scholarly journals Endonuclease-based genotyping of the RBM, a first-line method for the surveillance of emergence or evolution of SARS-CoV-2 Variants.

2021 ◽  
Author(s):  
Eva Lopez ◽  
Margot Barthelemy ◽  
Cecile Baronti ◽  
Shirley Masse ◽  
Alessandra Falchi ◽  
...  
Keyword(s):  
Region Of Interest ◽  
Selective Advantage ◽  
Partial Identification ◽  
Functional Adaptation ◽  
Clinical Samples ◽  
Full Genome Sequencing ◽  
First Line ◽  
Set Up ◽  
Detection Of Mutations ◽  
Human Receptor

Since the beginning of the Covid-19 pandemics, variants have emerged. Whereas most of them have no to limited selective advantage, some display increased transmissibility and/or resistance to immune response. To date, most of the mutations involved in the functional adaptation are found in the Receptor Binding Module (RBM), close to the interface with the human receptor ACE2. In this study, we thus developed and validated a fast and simple molecular assay allowing the detection and partial identification of the mutations in the RBM coding sequence. After the amplification of the region of interest, the amplicon is heat-denatured and hybridized with an amplicon of reference. The presence of a mutation in the heteroduplex can be cleaved by a mismatch-specific endonuclease and the cleavage pattern is analysed by capillary electrophoresis. The approach was first validated on viral RNA purified different SARS-CoV-2 variants produced in the lab before being implemented for clinical samples. The results highlighted the performance of the assay for the detection of mutations in the RBM from clinical samples. The procedure can be easily set up for high throughput identification of the presence of mutations and serve as a first-line screening to select the samples for full genome sequencing.

scholarly journals Circulation and Molecular Epidemiology of Enteroviruses in Paralyzed, Immunodeficient and Healthy Individuals in Tunisia, a Country with a Polio-Free Status for Decades

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 380
Author(s):  
Anissa Chouikha ◽  
Dorra Rezig ◽  
Nadia Driss ◽  
Ichrak Abdelkhalek ◽  
Ahlem Ben Yahia ◽  
...  
Keyword(s):  
Detection Rate ◽  
Warning System ◽  
World Health ◽  
Clinical Samples ◽  
Nucleotide Sequencing ◽  
Comprehensive Picture ◽  
Set Up ◽  
Health Organization ◽  
Type 3

This report is an overview of enterovirus (EV) detection in Tunisian polio-suspected paralytic cases (acute flaccid paralysis (AFP) cases), healthy contacts and patients with primary immunodeficiencies (PID) during an 11-year period. A total of 2735 clinical samples were analyzed for EV isolation and type identification, according to the recommended protocols of the World Health Organization. Three poliovirus (PV) serotypes and 28 different nonpolio enteroviruses (NPEVs) were detected. The NPEV detection rate was 4.3%, 2.8% and 12.4% in AFP cases, healthy contacts and PID patients, respectively. The predominant species was EV-B, and the circulation of viruses from species EV-A was noted since 2011. All PVs detected were of Sabin origin. The PV detection rate was higher in PID patients compared to AFP cases and contacts (6.8%, 1.5% and 1.3% respectively). PV2 was not detected since 2015. Using nucleotide sequencing of the entire VP1 region, 61 strains were characterized as Sabin-like. Among them, six strains of types 1 and 3 PV were identified as pre-vaccine-derived polioviruses (VDPVs). Five type 2 PV, four strains belonging to type 1 PV and two strains belonging to type 3 PV, were classified as iVDPVs. The data presented provide a comprehensive picture of EVs circulating in Tunisia over an 11-year period, reveal changes in their epidemiology as compared to previous studies and highlight the need to set up a warning system to avoid unnoticed PVs.

scholarly journals Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling

2020 ◽  
Vol 21 (12) ◽  
pp. 4364
Author(s):  
Giuseppa De Luca ◽  
Barbara Cardinali ◽  
Lucia Del Mastro ◽  
Sonia Lastraioli ◽  
Franca Carli ◽  
...  
Keyword(s):  
Substantial Reduction ◽  
Breast Cancer Patients ◽  
Dna Templates ◽  
Molecular Tagging ◽  
Mutational Profiling ◽  
Optimized Protocol ◽  
Set Up ◽  
Detection Of Mutations ◽  
Almost All ◽  
Next Generation Sequencing Ngs

Molecular characterization of Circulating Tumor Cells (CTCs) is still challenging, despite attempts to minimize the drawbacks of Whole Genome Amplification (WGA). In this paper, we propose a Next-Generation Sequencing (NGS) optimized protocol based on molecular tagging technology, in order to detect CTCs mutations while skipping the WGA step. MDA-MB-231 and MCF-7 cell lines, as well as leukocytes, were sorted into pools (2–5 cells) using a DEPArray™ system and were employed to set up the overall NGS procedure. A substantial reduction of reagent volume for the preparation of libraries was performed, in order to fit the limited DNA templates directly derived from cell lysates. Known variants in TP53, KRAS, and PIK3CA genes were detected in almost all the cell line pools (35/37 pools, 94.6%). No additional alterations, other than those which were expected, were found in all tested pools and no mutations were detected in leukocytes. The translational value of the optimized NGS workflow is confirmed by sequencing CTCs pools isolated from eight breast cancer patients and through the successful detection of variants. In conclusion, this study shows that the proposed NGS molecular tagging approach is technically feasible and, compared to traditional NGS approaches, has the advantage of filtering out the artifacts generated during library amplification, allowing for the reliable detection of mutations and, thus, making it highly promising for clinical use.

Continuous Cultures of Plasmodium Falciparum Established in Tanzania From Patients With Acute Malaria

2021 ◽  
Author(s):  
Florence Humphrey Urio ◽  
Matilda Mkombachepa ◽  
Gration Rwegasira ◽  
Twilumba Makene ◽  
Billy Ngasala ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Continuous Culture ◽  
Drug Sensitivity ◽  
Culture Media ◽  
Experimental System ◽  
Dar Es Salaam ◽  
Clinical Samples ◽  
Host Cell Interactions ◽  
Set Up

Abstract BackgroundMalaria morbidity and mortality, almost entirely from Plasmodium falciparum, are still rampant in Africa: therefore, it is important to study the biology of the parasite and the parasite-host cell interactions. In vitro cultivation of Plasmodium falciparum is most useful for this purpose, as well as for investigating drug resistance and possible new therapies. Here we report that the Trager & Jensen continuous culture of P. falciparum can be established in a laboratory in Tanzania with minimal facilities and with modest expenditure.MethodsAn in vitro set-up of continuous culture of P. falciparum was carried out in 2016 to 2020 at Muhimbili university of health and allied sciences, Dar-es salaam. Parasite samples were obtained from patients with acute malaria, frozen parasites and live cultures. Data was collected and analyzed using GraphPad Prism version 8.ResultsWe have successfully achieved exponential growth of existing strains that are used worldwide, as well as of parasites in clinical samples from patients with acute malaria. In the aim to optimize growth we have compared human serum and bovine serum albumin as components of the culture media. In addition, culture synchronization has been achieved using sorbitol.ConclusionThis experimental system is now available to our institution and to researchers aiming at investigating drug sensitivity and mechanisms of protection against Plasmodium falciparum that accrue from various genes expressed in red cells.

scholarly journals Improving the performance of 241Am-Be for PGNAA applications using a proper shielding for neutron source and the NaI detector

2010 ◽  
Vol 25 (3) ◽  
pp. 205-211 ◽  
Author(s):  
Hamed Panjeh ◽  
Hashem Hakimabad ◽  
Lalle Motavalli
Keyword(s):  
Gamma Rays ◽  
Energy Region ◽  
Gamma Ray ◽  
Region Of Interest ◽  
High Rate ◽  
Prompt Gamma ◽  
Peak Resolution ◽  
Set Up ◽  
Gamma Ray Detector

The gamma ray spectrum resolution from a 241Am-Be source-based prompt gamma ray activation analysis set-up has been observed to increase in the energy region of interest with enclosing the NaI detector in a proper neutron and gamma ray shield. We have investigated the tact that the peak resolution of prompt gamma rays in the region of interest from the set-up depends on the source activity to the great extent, size and kind of the detector and the geometry of the detector shield. In order to see the role of a detector shield, five kinds of the detector shield were used and finally the proper kind was introduced. Since the detector shield has an important contribution in the reduction of the undesirable and high rate gamma rays coming to the gamma ray detector, a good design of a proper shield enables the elimination of the unwanted events, such as a pulse pile-up. By improving the shielding design, discrete and distinguishable photoelectric peaks in the energy region of interest have been observed in the spectrum of prompt gamma rays.

scholarly journals Molecular Characterization of Circulating DENV-2 During Outbreak in Northern Senegal, Rosso 2018

2021 ◽  
Author(s):  
Idrissa Dieng ◽  
Mignane Ndiaye ◽  
Marie Henriette Ndione ◽  
Safietou Sankhe ◽  
Moussa Moïse Diagne ◽  
...  
Keyword(s):  
Dengue Infection ◽  
West African ◽  
Full Genome Sequencing ◽  
African Countries ◽  
Qrt Pcr ◽  
The North ◽  
Virus Incidence ◽  
Sporadic Cases ◽  
Set Up ◽  
High Level

Globally 390 millions of people are at risk of dengue infection; over the past 50 years the virus incidence increased thirty-fold. In Senegal, an unprecedented occurrence of outbreaks and sporadic cases was noticed since 2017. In October 2018 an outbreak of DENV-2 was reported in Rosso area in the north of Senegal at the border with Mauritania. Out of the 187 blood specimen samples collected, 27 were positives by qRT-PCR and 8 were serologically positive for DENV IgM. Serotyping using qRT-PCR reveals that isolates were positive for DENV-2. A subset of DENV-2 positives samples was selected and subjected to full genome sequencing followed by phylogenetic analysis. Analysis of 06 nearly completed genome sequences (n= 6) revealed that isolates belong to the cosmopolitan genotype and are closely related to the Mauritanian strains detected between 2017 and 2018 and those detected in many West African countries such as Burkina Faso or Cote d’Ivoire. Our results suggest a transboundary circulation of the DENV-2 cosmopolitan genotype between Senegal and Mauritania and call for a need of coordinated surveillance of arboviruses between these two countries. Interestingly, high level of homology between West African isolates highlights endemicity and call for a set-up of sub-regional viral genomic surveillance which will lead to a better understanding of viral dynamic, transmission and spread across Africa.

scholarly journals Complete Genome Sequences of Three African Foot-and-Mouth Disease Viruses from Clinical Samples Isolated in 2009 and 2010

2016 ◽  
Vol 4 (3) ◽  
Author(s):  
Steven Van Borm ◽  
Toon Rosseel ◽  
Andy Haegeman ◽  
Mpolokang Elliot Fana ◽  
Latoa Seoke ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Complete Genome ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Clinical Samples ◽  
Full Genome ◽  
Full Genome Sequencing ◽  
Genome Sequences ◽  
Foot And Mouth

The complete genome sequences of three foot-and-mouth disease viruses (one virus of each serotype SAT1, SAT2 and O) were directly sequenced from RNA extracted from clinical bovine samples, demonstrating the feasibility of full-genome sequencing from strong positive samples taken from symptomatic animals.

scholarly journals An estimate of heterosis in Drosophila melanogaster

1971 ◽  
Vol 18 (1) ◽  
pp. 97-105 ◽  
Author(s):  
J. A. Sved
Keyword(s):  
Drosophila Melanogaster ◽  
High Frequency ◽  
Marker Chromosome ◽  
Selective Advantage ◽  
Wild Type ◽  
Set Up

SUMMARYTwenty-five population cages of D. melanogaster were set up, each containing a different wild-type second chromosome and the marker chromosome Cy. In all but one case where contamination apparently occurred, the Cy chromosome persisted in the population at high frequency, showing a selective advantage of Cy/ + heterozygotes over wild-type homozygotes. Overall, the results indicate that homozygosity of the entire second chromosome causes a depression in fitness of the order of 85%.

Diagnostic techniques in the assessment of haematological malignancies

2020 ◽  
pp. 5181-5188
Author(s):  
Wendy N. Erber
Keyword(s):  
Bone Marrow ◽  
Blood Count ◽  
Molecular Genetic ◽  
Diagnostic Techniques ◽  
Blood Film ◽  
Clinical Samples ◽  
Clinical Relapse ◽  
Haematological Malignancies ◽  
Prognostic Prediction ◽  
Detection Of Mutations

The diagnosis of haematological malignancies requires an understanding of the diseases and the uses and limitations of the range of available investigations. The relative importance of different investigations varies by disease entity. The blood count is one of the most widely used tests in all of medicine and often the first indication of an underlying haematological malignancy. Some blood count features are ‘diagnostic’ and others may give an indication of a bone marrow defect. Morphological assessment of a stained blood film adds value to an abnormal blood count. It may identify abnormal morphology of red cells, leucocytes, or platelets which may be specific and diagnostic, or give clues suggesting a diagnosis. Bone marrow aspirate (liquid sample) gives cytological detail, and trephine biopsy provides information about marrow cellularity, architecture, cellular distribution, and extent of fibrosis. Immunophenotyping detects cellular antigens in clinical samples and is essential in the diagnosis and classification of haematological malignancies. It is also used for disease staging and monitoring, to detect surrogate markers of genetic aberrations, identify potential immunotherapeutic targets, and to aid prognostic prediction. Cytogenetics assesses the number and structure of whole chromosomes and chromosomal regions in neoplastic cells and is performed to diagnose and classify some haematological malignancies. Molecular genetic methods facilitate the detection of mutations, rearrangements, or translocations in genes. Applications in malignant haematology include confirming clonality, detecting disease-associated genotypes, determining prognosis, disease monitoring following therapy, predicting imminent clinical relapse, and identifying patients who are likely (or not) to respond to new targeted inhibitor therapies.

scholarly journals The BRAF V600E Mutation Detection by quasa Sensitive Real-Time PCR Assay in Northeast Romania Melanoma Patients

2021 ◽  
Vol 11 (20) ◽  
pp. 9511
Author(s):  
Elena Porumb-Andrese ◽  
Ramona Gabriela Ursu ◽  
Iuliu Ivanov ◽  
Irina-Draga Caruntu ◽  
Vlad Porumb ◽  
...  
Keyword(s):  
Malignant Melanoma ◽  
Target Therapy ◽  
Braf Inhibitor ◽  
Braf V600e Mutation ◽  
Braf V600e ◽  
Clinical Samples ◽  
Primary Cutaneous Melanoma ◽  
Purification Method ◽  
Detection Of Mutations ◽  
National Reporting System

Background: The prevalence of melanoma in Romanian patients is underestimated. There is a need to identify the BRAF V600E mutation to accurately treat patients with the newest approved BRAF inhibitor therapy. This is a pilot study in which we first aimed to choose the optimal DNA purification method from formalin fixation and paraffin embedding (FFPE) malignant melanoma skin samples to assess the BRAF mutation prevalence and correlate it with clinical pathological parameters. Methods: 30 FFPE samples were purified in parallel with two DNA extraction kits, a manual and a semi-automated kit. The extracted DNA in pure and optimum quantity was tested for the BRAF V600E mutation using the quantitative allele-specific amplification (quasa) method. quasa is a method for the sensitive detection of mutations that may be present in clinical samples at low levels. Results: The BRAF V600E mutation was detected in 60% (18/30) samples in patients with primary cutaneous melanoma of the skin. BRAFV600E mutation was equally distributed by gender and was associated with age >60, nodular melanoma, and trunk localization. Conclusions: The high prevalence of BRAF V600E mutations in our study group raises awareness for improvements to the national reporting system and initiation of the target therapy for patients with malignant melanoma of the skin.

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