scholarly journals New Histoplasma Diagnostic Assays Designed via Whole Genome Comparisons

2021 ◽  
Vol 7 (7) ◽  
pp. 544
Author(s):  
Juan E. Gallo ◽  
Isaura Torres ◽  
Oscar M. Gómez ◽  
Lavanya Rishishwar ◽  
Fredrik Vannberg ◽  
...  
Keyword(s):  
Pathogenic Fungi ◽  
Positive Control ◽  
Detection Sensitivity ◽  
Analytical Sensitivity ◽  
Protein Coding ◽  
Control Plasmid ◽  
Formalin Fixed Paraffin Embedded ◽  
Pcr Assays ◽  
Primer Sets

Histoplasmosis is a systemic fungal disease caused by the pathogen Histoplasma spp. that results in significant morbidity and mortality in persons with HIV/AIDS and can also affect immunocompetent individuals. Although some PCR and antigen-detection assays have been developed, conventional diagnosis has largely relied on culture, which can take weeks. Our aim was to provide a proof of principle for rationally designing and standardizing PCR assays based on Histoplasma-specific genomic sequences. Via automated comparisons of aligned genome contigs/scaffolds and gene (sub)sequences, we identified protein-coding genes that are present in existing sequences of Histoplasma strains but not in other genera. Two of the genes, PPK and CFP4, were used for designing primer sets for conventional and real-time PCR assays. Both resulted in a 100% analytical specificity in vitro and detected 62/62 H. capsulatum isolates using purified DNA. We also obtained positive detections of 2/2 confirmed H. capsulatum clinical FFPE (formalin-fixed paraffin-embedded) samples using both primer sets. Positive control plasmid 10-fold serial dilutions confirmed the analytical sensitivity of the assays. The findings suggest that these novel primer sets should allow for detection sensitivity and reduce false positive results/cross-reactions. New assays for detecting pathogenic fungi, constructed along these lines, could be simple and affordable to implement.

scholarly journals In vitro antifungal and demelanizing activity of Nepeta rtanjensis essential oil against the human pathogen Bipolaris spicifera

2011 ◽  
Vol 63 (3) ◽  
pp. 897-905 ◽  
Author(s):  
Milica Ljaljevic-Grbic ◽  
M. Stupar ◽  
Jelena Vukojevic ◽  
D. Grubisic
Keyword(s):  
Essential Oil ◽  
Pathogenic Fungus ◽  
Pathogenic Fungi ◽  
Positive Control ◽  
Alternative Source ◽  
Conidia Germination ◽  
Growth Assay ◽  
Human Pathogenic Fungus ◽  
Bipolaris Spicifera

The antifungal activity of Nepeta rtanjensis Diklic & Milojevic essential oil was tested against the human pathogenic fungus Bipolaris spicifera (Bainier) Subramanian via mycelial growth assay and conidia germination assay. The minimally inhibitory concentration (MIC) of the oil was determined at 1.0 ?g ml-1, while the MIC for the antifungal drug Bifonazole in a positive control was determined at 10.0 ?g ml-1. The maximum of conidia germination inhibition was accomplished at 0.6 ?g ml-1. In addition, at 0.6 ?g ml-1 and 0.8 ?g ml-1 the oil was able to cause morphophysiological changes in B. spicifera. The most significant result is the bleaching effect of the melanized conidial apparatus of the test fungi, since the melanin is the virulence factor in human pathogenic fungi. These results showed the strong antifungal properties of N. rtanjensis essential oil, supporting its possible rational use as an alternative source of new antifungal compounds.

scholarly journals A Method to evaluate analytical sensitivity of qRT-PCR kits for SARS-CoV-2 detection

2020 ◽  
Author(s):  
Di Wang ◽  
Zhidong Wang ◽  
Xiao Wu ◽  
Yongzhuo Zhang ◽  
Yunhua Gao
Keyword(s):  
Reference Material ◽  
Limit Of Detection ◽  
False Negative ◽  
Detection Sensitivity ◽  
Amplification Efficiency ◽  
Analytical Sensitivity ◽  
Limit Of Quantification ◽  
Qrt Pcr ◽  
Pcr Products ◽  
Pcr Assays

Abstract Background: The ongoing Coronavirus disease 2019 (COVID-19) pandemic has spread across the globe and is representing a huge challenge for all human population. Many commercial qRT-PCR assays have been developed to detect SARS-CoV-2, but related method validation especially the sensitivity evaluation has been insufficient, resulting in some false-negative cases have been reported. Methods: The analytical sensitivity of nine brands of qRT-PCR kits for detecting SARS-CoV-2 was evaluated in parallel based on a newly developed certified reference material, which was derived from genomic RNA of SARS-CoV-2 from clinical positive specimens. After validation of the the reference material by digital PCR, the detection sensitivity of these kits was preliminarily tested using the serially diluted reference material, resulting in three kits with two significantly different sensitivity levels were selected for further evaluation. We sequenced the qRT-PCR products for assay specificity evaluation, and used serial dilutions of the reference material to calculate amplification efficiency and estimate the limit of quantification as well as 95% limit of detection..Results: The results indicated that the analytical sensitivity varied markedly among these kits. For the three types of qRT-PCR kits (Kit-1, Kit-2 and Kit-7), specificity of the PCR products was confirmed by sequence alignment, in which the target amplicons completely matched the corresponding parts of the genome of SARS-CoV-2. The resulting limit of detection from replicate tests for the Kit-1 and Kit-2 was 5.6 copies (N), 3.5 copies (ORF 1ab), and 6.4 copies (N), 4.6 copies (ORF 1ab), respectively, at 95% probability. Compared with Kit-7, the limit of detection as well as limit of quantification of Kit-1 and Kit-2 were significantly lower, further supporting that the both kits worked well to detect low abundance of SARS-CoV-2.Conclusions: Considering that most of the tested kits have been approved for in vitro diagnostics (IVD) in China, the established method here provides a reliable tool to evaluate the sensitivity performance of various qRT-PCR kits for SARS-CoV-2 detection and thus enhance quality control of qRT-PCR assays, improving the laboratory diagnostic capability for fighting the COVID-19 pandemic.

scholarly journals Succeptibility of some fungi to Boswellia carteri Birdw. essential oil

2016 ◽  
pp. 19-27 ◽  
Author(s):  
Milos Stupar ◽  
Marina Kostic ◽  
Zeljko Savkovic ◽  
Nikola Unkovic ◽  
Milica Ljaljevic-Grbic ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Essential Oil ◽  
Pathogenic Fungi ◽  
Positive Control ◽  
Stachybotrys Chartarum ◽  
Commercial Sample ◽  
Mycelia Growth ◽  
Susceptible Isolate ◽  
Oil Concentration

Antifungal activity of commercial sample of Boswellia carteri essential oil against selected micromycetes was evaluated in vitro using a microatmosphere method. When compared with biocide Sanosil S003, used as positive control, the tested essential oil showed moderate antifungal activity. The most susceptible fungi to oil treatment were Stachybotrys chartarum and Trichotecium roseum. For both fungi, mycelia growth inhibition of 85% was recorded at oil concentration of 100 ?L mL-1. The tested essential oil caused inhibition of S. chartarum sporulation as well as depigmentation of conidi?, which is very significant since melanin contributes to virulence, survival and endurance of pathogenic fungi spores. Aspergillus niger was the least susceptible isolate to essential oil treatment. Mycelial growth of this fungus was not inhibited by any oil concentrations used in the experiment.

scholarly journals Cross-Platform Evaluation of Commercial Real-Time SYBR Green RT-PCR Kits for Sensitive and Rapid Detection of European BatLyssavirusType 1

2015 ◽  
Vol 2015 ◽  
pp. 1-18 ◽  
Author(s):  
Evelyne Picard-Meyer ◽  
Carine Peytavin de Garam ◽  
Jean Luc Schereffer ◽  
Clotilde Marchal ◽  
Emmanuelle Robardet ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Evaluation Criteria ◽  
Sybr Green ◽  
Detection Sensitivity ◽  
Analytical Sensitivity ◽  
Reaction Efficiency ◽  
Qrt Pcr ◽  
One Step ◽  
Pcr Assays

This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency,R2values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes.

Tulbaghia violacea L. II: In vivo antifungal properties towards plant pathogens

2006 ◽  
Vol 57 (5) ◽  
pp. 517 ◽  
Author(s):  
Leeto Nteso ◽  
Johan C. Pretorius
Keyword(s):  
Crude Extract ◽  
Seed Treatment ◽  
Plant Pathogens ◽  
Disease Incidence ◽  
Ascochyta Blight ◽  
Pathogenic Fungi ◽  
Positive Control ◽  
Mycosphaerella Pinodes

In vitro antifungal activity of crude extracts from Tulbaghia violacea against 6 economically important plant pathogenic fungi was previously reported. The in vivo control of Mycosphaerella pinodes, causative of Ascochyta blight, by different concentrations of a crude aerial part extract of T. violacea was subsequently followed qualitatively and quantitatively in terms of lesions that developed over a 6-day period at 20°C on detached pea (Pisum sativum) leaves. Infection by M. pinodes spores was prevented when the extract was applied both before and after inoculation, confirming complete inhibition of spore germination, whereas no phytotoxic effect was observed on the leaves. Additionally, the control of sorghum covered (Sporisorium sorghi) and loose (S. cruentum) kernel smuts by seed treatment with the crude extract was tested under field conditions. Before planting, different sorghum seed lots were inoculated separately with spores from the 2 pathogens at a rate of 0.5% per kg (w/w), followed by treatment with the crude extract, at a concentration of 2.0 mg/mL, 24 h later. A standard fungicide, Thiram (65 W), was applied as a positive control at a rate of 0.25% per kg (v/w). Disease incidence was quantified during harvest and expressed as percentage infected plants. Seed treatment with the extract significantly (P < 0.05) reduced the incidence of both sorghum loose and covered smut diseases, compared favourably with the standard fungicide, and resulted in significant yield increases compared to the untreated control.

scholarly journals A specific real-time PCR assay for the detection of Bordetella pertussis

2007 ◽  
Vol 56 (7) ◽  
pp. 918-920 ◽  
Author(s):  
Benoit Vincart ◽  
Ricardo De Mendonça ◽  
Sylvianne Rottiers ◽  
Françoise Vermeulen ◽  
Marc J. Struelens ◽  
...  
Keyword(s):  
Real Time ◽  
Bordetella Pertussis ◽  
Real Time Pcr ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Pcr Assays ◽  
Reference Strains ◽  
Respiratory Specimens

A novel real-time PCR (RT-PCR) assay was developed for detection of Bordetella pertussis in respiratory specimens by targeting the pertactin gene. In vitro evaluation with reference strains and quality control samples showed analytical sensitivity equivalent to and specificity superior to those of PCR assays which target the IS481 element. The pertactin-based RT-PCR assay offers better discrimination between B. pertussis and other Bordetella species than previously described assays.

scholarly journals Custom long non-coding RNA capture enhances detection sensitivity in different human sample types

2021 ◽  
Author(s):  
Annelien Morlion ◽  
Celine Everaert ◽  
Justine Nuytens ◽  
Eva Hulstaert ◽  
Jo Vandesompele ◽  
...  
Keyword(s):  
Expression Patterns ◽  
Detection Sensitivity ◽  
Specific Expression ◽  
Protein Coding ◽  
Cellular Processes ◽  
Non Coding Rna ◽  
Formalin Fixed Paraffin ◽  
Human Sample ◽  
Formalin Fixed Paraffin Embedded ◽  
Regulatory Functions

AbstractLong non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have emerged as potential diagnostic and therapeutic targets. The accurate characterization of lncRNAs in bulk transcriptome data remains challenging due to their low abundance compared to protein coding genes. To tackle this issue, we describe a unique short-read custom lncRNA capture sequencing approach that relies on a comprehensive set of 565,878 capture probes for 49,372 human lncRNA genes. This custom lncRNA capture approach was evaluated on various sample types ranging from artificial high-quality RNA mixtures to more challenging formalin-fixed paraffin-embedded tissue and biofluid material. The custom enrichment approach allows the detection of a more diverse repertoire of lncRNAs, with better reproducibility and higher coverage compared to classic total RNA-sequencing.

Inhibitory effects of bioaccessible anthocyanins and procyanidins from apple, red grape, cinnamon on α-amylase, α-glucosidase and lipase

2020 ◽  
pp. 1-9
Author(s):  
Pınar Ercan ◽  
Sedef Nehir El
Keyword(s):  
Inhibitory Activity ◽  
Digestive Enzymes ◽  
Positive Control ◽  
Anthocyanin Content ◽  
Inhibitory Effects ◽  
Total Anthocyanin ◽  
Total Anthocyanin Content ◽  
Before And After ◽  
Red Grape

Abstract. The goals of this study were to determine and evaluate the bioaccessibility of total anthocyanin and procyanidin in apple (Amasya, Malus communis), red grape (Papazkarası, Vitis vinifera) and cinnamon (Cassia, Cinnamomum) using an in vitro static digestion system based on human gastrointestinal physiologically relevant conditions. Also, in vitro inhibitory effects of these foods on lipid (lipase) and carbohydrate digestive enzymes (α-amylase and α-glucosidase) were performed with before and after digested samples using acarbose and methylumbelliferyl oleate (4MUO) as the positive control. While the highest total anthocyanin content was found in red grape (164 ± 2.51 mg/100 g), the highest procyanidin content was found in cinnamon (6432 ± 177.31 mg/100 g) (p < 0.05). The anthocyanin bioaccessibilities were found as 10.2 ± 1%, 8.23 ± 0.64%, and 8.73 ± 0.70% in apple, red grape, and cinnamon, respectively. The procyanidin bioaccessibilities of apple, red grape, and cinnamon were found as 17.57 ± 0.71%, 14.08 ± 0.74% and 18.75 ± 1.49%, respectively. The analyzed apple, red grape and cinnamon showed the inhibitory activity against α-glucosidase (IC50 544 ± 21.94, 445 ± 15.67, 1592 ± 17.58 μg/mL, respectively), α-amylase (IC50 38.4 ± 7.26, 56.1 ± 3.60, 3.54 ± 0.86 μg/mL, respectively), and lipase (IC50 52.7 ± 2.05, 581 ± 54.14, 49.6 ± 2.72 μg/mL), respectively. According to our results apple, red grape and cinnamon have potential to inhibit of lipase, α-amylase and α-glucosidase digestive enzymes.

Aktivitas Antifungi Air Perasan Bawang Merah (Allium ascalonicum L.) Terhadap Candida albicans Secara In Vitro

2017 ◽  
Vol 9 (2) ◽  
pp. 71
Author(s):  
Nurhasanah Nurhasanah ◽  
Fauzia Andrini ◽  
Yulis Hamidy
Keyword(s):  
Candida Albicans ◽  
Antifungal Activity ◽  
Allium Sativum ◽  
Fungicidal Activity ◽  
Positive Control ◽  
Negative Control ◽  
Randomized Design ◽  
Significant Difference ◽  
Completely Randomized Design

Shallot (Allium ascalonicum L.) has been known as traditional medicine. Shallot which has same genus with garlic(Allium sativum L.) contains allicin that is also found in garlic and has been suspected has fungicidal activity toCandida albicans. It is supported by several researches. Therefore, shallot is suspected has antifungal activity too.The aim of this research was to know antifungal activity of shallot’s water extortion againsts Candida albicans invitro. This was a laboratory experimental research which used completely randomized design, with diffusion method.Shallot’s water extortion was devided into three concentrations, there were 50%, 100% and 200%. Ketoconazole 2%was positive control and aquadest was negative control. The result of this research based on analysis of varians(Anova), there was significant difference between several treatments and was confirmed with Duncan New MultipleRange Test (DNMRT) p<0,05, there was significant difference between 100% shallot’s water extortion with othertreatments, but there was no significant difference between 50% shallot’s water extortion with 200% shallot’s. Theconclusion was shallot’s water extortion had antifungal activity againsts Candida albicans with the best concentration100%, but it was lower than ketoconazole 2%.

Improved Production of Two Anti-Candida Lipopeptide Homologues Co- Produced by the Wild-Type Bacillus subtilis RLID 12.1 under Optimized Conditions

2020 ◽  
Vol 21 (5) ◽  
pp. 438-450
Author(s):  
Ramya Ramchandran ◽  
Swetha Ramesh ◽  
Anviksha A ◽  
RamLal Thakur ◽  
Arunaloke Chakrabarti ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Pathogenic Fungi ◽  
Fold Increase ◽  
Production Yield ◽  
Yield Enhancement ◽  
Bioactive Metabolites ◽  
Candida Auris ◽  
Media Formulation ◽  
Static Conditions

Background:: Antifungal cyclic lipopeptides, bioactive metabolites produced by many species of the genus Bacillus, are promising alternatives to synthetic fungicides and antibiotics for the biocontrol of human pathogenic fungi. In a previous study, the co- production of five antifungal lipopeptides homologues (designated as AF1, AF2, AF3, AF4 and AF5) by the producer strain Bacillus subtilis RLID 12.1 using unoptimized medium was reported; though the two homologues AF3 and AF5 differed by 14 Da and in fatty acid chain length were found effective in antifungal action, the production/ yield rate of these two lipopeptides determined by High-Performance Liquid Chromatography was less in the unoptimized media. Methods:: In this study, the production/yield enhancement of the two compounds AF3 and AF5 was specifically targeted. Following the statistical optimization (Plackett-Burman and Box-Behnken designs) of media formulation, temperature and growth conditions, the production of AF3 and AF5 was improved by about 25.8- and 7.4-folds, respectively under static conditions. Results:: To boost the production of these two homologous lipopeptides in the optimized media, heat-inactivated Candida albicans cells were used as a supplement resulting in 34- and 14-fold increase of AF3 and AF5, respectively. Four clinical Candida auris isolates had AF3 and AF5 MICs (100 % inhibition) ranging between 4 and 16 μg/ml indicating the lipopeptide’s clinical potential. To determine the in vitro pharmacodynamic potential of AF3 and AF5, time-kill assays were conducted which showed that AF3 (at 4X and 8X concentrations) at 48h exhibited mean log reductions of 2.31 and 3.14 CFU/ml of C. albicans SC 5314, respectively whereas AF5 at 8X concentration showed a mean log reduction of 2.14 CFU/ml. Conclusion:: With the increasing threat of multidrug-resistant yeasts and fungi, these antifungal lipopeptides produced by optimized method promise to aid in the development of novel antifungal that targets disease-causing fungi with improved efficacy.

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