Custom amplicon

BACKGROUND: While Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories...

BACKGROUND: Asthma is a complex disease and a severe global public health problem resulting from interactions between genetic background and environmental exposures. It has been suggested that gut microbiota may be related to asthma development; however, such relationships needs further investigation. OBJECTIVE: This study aimed to characterize the gut microbiota as well as the nasal lavage cytokine profile of asthmatic and nonasthmatic individuals. METHODS: Stool and nasal lavage samples were collected from 29 children and adolescents with type 2 asthma and 28 children without asthma in Brazil...

Genetically modified crops (GMCs) have been discussed due to unknown safety, and thus, it is imperative to develop an effective detection technology. CRISPR/Cas is deemed a burgeoning technology for nucleic acid detection. Herein, we developed a novel detection method for the first time, which combined thermostable Cas12b with loop-mediated isothermal amplification (LAMP), to detect genetically modified (GM) soybeans in a customized one-pot vessel. In our method, LAMP-specific primers were used to amplify the cauliflower mosaic virus 35S promoter (CaMV35S) of the GM soybean samples...

In this study, we evaluated the implementation of a second-tier genetic screening test using an amplicon-based next-generation sequencing (NGS) panel in our laboratory during the period of 1 September 2021 to 31 August 2022 for the newborn screening (NBS) of six conditions for inborn errors of metabolism: citrullinemia type II (MIM #605814), systemic primary carnitine deficiency (MIM #212140), glutaric acidemia type I (MIM #231670), beta-ketothiolase deficiency (#203750), holocarboxylase synthetase deficiency (MIM #253270) and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (MIM # 246450)...

While the gut microbiota is known to be influenced by habitual food intake, this relationship is seldom explored in type 2 diabetes patients. This study aims to investigate the relationship between dietary patterns and gut microbial species abundance in 113 type 2 diabetes patients (mean age, 58 years; body mass index, 29.1; glycohemoglobin [HbA1c], 8.1%). We analyzed the gut microbiota using 16S amplicon sequencing, and all patients were categorized into either the Bacteroides enterotype (57.5%, n = 65) or the Prevotella enterotype (42...

16S rRNA targeted amplicon sequencing is an established standard for elucidating microbial community composition. While high-throughput short-read sequencing can elicit only a portion of the 16S rRNA gene due to their limited read length, third generation sequencing can read the 16S rRNA gene in its entirety and thus provide more precise taxonomic classification. Here, we present a protocol for generating full-length 16S rRNA sequences with Oxford Nanopore Technologies (ONT) and a microbial community profile with Emu...

BACKGROUND: The emergence of drug-resistant clones of Plasmodium falciparum is a major public health concern, and the ability to detect and track the spread of these clones is crucial for effective malaria control and treatment. However, in endemic settings, malaria infected people often carry multiple P. falciparum clones simultaneously making it likely to miss drug-resistant clones using traditional molecular typing methods. OBJECTIVES: Our goal was to develop a bioinformatics pipeline for compositional profiling in multiclonal P...

In order to realize portable pathogen diagnostics with easier quantitation, digitization and integration, we develop a ready-to-use electrochemical sensing strategy (Iso-E-Codelock) for real-time detection of isothermal nucleic acid amplification. Bridged by a branch DNA as codelock, the isothermal amplicon is transduced into increased current of an electrochemical probe, holding multiple advantages of high sensitivity, high selectivity, signal-on response, "zero" background and one-pot operation. Through a self-designed portable instrument (BioAlex PHE-T), the detection can implement on a multichannel microchip and output real-time amplification curves just like an expensive commercial PCR machine...

The production of gueuze beers through refermentation and maturation of blends of lambic beer in bottles is a way for lambic brewers to cope with the variability among different lambic beer batches. The resulting gueuze beers are more carbonated than lambic beers and are supposed to possess a unique flavor profile that varies over time. To map this refermentation and maturation process for gueuze production, a blend of lambic beers was made and bottled, whereby one of them was produced with the old wheat landrace Zeeuwse Witte ...

Strawberry (Fragaria × ananassa Duch.) was introduced in Sicily (Italy) in the 1930s in the small town of Maletto, on the slopes of Etna volcan, where it's currently cultivated in a total area of 30 ha. The French cv. 'Madame Moutot', appreciated for its unique flavor and intense fragrance, was there propagated vegetatively and after decades, the distinctive 'Etna ecotype' originated by adaptation to the peculiar environmental conditions of the area (Milella et al., 2006). In May 2023, in a 0.5 ha "Etna ecotype" strawberry field, virus-like symptoms were observed in approximately 50% of the plants...

Simultaneous in situ detection of transcript and protein markers at the single-cell level is essential for gaining a better understanding of tumor heterogeneity and for predicting and monitoring treatment responses. However, the limited accessibility to advanced 3D imaging techniques has hindered their rapid implementation. Here, we present a 3D single-cell imaging technique, termed 3D digital rolling circle amplification (4DRCA), capable of the multiplexed and amplified simultaneous digital quantification of single-cell RNAs and proteins using standard fluorescence microscopy and off-the-shelf reagents...

UNLABELLED: We report a deep next-generation sequencing analysis of 13 sequentially obtained tumor samples, eight sequentially obtained circulating tumor DNA (ctDNA) samples and three germline DNA samples over the life history of 3 patients with triple-negative breast cancer (TNBC), 2 of whom had germline pathogenic BRCA1 mutation, to unravel tumor evolution. Tumor tissue from all timepoints and germline DNA was subjected to whole-exome sequencing (WES), custom amplicon deep sequencing (30,000X) of a WES-derived somatic mutation panel, and SNP arrays for copy-number variation (CNV), while whole transcriptome sequencing (RNA-seq) was performed only on somatic tumor...

Although it is known that the relative abundance of Akkermansia, a bacterial genus commonly associated with health, increases in the gut microbiota of Parkinson's disease (PD) patients, the exact reason for this increase remains unclear. This study was aimed to identify potential changes in Akkermansia within the gut microbiota of PD patients in Türkiye. For this purpose, shotgun metagenomics and a novel Akkermansia genus-specific amplicon sequencing technique was used to investigate the presence of specific Akkermansia strains associated with cognitive impairment (CI) stages in PD and to examine potential genes within these strains...

BACKGROUND: Recent advancements in next-generation sequencing (NGS) technology have ushered in significant improvements in sequencing speed and data throughput, thereby enabling the simultaneous analysis of a greater number of samples within a single sequencing run. This technology has proven particularly valuable in the context of microbial community profiling, offering a powerful tool for characterizing the microbial composition at the species level within a given sample. This profiling process typically involves the sequencing of 16S ribosomal RNA (rRNA) gene fragments...

Botrytis cinerea is a well-known plant pathogen responsible for grey mould disease infecting more than 500 plant species. It is listed as the second most important plant pathogen scientifically and economically. Its impact is particularly severe in grapes since it affects both the yield of grape berries and the quality of wines. While various methods for detecting B. cinerea have been investigated, the application of Oxford Nanopore Technology (ONT) for complete ribosomal operon sequencing, which has proven effective in human and animal fungal research and diagnostics, has not yet been explored in grapevine ( Vitis vinifera ) disease research...

BACKGROUND: The current therapeutic algorithm for Advanced Stage Melanoma comprises of alternating lines of Targeted and Immuno-therapy, mostly via Immune-Checkpoint blockade. While Comprehensive Genomic Profiling of solid tumours has been approved as a companion diagnostic, still no approved predictive biomarkers are available for Melanoma aside from BRAF mutations and the controversial Tumor Mutational Burden. This study presents the results of a Multi-Centre Observational Clinical Trial of Comprehensive Genomic Profiling on Target and Immuno-therapy treated advanced Melanoma...

BACKGROUND: Insecticide resistance is reducing the efficacy of vector control interventions, consequently threatening efforts to control vector-borne diseases, including malaria. Investigating the prevalence of molecular markers of resistance is a useful tool for monitoring the spread of insecticide resistance in disease vectors. The Bijagós Archipelago (Bijagós) in Guinea-Bissau is a region of stable malaria transmission where insecticide-treated nets are the mainstay for malaria control...

Ever since the outbreak of COVID-19 disease in Wuhan, China, different variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been identified. Wastewater-based epidemiology (WBE), an approach that has been successfully applied in numerous case studies worldwide, offers a cost-effective and rapid way for monitoring trends of SARS-Cov-2 in the community level without selection bias. Despite being a gold-standard procedure, WBE is a challenging approach due to the sample instability and the moderate efficiency of SARS-CoV-2 concentration in wastewater...

The continual investigation of novel genetic markers has yielded promising solutions for addressing the challenges encountered in forensic DNA analysis. In this study, we have introduced a custom-designed panel capable of simultaneously amplifying 41 novel Multi-insertion/deletion (Multi-InDel) markers and an amelogenin locus using the capillary electrophoresis platform. Through a developmental validation study conducted in accordance with guidelines recommended by the Scientific Working Group on DNA Analysis Methods, we demonstrated that the new Multi-InDel system exhibited the sensitivity to produce reliable genotyping profiles with as little as 62...

Analysis of the bacterial community from a 16S rRNA gene sequencing technologies requires comparing the reads to a reference database. The challenging task involved in annotation relies on the currently available tools and 16S rRNA databases: SILVA, Greengenes and RDP. A successful annotation depends on the quality of the database. For instance, Greengenes and RDP have not been updated since 2013 and 2016, respectively. In addition, the nature of 16S sequencing technologies (short reads) focuses mainly on the V3-V4 hypervariable region sequencing and hinders the species assignment, in contrast to whole shotgun metagenome sequencing...