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Cureus Aug 2023The main aim/objective of this study was to detect and characterize the speciesfrom patients having complaints of joint pain and also to know the potential causes of...
AIM
The main aim/objective of this study was to detect and characterize the speciesfrom patients having complaints of joint pain and also to know the potential causes of human brucellosis. In our study, we focused on joint pain symptoms that may be due to arthralgia or arthritis. Introduction: Brucellosis is a neglected zoonotic disease that affects both humans and animals. In humans, brucellosis begins with chronic illness leading to great financial losses from not being able to work well and continued treatment costs, but few such studies have come from northern India. Joint pain is the common presentation of brucellosis and there are several risk factors associated with brucellosis.
METHODS
A total of 200 blood samples were collected from the participants having joints pain from September 2019 to September 2021 at Gandhi Memorial & Associated Hospitals of King George's Medical University, Lucknow, India, and tested by serology for antiIgM and IgG enzyme-linked immunosorbent assay (ELISA), moleculartests byreverse transcriptase-polymerase chain reaction (RT-PCR),conventional polymerase chain reaction (PCR), and automated blood culture system. The anti- IgM and IgG ELISA were performed using the kit from NovaTec Immundiagnostica GmbH (Dietzenbach, Germany). Isolation of DNA was carried out using the QIAamp DNA Mini kit (QIAGEN, Hilden, Germany), and the primers and probes specific for targeted regions ( gene) in the genome were procured from Eurofins Scientific SE (Luxembourg, France), and for internal control from CDC.
RESULT
The study showed 19 (9.5%) and 23 (11.5%) positive results by anti-IgM ELISA and antiIgG, respectively, and of these, one (0.5%) was positive for both anti- IgM and anti- IgG ELISA. Out of 19 anti- IgM ELISA positive, eight (4%) samples were positive for PCR/RT-PCR and that was negative for anti- IgG ELISA. All blood culture reports of all patients were negative. Conclusion: Anti- IgM ELISA was more accurate than anti- IgG ELISA in detecting human brucellosis. Consumption of animal products (i.e. milk, a dairy product of cow, buffalo, goat, and meat of goat) and contact with animals were the main risk factors that were identified for disease.
PubMed: 37671228
DOI: 10.7759/cureus.42980 -
Scientific Reports Sep 2023Climate changes and anthropogenic pressures are causing a biodiversity decline in terms of species number and genetic diversity, reducing the adaptability and...
Climate changes and anthropogenic pressures are causing a biodiversity decline in terms of species number and genetic diversity, reducing the adaptability and evolvability of natural communities. Transitional water ecosystems are more sensitive to habitat reduction and degradation and, thus, are more exposed to biodiversity declines requiring biodiversity monitoring programs for their conservation. Environmental DNA (eDNA) metabarcoding represents a high-throughput tool for biodiversity assessment that is facilitating data collection for biodiversity monitoring. In this study, we applied, for the first time, eDNA metabarcoding in a Mediterranean coastal lagoon to assess the ecological features of eukaryotic phytoplankton communities. We sampled water in seven different lagoon sites and amplified the extracted DNA with primers targeting the variable region 4 (V4) of the 18S rRNA gene marker. The results demonstrated the validity of eDNA studies to provide insights into lagoon phytoplankton composition, establish the structure and spatial variation of phytoplankton communities, and evaluate its correlation to abiotic factors. Finally, the genetic distances analysis suggests that the different spatial distribution of OTUs, at least for the Tetraselmis genus, reflects the genetic background.
Topics: DNA, Environmental; Phytoplankton; Ecosystem; Biodiversity; Water
PubMed: 37709858
DOI: 10.1038/s41598-023-42389-3 -
Parasites & Vectors Mar 2024Schistosomiasis, a neglected tropical disease, remains an important public health problem. Although there are various methods for diagnosing schistosomiasis, many...
BACKGROUND
Schistosomiasis, a neglected tropical disease, remains an important public health problem. Although there are various methods for diagnosing schistosomiasis, many limitations still exist. Early diagnosis and treatment of schistosomiasis can significantly improve survival and prognosis of patients.
METHODOLOGY
Circulating cell-free (cf)DNA has been widely used in the diagnosis of various diseases. In our study, we evaluated the diagnostic value of circulating cfDNA for schistosomiasis caused by Schistosoma japonicum. We focused on the tandem sequences and mitochondrial genes of S. japonicum to identify highly sensitive and specific targets for diagnosis of Schistosomiasis japonica.
RESULTS
Through data screening and analysis, we ultimately identified four specific tandem sequences (TD-1, TD-2, TD-3. and TD-4) and six mitochondrial genes (COX1(1), COX1(2), CYTB, ATP6, COX3, and ND5). We designed specific primers to detect the amount of circulating cfDNA in S. japonicum-infected mouse and chronic schistosomiasis patients. Our results showed that the number of tandem sequences was significantly higher than that of the mitochondrial genes. A S. japonicum infection model in mice suggested that infection of S. japonicum can be diagnosed by detecting circulating cfDNA as early as the first week. We measured the expression levels of circulating cfDNA (TD-1, TD-2, and TD-3) at different time points and found that TD-3 expression was significantly higher than that of TD-1 or TD-2. We also infected mice with different quantities of cercariae (20 s and 80 s). The level of cfDNA (TD-3) in the 80 s infection group was significantly higher than in the 20 s infection group. Additionally, cfDNA (TD-3) levels increased after egg deposition. Meanwhile, we tested 42 patients with chronic Schistosomiasis japonica and circulating cfDNA (TD-3) was detected in nine patients.
CONCLUSIONS
We have screened highly sensitive targets for the diagnosis of Schistosomiasis japonica, and the detection of circulating cfDNA is a rapid and effective method for the diagnosis of Schistosomiasis japonica. The levels of cfDNA is correlated with cercariae infection severity. Early detection and diagnosis of schistosomiasis is crucial for patient treatment and improving prognosis.
Topics: Humans; Animals; Mice; Schistosomiasis japonica; Biomarkers; Schistosoma japonicum; Cell-Free Nucleic Acids; Cercaria
PubMed: 38449022
DOI: 10.1186/s13071-024-06203-x -
Microbiology Spectrum Jun 2024is a diverse and ubiquitous strain of both commensal and pathogenic bacteria. In this study, we propose the use of multiplex polymerase chain reaction (PCR), using...
UNLABELLED
is a diverse and ubiquitous strain of both commensal and pathogenic bacteria. In this study, we propose the use of multiplex polymerase chain reaction (PCR), using amplification of three genes (, and ), as a method for determining the affiliation of the tested strains to the species. The novelty of the method lies in the small number of steps needed to perform the diagnosis and, consequently, in the small amount of time needed to obtain it. This method, like any other, has some limitations, but its advantage is fast, cheap, and reliable identification of the presence of . Sequences of the indicated genes from 1,171 complete genomes in the NCBI database were used to prepare the primers. The developed multiplex PCR was tested on 47,370 different genomes using PCR. The sensitivity and specificity of the developed test were 95.76% and 99.49%, respectively. Wet laboratory analyses confirmed the high specificity, repeatability, reproducibility, and reliability of the proposed test. Because of the detection of three genes, this method is very cost and labor-effective, yet still highly accurate, specific, and sensitive in comparison to similar methods.
IMPORTANCE
Detection of from environmental or clinical samples is important due to the common occurrence of this species of bacteria in all human and animal environments. As commonly known, these bacteria strains can be commensal and pathogenic, causing numerous infections of clinical importance, including infections of the digestive system, urinary, respiratory, and even meninges, particularly dangerous for newborns. The developed multiplex polymerase chain reaction test, confirming the presence of in samples, can be used in many laboratories. The test provides new opportunities for quick and cheap analyses, detecting using only three pairs of primers (analysis of the presence of three genes) responsible for metabolism and distinguishing from other pathogens from the Enterobacteriaceae family. Compared to other tests previously described in the literature, our method is characterized by high specificity and sensitivity.
Topics: Multiplex Polymerase Chain Reaction; Escherichia coli; Humans; Sensitivity and Specificity; Escherichia coli Infections; Reproducibility of Results; Genome, Bacterial; Escherichia coli Proteins; DNA, Bacterial; DNA Primers
PubMed: 38687052
DOI: 10.1128/spectrum.03773-23 -
Plant Disease Nov 2023is growing in popularity amongst consumers in cut-flower and pop-flower market as an ornamental woody plant for its florid berry and colorful flower. In August 2019,...
is growing in popularity amongst consumers in cut-flower and pop-flower market as an ornamental woody plant for its florid berry and colorful flower. In August 2019, a new leaf spot disease was observed on in three commercial nurseries in Kunming (25°05'N, 102°72'E), Yunnian province, China. Disease symptoms were observed on approximately 40% of the plants one year after planting and 30% of the leaves were infected. Leaf symptoms began as small, water-soaked lesions on young leaves which later became larger, dark brown and necrotic. The lesion size ranged from 0.2 to 2.8 cm in diameter. For pathogen isolation, three samples of symptomatic leaves were collected from four different nurseries. The leaves were cut into 0.5 mm pieces, surface sterilized using 70% ethanol for 30 s, and 3% NaOCl for 5 min, rinsed three times in sterilized distilled water and plated on potato dextrose agar (PDA) (Zhou et al. 2023). The plates were incubated at 26°C in the dark for 3 days. Eight isolates with comparable morphological characteristics were obtained. Initially, colonies produced pale gray to white aerial mycelia, turning dark gray after 5 days. The isolates produced hyaline, single celled, straight and cylindrical conidia, with mean size 9.7 to 14.8 μm long × 3.7 to 5.6 μm wide ( = 100). Morphological characteristics were consistent with sp. (Bailey and Jeger 1992). For molecular analysis, genomic DNA was extracted from three representative isolates (XSD1, XSD3 and XSD5), amplified using the primers ITS1/ITS4 (Yin et al. 2012) and T1/Bt2b (Glass and Donaldson 1995) and submitted to sequencing (Weir et al. 2012). DNA sequences of the isolates XSD2, XSD3 and XSD8 were identical. DNA sequences of a representative isolate XSD2 were deposited in GenBank (accession no. MW202334 for ITS, and OR347007 for ). MegaBLAST analysis of the ITS and TUB2 sequences showed 99.5% and 99.3% similarity with C. kahawae strain ICMP 18539 (accession no. NR_120138.1 for ITS) and strain IMI319418 (JX145227.1 for ). Pathogenicity tests were conducted by inoculating the pathogen on healthy mature leaves of in the field. Ten leaves (two leaves/plant) were inoculated by spraying conidial suspension (10 spores/ml) of isolates XSD1, XSD3 and XSD5, and covered with plastic bags to maintain high humidity for 48 hours, respectively. Leaves treated with sterile distilled water served as a control. All inoculated leaves showed symptoms similar to those observed in the field at 23±5°C 10 days after inoculation. No symptoms developed on non-inoculated leaves. The pathogen was re-isolated from inoculated diseased leaves and identified as based on morphological and molecular characters. C. kahawae has been reported to cause leaf spot on cultivated rocket in Italy (Garibaldi et al. 2016), and anthracnose disease on tree tomato in Colombia (Rojas et al. 2018), to our knowledge, this is the first report of causing anthracnose on worldwide. Due to important ornamental and economic value of , the distribution of needs to be investigated and monitored for effective disease management strategies to be developed.
PubMed: 38035788
DOI: 10.1094/PDIS-08-23-1496-PDN -
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 -
Tropical Parasitology 2023Periodontal disease is often caused by bacterial plaque. However, there are indications that some infective agents, including parasites, may play important roles in the...
BACKGROUND
Periodontal disease is often caused by bacterial plaque. However, there are indications that some infective agents, including parasites, may play important roles in the pathogenesis of the disease.
AIM
This study aimed at assessing the prevalence of gingivitis and periodontitis, as well as the prevalence of and , in the oral biofilm of individuals with periodontal diseases using polymerase chain reaction.
MATERIALS AND METHODS
One hundred and six respondents with periodontal disease participated in the study. All study participants had a full-mouth examination, and dental plaque samples were collected with a sterile curette. Samples were transported to the laboratory in a cold chain and stored frozen till analyzed. DNA was extracted from the samples and amplified using EGO and ENTAM primers for and TGBK primers for .
RESULTS
The mean age of respondents was 45 ± 16.3 years, with none using tobacco. The prevalence of gingivitis and periodontitis obtained from this study was 84.9% and 15.1%. The prevalence obtained for and was 0.9% each; however, no participant had both parasites. The positive samples were from male participants with moderate gingivitis.
CONCLUSION
Gingivitis was more prevalent than periodontitis, though with a high preponderance in females. and may not be of etiologic importance in periodontal disease as they occurred sparsely in the studied population.
PubMed: 37860615
DOI: 10.4103/tp.tp_8_23 -
Food Chemistry. Molecular Sciences Jul 2024The objective of this study was to develop a DNA-based method for the identification and tracking of edible oils, which is important for health management. Three...
Quantitative and qualitative analysis of three DNA extraction methods from soybean, maize, and canola oils and investigation of the presence of genetically modified organisms (GMOs).
The objective of this study was to develop a DNA-based method for the identification and tracking of edible oils, which is important for health management. Three different DNA extraction methods (CTAB, MBST kit, and manual hexane-based method) were used to obtain high-purity DNA from crude and refined soybean, maize, and canola oils. PCR was then conducted using specific primers to identify the presence of genes related to each oil type and to assess transgenicity. The results showed that DNA was present in crude and refined oils, but in very low amounts. However, using method 3 for DNA extraction provided sufficient quantity and quality of DNA for successful PCR amplification. The study concluded that the main challenge in DNA extraction from oils is the presence of PCR inhibitors, which can be overcome using the manual hexanebased method. Also, the examination of protein presence in the oils using SDS-PAGE did not indicate any protein bands.
PubMed: 38577346
DOI: 10.1016/j.fochms.2024.100201 -
ACS Omega Sep 2023A fast, easy-to-implement, highly sensitive, and point-of-care (POC) detection system for frog virus 3 (FV3) is proposed. Combining recombinase polymerase amplification...
A fast, easy-to-implement, highly sensitive, and point-of-care (POC) detection system for frog virus 3 (FV3) is proposed. Combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a, a limit of detection (LoD) of 100 aM (60.2 copies/μL) is achieved by optimizing RPA primers and CRISPR RNAs (crRNAs). For POC detection, smartphone microscopy is implemented, and an LoD of 10 aM is achieved in 40 min. The proposed system detects four positive animal-derived samples with a quantitation cycle (Cq) value of quantitative PCR (qPCR) in the range of 13 to 32. In addition, deep learning models are deployed for binary classification (positive or negative samples) and multiclass classification (different concentrations of FV3 and negative samples), achieving 100 and 98.75% accuracy, respectively. Without temperature regulation and expensive equipment, the proposed RPA-CRISPR/Cas12a combined with smartphone readouts and artificial-intelligence-assisted classification showcases the great potential for FV3 detection, specifically POC detection of DNA virus.
PubMed: 37720737
DOI: 10.1021/acsomega.3c02929 -
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