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ACS Omega Aug 2023Adulteration and substitution of medicinal plants have become a matter of great concern in recent years. is one such medicinal plant that has gained importance but is...
Adulteration and substitution of medicinal plants have become a matter of great concern in recent years. is one such medicinal plant that has gained importance but is often confused with other plants of the same species. In order to address this issue, this study aimed to conduct a conventional and molecular pharmacognostic study for the identification of the root of . The root of the plant was studied for the macroscopic observations, and then, the root was ground into coarse powder for microscopic studies and to determine the physiochemical properties. The powder was subjected to extraction with solvents such as ethanol, ethanol/water (1:1), hexane, and ethyl acetate. The extracts were then used for qualitative and quantitative (phenol, alkaloids, and flavonoids) phytochemical analysis. The molecular study was performed with the DNA barcoding technique. The DNA was extracted from the root of the plant, and its purity was examined by gel electrophoresis (1% w/v). The DNA was then amplified using an Applied Biosystems 2720 thermal cycler for the rbcL, matK, and ITS primers. The amplified primers were sequenced with a 3130 Genetic Analyzer, and the generated sequences were searched for similarity in the GenBank Database using the nucleotide BLAST analysis. The micro- and macroscopic studies revealed the morphological and organoleptic characters as well as the presence of medullary rays, fiber, cork, sclereids, parenchymal cells, and scalariform vessels. The physiochemical properties were found within the limit. The phytochemical analysis revealed the presence of terpenoids, flavonoids, saponins, and alkaloids. In addition, the alkaloidal content was high in the ethanol extract (63.04 ± 3.08 mg At E/g), while the phenol content was high in the hexane extract (10.26667 ± 1.77 mg At E/g), and the flavonoid content was high in the ethyl acetate extract (41.458 ± 1.33 mg At E/g). After the BLAST analysis from the GenBank database, the rbcL, ITS, and matK primers showed a similarity percentage of 99.83, 99.84, and 100. The phylogenetic tree for the species closest to each primer was generated using the MEGA 6 software. The matK loci had the highest percentage similar to the rbcL and ITS loci, indicating that the matK loci can be used to identify the root of as a standalone. The results from this study can be used to establish a quality standard for that will ensure its quality and purity.
PubMed: 37599932
DOI: 10.1021/acsomega.3c02543 -
Scientific Reports Oct 2023The microbial community composition of five distinct thermophilic hot springs was effectively described in this work, using broad-coverage nanopore sequencing (ONT...
The microbial community composition of five distinct thermophilic hot springs was effectively described in this work, using broad-coverage nanopore sequencing (ONT MinION sequencer). By examining environmental samples from the same source, but from locations with different temperatures, bioinformatic analysis revealed dramatic changes in microbial diversity and archaeal abundance. More specifically, no archaeal presence was reported with universal bacterial primers, whereas a significant archaea presence and also a wider variety of bacterial species were reported. These results revealed the significance of primer preference for microbiomes in extreme environments. Bioinformatic analysis was performed by aligning the reads to 16S microbial databases for identification using three different alignment methods, Epi2Me (Fastq 16S workflow), Kraken, and an in-house BLAST tool, including comparison at the genus and species levels. As a result, this approach to data analysis had a significant impact on the genera identified, and thus, it is recommended that use of multiple analysis tools to support findings on taxonomic identification using the 16S region until more precise bioinformatics tools become available. This study presents the first compilation of the ONT-based inventory of the hydrogen producers in the designated hot springs in Türkiye.
Topics: Archaea; Nanopore Sequencing; Bacteria; Microbiota; RNA, Ribosomal, 16S; Phylogeny; Sequence Analysis, DNA
PubMed: 37813931
DOI: 10.1038/s41598-023-44357-3 -
Plant Disease Nov 2023(de Bary) Abad, de Cock, Bala, Robideau, A. M. Lodhi & Levesque is an important waterborne and soil-inhabiting oomycete pathogen causing root and crown rot of various...
(de Bary) Abad, de Cock, Bala, Robideau, A. M. Lodhi & Levesque is an important waterborne and soil-inhabiting oomycete pathogen causing root and crown rot of various plants including certain woody ornamentals, fruit, and forest trees. Early and accurate detection of in the nursery production system is critical, as this pathogen is quickly transported to neighboring healthy plants through the irrigation system. Conventional methods for the detection of this pathogen are tedious, frequently inconclusive, and costly. Hence, a specific, sensitive, and rapid molecular diagnostic method is required to overcome the limitations of traditional identification. In the current study, loop-mediated isothermal amplification (LAMP) for DNA amplification was developed for the identification of . It was evaluated using real-time and colorimetric assays. Several sets of LAMP primers were designed and screened, but PVLSU2 was found to be specific to as it did not amplify other closely related oomycetes, fungi, and bacteria. Moreover, the developed assays were sensitive enough to amplify DNA up to 102 fg per reaction. The real-time LAMP assay was more sensitive than traditional PCR and culture-based methods to detect infected plant samples. In addition, both LAMP assays detected as few as 100 zoospores suspended in 100 ml water. These LAMP assays are anticipated to save time in detection by disease diagnostic laboratories and research institutions and enable early preparedness in the event of disease outbreaks.
Topics: Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; DNA; Oomycetes
PubMed: 37018213
DOI: 10.1094/PDIS-08-22-1944-RE -
Journal of Human Genetics May 2024Cell-type-specific regulatory elements, cataloged through extensive experiments and bioinformatics in large-scale consortiums, have enabled enrichment analyses of... (Review)
Review
Cell-type-specific regulatory elements, cataloged through extensive experiments and bioinformatics in large-scale consortiums, have enabled enrichment analyses of genetic associations that primarily utilize positional information of the regulatory elements. These analyses have identified cell types and pathways genetically associated with human complex traits. However, our understanding of detailed allelic effects on these elements' activities and on-off states remains incomplete, hampering the interpretation of human genetic study results. This review introduces machine learning methods to learn sequence-dependent transcriptional regulation mechanisms from DNA sequences for predicting such allelic effects (not associations). We provide a concise history of machine-learning-based approaches, the requirements, and the key computational processes, focusing on primers in machine learning. Convolution and self-attention, pivotal in modern deep-learning models, are explained through geometrical interpretations using dot products. This facilitates understanding of the concept and why these have been used for machine learning for DNA sequences. These will inspire further research in this genetics and genomics field.
PubMed: 38730006
DOI: 10.1038/s10038-024-01256-3 -
Virology Journal Jul 2023Originating in Africa, African swine fever (ASF) was introduced to China in 2018. This acute and highly virulent infectious disease affects domestic pigs. The World...
BACKGROUND
Originating in Africa, African swine fever (ASF) was introduced to China in 2018. This acute and highly virulent infectious disease affects domestic pigs. The World Organization for Animal Health has listed it as a statutory reportable disease, and China has listed it as a category A infectious disease.
METHODS
Primers and probes were designed for four ASFV genes (B646L, EP402R, MGF505-3R, and A137R). The primers/probes were highly conserved compared with the gene sequences of 21 ASFV strains.
RESULTS
After optimization, the calibration curve showed good linearity (R > 0.99), the minimum concentration of positive plasmids that could be detected was 50 copies/µL, and the minimum viral load detection limit was 10 HAD/mL. Furthermore, quadruple quantitative polymerase chain reaction (qPCR) with nucleic acids from three porcine-derived DNA viruses and cDNAs from eight RNA viruses did not show amplification curves, indicating that the method was specific. In addition, 1 × 10, 1 × 10, and 1 × 10 copies/µL of mixed plasmids were used for the quadruple qPCR; the coefficient of variation for triplicate determination between groups was < 2%, indicating the method was reproducible.
CONCLUSIONS
The results obtained by testing clinical samples containing detectable EP402R, MGF505-3R, and A137R strains with different combinations of gene deletions were as expected. Therefore, the established quadruple qPCR method was validated for the molecular diagnosis of ASF using gene-deleted ASFV strains.
Topics: Swine; Animals; African Swine Fever; African Swine Fever Virus; Viral Proteins; Sus scrofa; Polymerase Chain Reaction; DNA Primers
PubMed: 37452402
DOI: 10.1186/s12985-023-02111-1 -
International Journal of Molecular... May 2024The Chinese giant salamander (), listed as an endangered species under "secondary protection" in China, faces significant threats due to ecological deterioration and the...
The Chinese giant salamander (), listed as an endangered species under "secondary protection" in China, faces significant threats due to ecological deterioration and the expansion of human activity. Extensive field investigations are crucial to ascertain the current status in the wild and to implement effective habitat protection measures to safeguard this species and support its population development. Traditional survey methods often fall short due to the elusive nature of the , presenting challenges that are time-consuming and generally ineffective. To overcome these obstacles, this study developed a real-time monitoring method that uses environmental DNA (eDNA) coupled with recombinase polymerase amplification and lateral flow strip (RPA-LFD). We designed five sets of species-specific primers and probes based on mitochondrial genome sequence alignments of and its close relatives. Our results indicated that four of these primer/probe sets accurately identified distinguishing it from other tested caudata species using both extracted DNA samples and water samples from a tank housing an individual. This method enables the specific detection of genomic DNA at concentrations as low as 0.1 ng/mL within 50 min, without requiring extensive laboratory equipment. Applied in a field survey across four sites in Huangshan City, Anhui Province, where is known to be distributed, the method successfully detected the species at three of the four sites. The development of these primer/probe sets offers a practical tool for field surveying and monitoring, facilitating efforts in population recovery and resource conservation for .
Topics: Animals; Urodela; China; Endangered Species; DNA, Environmental; DNA, Mitochondrial; Genome, Mitochondrial
PubMed: 38732163
DOI: 10.3390/ijms25094946 -
Analytica Chimica Acta Jul 2023In the context of personalized and cost-effective treatment, knowledge of the mutational status of specific genes is advantageous to predict which patients are...
In the context of personalized and cost-effective treatment, knowledge of the mutational status of specific genes is advantageous to predict which patients are responsive to therapies. As an alternative to one-by-one detection or massive sequencing, the presented genotyping tool determines multiple polymorphic sequences that vary a single nucleotide. The biosensing method includes an effective enrichment of mutant variants and selective recognition by colorimetric DNA arrays. The proposed approach is the hybridization between sequence-tailored probes and products from PCR with SuperSelective primers to discriminate specific variants in a single locus. A fluorescence scanner, a documental scanner, or a smartphone captured the chip images to obtain spot intensities. Hence, specific recognition patterns identified any single-nucleotide change in the wild-type sequence overcoming qPCR methods and other array-based approaches. Studied mutational analyses applied to human cell lines provided high discrimination factors, the precision was 95%, and the sensitivity was 1% mutant of total DNA. Also, the methods showed a selective genotyping of the KRAS gene from tumorous samples (tissue and liquid biopsy), corroborating results by NGS. The developed technology supported on low-cost robust chips and optical reading provides an attractive pathway toward implementing fast, cheap, reproducible discrimination of oncological patients.
Topics: Humans; Nucleotides; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; DNA; Mutation
PubMed: 37230582
DOI: 10.1016/j.aca.2023.341343 -
BMC Microbiology Sep 2023Mycobacterium leprae (ML) is the pathogen that causes leprosy, which has a long history and still exists today. ML is an intracellular mycobacterium that dominantly...
BACKGROUND
Mycobacterium leprae (ML) is the pathogen that causes leprosy, which has a long history and still exists today. ML is an intracellular mycobacterium that dominantly induces leprosy by causing permanent damage to the skin, nerves, limbs and eyes as well as deformities and disabilities. Moreover, ML grows slowly and is nonculturable in vitro. Given the prevalence of leprosy, a highly sensitive and rapid method for the early diagnosis of leprosy is urgently needed.
RESULTS
In this study, we devised a novel tool for the diagnosis of leprosy by combining restriction endonuclease, real-time fluorescence analysis and multiple cross displacement amplification (E-RT-MCDA). To establish the system, primers for the target gene RLEP were designed, and the optimal conditions for E-RT-MCDA at 67 °C for 36 min were determined. Genomic DNA from ML, various pathogens and clinical samples was used to evaluate and optimize the E-RT-MCDA assay. The limit of detection (LoD) was 48.6 fg per vessel for pure ML genomic DNA, and the specificity of detection was as high as 100%. In addition, the detection process could be completed in 36 min by using a real-time monitor.
CONCLUSION
The E-RT-MCDA method devised in the current study is a reliable, sensitive and rapid technique for leprosy diagnosis and could be used as a potential tool in clinical settings.
Topics: Humans; Mycobacterium leprae; Sensitivity and Specificity; Leprosy; Skin; DNA; DNA, Bacterial; Nucleic Acid Amplification Techniques
PubMed: 37770823
DOI: 10.1186/s12866-023-03004-7 -
International Journal of Molecular... Dec 2023The increasing number of patients with chronic wounds requires the development of quick and accurate diagnostics methods. One of the key and challenging aspects of... (Review)
Review
The increasing number of patients with chronic wounds requires the development of quick and accurate diagnostics methods. One of the key and challenging aspects of treating ulcers is to control wound infection. Early detection of infection is essential for the application of suitable treatment methods, such as systemic antibiotics or other antimicrobial agents. Clinically, the most frequently used method for detecting microorganisms in wounds is through a swab and culture on appropriate media. This test has major limitations, such as the long bacterial growth time and the selectivity of bacterial growth. This article presents an overview of molecular methods for detecting bacteria in wounds, including real-time polymerase chain reaction (rtPCR), quantitative polymerase chain reaction (qPCR), genotyping, next-generation sequencing (NGS), and loop-mediated isothermal amplification (LAMP). We focus on the LAMP method, which has not yet been widely used to detect bacteria in wounds, but it is an interesting alternative to conventional detection methods. LAMP does not require additional complicated equipment and provides the fastest detection time for microorganisms (approx. 30 min reaction). It also allows the use of many pairs of primers in one reaction and determination of up to 15 organisms in one sample. Isothermal amplification of DNA is currently the easiest and most economical method for microbial detection in wound infection. Direct visualization of the reaction with dyes, along with omitting DNA isolation, has increased the potential use of this method.
Topics: Humans; DNA; DNA Primers; Wound Infection; Bacteria; Nucleic Acid Amplification Techniques; Molecular Diagnostic Techniques
PubMed: 38203582
DOI: 10.3390/ijms25010411 -
Analytical Biochemistry Jan 2024Nucleic acids amplification is a widely used technique utilized for different manipulations with DNA and RNA. Although, polymerase chain reaction (PCR) remains the most...
Nucleic acids amplification is a widely used technique utilized for different manipulations with DNA and RNA. Although, polymerase chain reaction (PCR) remains the most popular amplification method, isothermal approaches are gained more attention last decades. Among these, loop-mediated isothermal amplification (LAMP) became an excellent alternative to PCR. LAMP requires an increased number of primers and, therefore, is considered a highly specific amplification reaction compared to PCR. LAMP primers design is still a non-trivial task, and all niceties should be taken into account during their selection. Here, we report on a new program called LAMPrimers iQ destined for high-quality LAMP primers design. LAMPrimers iQ is based on an original algorithm considering rigorous criteria for primers selection. Unlike alternative programs, LAMPrimers iQ can process long DNA or RNA sequences, and completely avoid primers that can form homo- and heterodimers. The quality of the primers designed was checked using SARS-CoV-2 coronavirus RNA as a model target. It was shown that primers selected with LAMPrimers iQ provide higher specificity and reliable detection of viral RNA compared to those obtained by alternative programs. The program is available at https://github.com/Restily/LAMPrimers-iQ.
Topics: Sensitivity and Specificity; Nucleic Acid Amplification Techniques; DNA; Software; RNA
PubMed: 37924966
DOI: 10.1016/j.ab.2023.115376