/MS), as well as the improvement in methods for the extraction of oxylipins from biological samples. In this review, we systematize and compare information on sample preparation procedures, including solid-phase extraction, liquid–liquid extraction from different biological tissues.

Analytical Methodology for the Determination of Organochlorine Pesticides in Vegetation

2012 ◽  
Vol 95 (5) ◽  
pp. 1291-1310 ◽  
Author(s):  
Elisa Beceiro-González ◽  
María J González-Castro ◽  
Soledad Muniategui-Lorenzo ◽  
Purificación López-Mahía ◽  
Darío Prada-Rodríguez
Keyword(s):  
Sample Preparation ◽  
Solid Phase Extraction ◽  
Organochlorine Pesticides ◽  
Pesticide Residues ◽  
Solid Phase ◽  
Liquid Extraction ◽  
Stir Bar Sorptive Extraction ◽  
Phase Extraction ◽  
Chromatographic Techniques ◽  
Wide Range

Abstract Due to the extensive use of organochlorine pesticides (OCPs) for agricultural purposes and their high persistence and low biodegradability, they have become an important group of contaminants. Detection and quantification of pesticide residues in food, particularly fruits and vegetables, is of growing concern for producers, consumers, and governments. The most widely used pretreatment for the extraction of pesticides in plants is based on solvent extraction liquid-solid extraction (LSE). LSE can be carried out using Soxhlet, shake-flask, homogenization, sonication, and, more recently, microwave-assisted extraction, pressurized liquid extraction, and supercritical fluid extraction. Furthermore, new analytical procedures using the extraction with sorbents, such as solid-phase micro-extraction, stir bar sorptive extraction, and matrix solid-phase dispersion, have also been used. On the other hand, a wide range of cleanup methods (liquid–liquid extraction, solid-phase extraction, gel permeation chromatography, and dispersive solid-phase extraction; and chromatographic techniques with electron capture detector and mass spectrometry detector; and HPLC with a ultraviolet detector are reported in the literature. This article reviews the applicability, advantages, and disadvantages of various sample preparation techniques (traditional and new techniques) for the analysis of OCPs in different plants and plant materials. It covers more than 15 years of published methods in which pesticide residues have been determined in a wide range of vegetation samples (fruits, horticultural samples, medicinal plants, tree leaves, etc.) by the use of chromatographic techniques after various sample preparation steps. A great number of applications in different plant material are provided. To the best of the authors' knowledge, previously published reviews have not covered as wide and exhaustive range of vegetation matrixes as presented here. A summary of pesticide levels cited in the literature is included.

Group-specific differentiation of Rhizobium from clover species by PCR amplification of 23S rDNA sequences

1998 ◽  
Vol 44 (11) ◽  
pp. 1102-1105
Author(s):  
Mesfin Tesfaye ◽  
F Brian Holl
Keyword(s):  
Root Nodule ◽  
Bacterial Species ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Specific Detection ◽  
Rdna Sequences ◽  
Rhizobium Spp ◽  
Polymerase Chain ◽  
Inoculation Group ◽  
Identification Tool

Two 20-bp primers that provide group-specific detection of Rhizobium spp. by polymerase chain reaction (PCR) have been used to differentiate strains that belong to different effectiveness groups within the Rhizobium-Trifolium cross-inoculation group. The target for DNA amplification was a 370-bp fragment of the 23S rDNA region. Analysis of additional root-nodule forming, as well as root-associated bacterial species by PCR-primer assay revealed that variability within this 20-bp segment of the 23S rDNA region may be widespread and provide an effective identification tool. Our data suggest that strains of Rhizobium isolated from the perennial clover Trifolium semipilosum may be phylogenetically more closely related to Rhizobium etli.Key words: Rhizobium, Trifolium, detection, PCR, rDNA.

scholarly journals SP3 Protocol for Proteomic Plant Sample Preparation Prior LC-MS/MS

2021 ◽  
Vol 12 ◽  
Author(s):  
Kamil Mikulášek ◽  
Hana Konečná ◽  
David Potěšil ◽  
Renata Holánková ◽  
Jan Havliš ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Magnetic Particles ◽  
Protein Extraction ◽  
Solid Phase ◽  
Handling Time ◽  
Plant Sample ◽  
Preparation Methods ◽  
Wide Range ◽  
Contaminants Removal ◽  
Paramagnetic Beads

Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the filter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal and protein digestion from Arabidopsis thaliana (AT) lysate. Finally, the optimized carboxylated SP3 workflow was benchmarked closely against the routine FASP. Ultimately, LC-MS/MS analyses revealed that regarding the number of identifications, number of missed cleavages, proteome coverage, repeatability, reduction of handling time, and cost per assay, the SP3 on carboxylated magnetic particles proved to be the best alternative for SDS and other contaminants removal from plant sample lysate. A robust and efficient 2-h SP3 protocol for a wide range of protein input is presented, benefiting from no need to adjust the amount of beads, binding and rinsing conditions, or digestion parameters.

scholarly journals Simultaneous detection of multiple fly-borne bacterial pathogenic microorganisms by the reverse line blot hybridization assay

2020 ◽  
Author(s):  
Yonghua Ma ◽  
Huitian Gou ◽  
Xiaolin Sun ◽  
Zexiang Wang ◽  
Mei Li ◽  
...  
Keyword(s):  
Detection Efficiency ◽  
Bacterial Species ◽  
Blot Hybridization ◽  
Simultaneous Detection ◽  
Pathogenic Microorganisms ◽  
Bacterial Strains ◽  
Bacterial Diseases ◽  
Wide Range ◽  
Species Specific ◽  
And Control

Abstract Background: As a widespread health pest, flies can carry more than 100 kinds of pathogenic microbes to threat human health, resulting in a wide range of disease infection and transmission. The aim of this study was to develop a sensitive, reliable and rapid method for the simultaneous detection of multiple fly-borne bacterial pathogenic microorganisms, in order to effectively prevent and control fly-borne bacterial diseases.Results: PCR-RLB method could directly and accurately detect fly-borne bacteria species corresponding of 7 species-specific probes. At the same time, the membrane binding oligonucleotide species-specific probes prepared in RLB detection technology can be reused for detection of bacteria after washing with 0.5 M EDTA, which greatly improves the detection efficiency. In 106 groups of samples, the numbers of samples carrying seven different bacterial strains were 2 (S. aureus), 52 (S. flexneri), 0 (A. caviae), 3% (V. vulnificus), 56 (S. enterica), 1 (P. vulgaris) and 33 (Y. enterocolitica), respectively. Their proportions of 7 bacterial strains carried by houseflies were 1.23% (S. aureus), 32.1% (S. flexneri), 0% (A. caviae), 1.85% (V. vulnificus), 34.57% (S. enterica), 0.62% (P. vulgaris) and 20.37% (Y. enterocolitica), respectively. It was found that the worse the hygienic condition, the higher the bacteria carrying rate of houseflies was. S. enterica, S. flexneri and Y. enterocolitica accounted for the overwhelming majority of the seven pathogenic strains carried by houseflies from four different environments in Lanzhou. This indicated that houseflies played an important role in the transmission of intestinal diseases, which was mainly related to the breeding and reproduction of houseflies in feces, carrion and food. S. aureus was carried by houseflies in the hospital area indicates that hospitals should do well in killing and controlling flies and further strengthen the prevention and control of fly-borne bacterial diseases. Conclusion: The RLB assay appeared to have potential clinical application in the simultaneous detection of fly-borne bacterial species.

scholarly journals Multivariate Assessment of Procedures for Molecularly Imprinted Polymer Synthesis for Pesticides Determination in Environmental and Agricultural Samples

Materials ◽  
2021 ◽  
Vol 14 (22) ◽  
pp. 7078
Author(s):  
Mariusz Marć ◽  
Marta Bystrzanowska ◽  
Katarzyna Pokajewicz ◽  
Marek Tobiszewski
Keyword(s):  
Sample Preparation ◽  
Solid Phase ◽  
Food Samples ◽  
Mechanical Resistance ◽  
Molecularly Imprinted ◽  
Innovative Strategies ◽  
Wide Range ◽  
Analytical Procedures ◽  
Pre Treatment ◽  
The Impact

In the case of quantitative and qualitative analysis of pesticides in environmental and food samples, it is required to perform a sample pre-treatment process. It allows to minimalize the impact of interferences on the final results, as well as increase the recovery rate. Nowadays, apart from routinely employed sample preparation techniques such as solid-phase extraction (SPE) or solid-phase microextraction (SPME), the application of molecularly imprinted polymers (MIPs) is gaining greater popularity. It is mainly related to their physicochemical properties, sorption capacity and selectivity, thermo-mechanical resistance, as well as a wide range of polymerization techniques allowing to obtain the desired type of sorption materials, adequate to a specific type of pesticide. This paper targets to summarize the most popular and innovative strategies since 2010, associated with the MIPs synthesis and analytical procedures for pesticides determination in environmental and food samples. Application of multi-criteria decision analysis (MCDA) allows for visualization of the most beneficial analytical procedures in case of changing the priority of each step of analysis (MIPs synthesis, sample preparation process—pesticides extraction, chromatographic analysis) bearing in mind metrological and environmental issues.

scholarly journals A review of recent developments and trends in the QuEChERS sample preparation approach

2015 ◽  
Vol 13 (1) ◽  
Author(s):  
Tomasz Rejczak ◽  
Tomasz Tuzimski
Keyword(s):  
Sample Preparation ◽  
Polybrominated Diphenyl Ethers ◽  
Flame Retardants ◽  
Fruits And Vegetables ◽  
Solid Phase ◽  
Salting Out ◽  
Pesticides Residues ◽  
Extraction Solvent ◽  
Wide Range ◽  
Recent Developments

AbstractA comprehensive review is presented on the recent developments and trends in the QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation approach. This technique involves liquid-liquid partitioning using acetonitrile and purifying the extract using dispersive solid-phase extraction (d-SPE). Originally, the QuEChERS was introduced for pesticides residues analysis in high moisture fruits and vegetables, but more recently it is gaining significant popularity in the analysis of broad spectrum of analytes in huge variety of samples. The wide range of the technique applications is possible due to introducing various modifications based on the use of different extraction solvent and salt formulation and buffer additions for salting-out partitioning step and the application of various d-SPE sorbents for clean-up step. Therefore, the QuEChERS approach is useful for analysis of, among others pesticides, veterinary drugs and other pharmaceuticals, mycotoxins, polycyclic aromatic hydrocarbons (PAHs), dyes, acrylamide, synthetic musks and UV filters, bisphenols, polybrominated diphenyl ethers and other flame retardants, endocrine disruptors, and other chemical compounds. Thanks to the QuEChERS approach, high-throughput multiresidue methods operate in a routine contaminant control of food products, feedstuff, and environmental samples.

scholarly journals The relationship between biofilm formation, genes of virulence and iron metabolism in Escherichia coli

2018 ◽  
Author(s):  
Lívia Handrová ◽  
Anna Čuvalová ◽  
Vladimír Kmeť
Keyword(s):  
Escherichia Coli ◽  
Iron Metabolism ◽  
Biofilm Formation ◽  
Bacterial Infections ◽  
Opportunistic Infections ◽  
Bacterial Species ◽  
Pcr Amplification ◽  
Bacterial Strains ◽  
E Coli ◽  
Wide Range

Escherichia coli is known as one of the bacterial species with the widest adaptability to variety of niches either within organisms or outside in environment. Most strains of E. coli are of low virulence and associated with opportunistic infections, whereas others are highly virulent. The success of E. coli in colonising such a wide range of hosts and environments is basically due to a noticeable ductility in exploiting the available resources. It is becoming increasingly clear that biofilms have an enormous impact on medicine because since 65% of animal and human bacterial infections involve biofilms. In present study, we isolated strains of E. coli from animals. 19 interesting isolates were selected and tested by PCR amplification to virulence – iutA, cvaC, iss, tsh, papC, kps, iha and iron metabolism genes – sitA, feoB, irp2, fyuA, iroN, ireA. The ability of biofilm formation was assessed in a quantitative assay using a microtiter-plate test. Bacterial strains were grown on BHI. We divided isolates of E. coli into four classes: very weak (63.0%), weak (10.5%), moderate (10.5%) and strong (16.0%) biofilm producers. Representation genes of virulence were highly in isolates from very weak biofilm producers – from 7 genes were 6 highly; only papC (P fimbrial adhesin) was low. Genes of iron metabolism were different. Genes – sitA, fyuA, ireA in strong isolates producing biofilm and feoB, irp2, iroN in weak producers were most represented. The results show possible relation between presence virulence factor and low biofilm formation.

Sign in / Sign up

Export Citation Format

Share Document