scholarly journals Molecular tools for phytoplankton monitoring samples

2018 ◽  
Author(s):  
Bárbara Frazão ◽  
Alexandra Silva
Keyword(s):  
Dna Extraction ◽  
Dna Polymerases ◽  
Life Sciences ◽  
Dna Amplification ◽  
Cell Number ◽  
Molecular Tools ◽  
Direct Pcr ◽  
Tissue Samples ◽  
Thermofisher Scientific ◽  
Phytoplankton Monitoring

AbstractHABs can have severe impacts in fisheries or human health by the consumption of contaminated bivalves. Monitoring assessment (quantitative and qualitative identification) of these organisms, is routinely accomplished by microscopic identification and counting of these organisms. Nonetheless, molecular biology techniques are gaining relevance, once these approaches can easily identify phytoplankton organisms at species level and even cell number quantifications. This work tests 12 methods/kits for genomic DNA extraction and seven DNA polymerases to determine which is the best method for routinely use in a common molecular laboratory, for phytoplankton monitoring samples analyses. From our work, Direct PCR master mix for tissue samples, proved to be the most adequate by its velocity of processivity, practicability, reproducibility, sensitiveness and robustness. However, brands such as Omega Biotek, GRISP, Qiagen and MP Biomedicals also showed good results for conventional DNA extraction as well as all the Taq brands tested (GRISP, GE Healthcare Life Sciences, ThermoFisher Scientific and Promega). Lugol’s solution, with our tested kits did not show negative interference in DNA amplification. The same can be said about mechanical digestion, with no significant differences among kits with or without this homogenization step.

scholarly journals DNA recovery from microhymenoptera using six non-destructive methodologies with considerations for subsequent preparation of museum slides

Genome ◽  
2017 ◽  
Vol 60 (1) ◽  
pp. 85-91 ◽  
Author(s):  
Adriana J. Guzmán-Larralde ◽  
Alba P. Suaste-Dzul ◽  
Adrien Gallou ◽  
Kenzy I. Peña-Carrillo
Keyword(s):  
Dna Extraction ◽  
Mitochondrial Gene ◽  
Dna Amplification ◽  
Morphological Identification ◽  
Direct Pcr ◽  
Dna Recovery ◽  
Require Time ◽  
Gene Coi ◽  
Permanent Slide ◽  
Non Destructive

Because of the tiny size of microhymenoptera, successful morphological identification typically requires specific mounting protocols that require time, skills, and experience. Molecular taxonomic identification is an alternative, but many DNA extraction protocols call for maceration of the whole specimen, which is not compatible with preserving museum vouchers. Thus, non-destructive DNA isolation methods are attractive alternatives for obtaining DNA without damaging sample individuals. However, their performance needs to be assessed in microhymenopterans. We evaluated six non-destructive methods: (A) DNeasy® Blood & Tissue Kit; (B) DNeasy® Blood & Tissue Kit, modified; (C) Protocol with CaCl2 buffer; (D) Protocol with CaCl2 buffer, modified; (E) HotSHOT; and (F) Direct PCR. The performance of each DNA extraction method was tested across several microhymenopteran species by attempting to amplify the mitochondrial gene COI from insect specimens of varying ages: 1 day, 4 months, 3 years, 12 years, and 23 years. Methods B and D allowed COI amplification in all insects, while methods A, C, and E were successful in DNA amplification from insects up to 12 years old. Method F, the fastest, was useful in insects up to 4 months old. Finally, we adapted permanent slide preparation in Canada balsam for every technique. The results reported allow for combining morphological and molecular methodologies for taxonomic studies.

Extraction of DNA from a Wide Range of Objects by Treatment with Ammonium Salts

2020 ◽  
Vol 36 (6) ◽  
pp. 98-106
Author(s):  
E.I. Levitin ◽  
B.V. Sviridov ◽  
O.V. Piksasova ◽  
T.E. Shustikova
Keyword(s):  
Dna Extraction ◽  
Dna Isolation ◽  
Tissue Samples ◽  
New Approach ◽  
Cell Wall Disruption ◽  
Wide Range ◽  
Low Salt ◽  
Mammalian Blood ◽  
Plasmid Extraction ◽  
Two Hybrid

Currently, simple, rapid, and efficient techniques for DNA isolation from a wide range of organisms are in demand in biotechnology and bioinformatics. A key (and often limiting) step is the cell wall disruption and subsequent DNA extraction from the disintegrated cells. We have developed a new approach to DNA isolation from organisms with robust cell walls. The protocol includes the following steps: treatment of cells or tissue samples with ammonium acetate followed by cell lysis in low-salt buffer with the addition of SDS. Further DNA extraction is carried out according to standard methods. This approach is efficient for high-molecular native DNA isolation from bacteria, ascomycetes, yeast, and mammalian blood; it is also useful for express analysis of environmental microbial isolates and for plasmid extraction for two-hybrid library screening. express method for DNA isolation; ammonium salt treatment (в русских ключевых такой порядок), osmotic breakage of cells This study was financially supported by the NRC "Kurchatov Institute"-GOSNIIGENETIKA Kurchatov Genomic Center.

scholarly journals Direct PCR from whole blood, without DNA extraction

1990 ◽  
Vol 18 (19) ◽  
pp. 5908-5909 ◽  
Author(s):  
B. Mercier ◽  
C. Gaucher ◽  
O. Feugeas ◽  
C. Mazurier
Keyword(s):  
Dna Extraction ◽  
Whole Blood ◽  
Direct Pcr

scholarly journals Detection of Bacteria in Environmental Samples by Direct PCR Without DNA Extraction

BioTechniques ◽  
2001 ◽  
Vol 31 (3) ◽  
pp. 598-607 ◽  
Author(s):  
Kimberly A. Fode-Vaughan ◽  
Charles F. Wimpee ◽  
Charles C. Remsen ◽  
Mary Lynne Perille Collins
Keyword(s):  
Dna Extraction ◽  
Environmental Samples ◽  
Direct Pcr

scholarly journals Cell-Tak coating may cause mis-normalization of Seahorse metabolic flux data

2021 ◽  
Author(s):  
Michal Šíma ◽  
Stanislava Martínková ◽  
Anežka Kafková ◽  
Jan Pala ◽  
Jan Trnka
Keyword(s):  
Metabolic Flux ◽  
Cell Number ◽  
Cell Counting ◽  
Fluorescent Detection ◽  
Data Normalization ◽  
Tissue Samples ◽  
Flux Data ◽  
Research Areas ◽  
Healthy State ◽  
A Cell

Metabolic flux investigations of cells and tissue samples are a rapidly advancing tool in diverse research areas. Reliable methods of data normalization are crucial for an adequate interpretation of results and to avoid a misinterpretation of experiments and incorrect conclusions. The most common methods for metabolic flux data normalization are to cell number, DNA and protein. Data normalization may be affected by a variety of factors, such as density, healthy state, adherence efficiency, or proportional seeding of cells. The mussel-derived adhesive Cell-Tak is often used to immobilize poorly adherent cells. Here we demonstrate that this coating may strongly affect the fluorescent detection of DNA leading to an incorrect and highly variable normalization of metabolic flux data. Protein assays are much less affected and cell counting can virtually completely remove the effect of the coating. Cell-Tak coating also affects cell shape in a cell line-specific manner and may change cellular metabolism. Based on these observations we recommend cell counting as a gold standard normalization method for Seahorse metabolic flux measurements with protein content as a reasonable alternative.

scholarly journals First report on the molecular detection, phylogeny, virological and pathological investigations of Avibacterium paragallinarum in chickens in Turkey

2020 ◽  
Vol 76 (03) ◽  
pp. 6378-2020
Author(s):  
SAJID UMAR ◽  
HASAN ONGOR ◽  
ERHAN BAYRAKTAR ◽  
HAZAL OZTURK GURGEN ◽  
BELGI DIREN SIGIRCI ◽  
...  
Keyword(s):  
Viral Infections ◽  
Control Strategies ◽  
Inflammatory Cells ◽  
The United States ◽  
Molecular Characteristics ◽  
Nasal Discharge ◽  
Direct Pcr ◽  
Tissue Samples ◽  
Avibacterium Paragallinarum ◽  
Species Specific

Avibacterium paragallinarum is an important pathogen affecting the respiratory tract of chickens. There is a paucity of information on the molecular characteristics and pathology of A. paragallinarum in Turkish poultry flocks. In the present study, broiler and layer flocks (n = 2) suspected of viral infections with serious respiratory signs and significant mortality were visited. Chickens showed various disease signs and necropsy lesions, including purulent nasal discharge, respiratory distress, facial edema, sticky eyes, mucoid tracheitis, hemorrhagic inflammation of the infraorbital sinuses along with fibrinous mass and conjunctivitis. Histopathological lesions included loss of cilia along with necrosis and exfoliation of the superficial mucosal epithelium of the trachea, facial cellulitis, dermatitis, fibrinous plasmatic edema and infiltration of inflammatory cells, especially heterophils. A. paragallinarum was detected in tissue samples by species-specific polymerase chain reaction (PCR). The sequencing and phylogenetic analysis of the core region of the hemagglutinin (HA) gene revealed that Turkish strains detected here belonged to serotype A (serovar A1). They were related to strains reported from India (VRDC), the United States (0083), and Japan (0221), which are representatives of serovar A1. A homology of 88-90% was found between Indian strains and the Turkish strains detected in this study. Surprisingly, only vaccine strains of infectious bronchitis virus (IBV) were detected as a co-infecting agent in all samples positive for A. paragallinarum. Our findings suggest that A. paragallinarum may be an emerging pathogen in Turkish poultry flocks, and direct PCR may facilitate rapid diagnosis of infectious coryza. These results will also help to develop control strategies for A. paragallinarum.

scholarly journals Hairpin structure facilitates high-fidelity DNA amplification reactions in both qPCR and high-throughput sequencing

2021 ◽  
Author(s):  
Kerou Zhang ◽  
Alessandro Pinto ◽  
Peng Dai ◽  
Michael Wang ◽  
Lauren Yuxuan Cheng ◽  
...  
Keyword(s):  
Dna Sequences ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Dna Polymerases ◽  
Dna Amplification ◽  
Cost Effective ◽  
High Fidelity ◽  
Resistance Mutations ◽  
Chain Reactions ◽  
Early Cancer Diagnosis

Effective polymerase chain reactions (PCR) are important in bio-laboratories. It is essential to detect rare DNA-sequence variants for early cancer diagnosis or for drug-resistance mutations identification. Some of the common detection quantitative PCR (qPCR) methods are restricted in the limit of detection (LoD) because of the high polymerase misincorporation rate in Taq DNA polymerases. High-fidelity (HiFi) DNA polymerases have a 50- to 250-fold higher fidelity. Yet, there are currently no proper designs for multiplexed HiFi qPCR reactions. Moreover, the popularity of targeting highly multiplex DNA sequences requires minimizing PCR side products, as the potential of dimerization grows quadratically as the plexes of primers increases. Efforts tried before were either an add-on step, or technology-specific, or requiring high-level computing skills. There lacks an easy-to-apply and cost-effective method for dimerization reduction. Here, we presented the Occlusion System, composed of a 5'-overhanged primer and a probe with a short-stem hairpin. We demonstrated that it allowed multiplexing high-fidelity qPCR reaction, it was also compatible with the current variant-enrichment method to improve the LoD by 10-fold. Further, we found that the Occlusion System reduced the dimerization up to 10-fold in highly multiplexed PCR. Thus, the Occlusion System satisfactorily improved both qPCR sensitivity and PCR efficiency.

Toxocara DNA amplification in serum and tissue samples in BALB/c mice

2021 ◽  
pp. 111429
Author(s):  
Gabriela Rodrigues e Fonseca ◽  
Gessica Baptista de Mello ◽  
Fabiana Martins de Paula ◽  
Fernanda Melo Malta ◽  
Ronaldo Cesar Borges Gryschek ◽  
...  
Keyword(s):  
Dna Amplification ◽  
Tissue Samples

scholarly journals Capacity of Nine Thermostable DNA Polymerases To Mediate DNA Amplification in the Presence of PCR-Inhibiting Samples

1998 ◽  
Vol 64 (10) ◽  
pp. 3748-3753 ◽  
Author(s):  
Waleed Abu Al-Soud ◽  
Peter Rådström
Keyword(s):  
Dna Polymerase ◽  
Biological Samples ◽  
Detection Method ◽  
Dna Polymerases ◽  
Dna Amplification ◽  
Nucleic Acid Amplification ◽  
Full Potential ◽  
Thermus Aquaticus ◽  
Pcr Inhibitors ◽  
Thermostable Dna Polymerase

ABSTRACT The PCR is an extremely powerful method for detecting microorganisms. However, its full potential as a rapid detection method is limited by the inhibition of the thermostable DNA polymerase fromThermus aquaticus by many components found in complex biological samples. In this study, we have compared the effects of known PCR-inhibiting samples on nine thermostable DNA polymerases. Samples of blood, cheese, feces, and meat, as well as various ions, were added to PCR mixtures containing various thermostable DNA polymerases. The nucleic acid amplification capacity of the nine polymerases, under buffer conditions recommended by the manufacturers, was evaluated by using a PCR-based detection method for Listeria monocytogenes in the presence of purified template DNA and different concentrations of PCR inhibitors. The AmpliTaqGold and the Taq DNA polymerases from Thermus aquaticus were totally inhibited in the presence of 0.004% (vol/vol) blood in the PCR mixture, while the HotTub, Pwo, rTth, andTfl DNA polymerases were able to amplify DNA in the presence of 20% (vol/vol) blood without reduced amplification sensitivity. The DNA polymerase from Thermotoga maritima(Ultma) was found to be the most susceptible to PCR inhibitors present in cheese, feces, and meat samples. When the inhibitory effect of K and Na ions was tested on the nine polymerases, HotTub from Thermus flavus and rTthfrom Thermus thermophilus were the most resistant. Thus, the PCR-inhibiting effect of various components in biological samples can, to some extent, be eliminated by the use of the appropriate thermostable DNA polymerase.

Clinical validation of qPCR Target Selector™ assays using highly specific switch-blockers for rare mutation detection

2020 ◽  
Vol 73 (10) ◽  
pp. 648-655
Author(s):  
Lyle Arnold ◽  
Vassilios Alexiadis ◽  
Tim watanaskul ◽  
Vahid Zarrabi ◽  
Jason Poole ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Dna Amplification ◽  
Resistance Mutations ◽  
Tissue Samples ◽  
Patients With Cancer ◽  
Non Invasive ◽  
Dna Mutations ◽  
Substantial Risk ◽  
Specific Amplification ◽  
Circulating Tumour Dna

AimsThe identification of actionable DNA mutations associated with a patient’s tumour is critical for devising a targeted, personalised cancer treatment strategy. However, these molecular analyses are typically performed using tissue obtained via biopsy, which involves substantial risk and is often not feasible. In addition, biopsied tissue does not always reflect tumour heterogeneity, and sequential biopsies to track disease progression (eg, emergence of drug resistance mutations) are not well tolerated. To overcome these and other biopsy-associated limitations, we have developed non-invasive ‘liquid biopsy’ technologies to enable the molecular characterisation of a patient’s cancer using peripheral blood samples.MethodsThe Target Selector ctDNA platform uses a real-time PCR-based approach, coupled with DNA sequencing, to identify cancer-associated genetic mutations within circulating tumour DNA. This is accomplished via a patented blocking approach suppressing wild-type DNA amplification, while allowing specific amplification of mutant alleles.ResultsTo promote the clinical uptake of liquid biopsy technologies, it is first critical to demonstrate concordance between results obtained via liquid and traditional biopsy procedures. Here, we focused on three genes frequently mutated in cancer: EGFR (Del19, L858, and T790), BRAF (V600) and KRAS (G12/G13). For each Target Selector assay, we demonstrated extremely high accuracy, sensitivity and specificity compared with results obtained from tissue biopsies. Overall, we found between 93% and 96% concordance to blinded tissue samples across 127 clinical assays.ConclusionsThe switch-blocker technology reported here offers a highly effective method for non-invasively determining the molecular signatures of patients with cancer.

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