scholarly journals Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma

2015 ◽  
Vol 53 (10) ◽  
pp. 3280-3285 ◽  
Author(s):  
Sarah A. Ahmed ◽  
Wendy W. J. van de Sande ◽  
Marie Desnos-Ollivier ◽  
Ahmed H. Fahal ◽  
Najwa A. Mhmoud ◽  
...  
Keyword(s):  
Morphological Characteristics ◽  
High Specificity ◽  
Its Region ◽  
Molecular Techniques ◽  
Isothermal Amplification ◽  
Content Type ◽  
Fungal Dna ◽  
Madurella Mycetomatis ◽  
Phenotypic Methods ◽  
Species Specific

Appropriate diagnosis and treatment of eumycetoma may vary significantly depending on the causative agent. To date, the most common fungus causing mycetoma worldwide isMadurella mycetomatis. This species fails to express any recognizable morphological characteristics, and reliable identification can therefore only be achieved with the application of molecular techniques. Recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) are proposed as alternatives to phenotypic methods. Species-specific primers were developed to target the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region ofM. mycetomatis. Both isothermal amplification techniques showed high specificity and sufficient sensitivity to amplify fungal DNA and proved to be appropriate for detection ofM. mycetomatis. Diagnostic performance of the techniques was assessed in comparison to conventional PCR using biopsy specimens from eumycetoma patients. RPA is reliable and easy to operate and has the potential to be implemented in areas where mycetoma is endemic. The techniques may be expanded to detect fungal DNA from environmental samples.

scholarly journals Effect of Incubation Temperature on the Detection of Thermophilic Campylobacter Species from Freshwater Beaches, Nearby Wastewater Effluents, and Bird Fecal Droppings

2013 ◽  
Vol 79 (24) ◽  
pp. 7639-7645 ◽  
Author(s):  
Izhar U. H. Khan ◽  
Stephen Hill ◽  
Eva Nowak ◽  
Thomas A. Edge
Keyword(s):  
Surface Water ◽  
Large Scale ◽  
Incubation Temperature ◽  
Its Region ◽  
23S Rrna ◽  
Rrna Gene ◽  
Content Type ◽  
C 32 ◽  
Species Specific ◽  
Incubation Temperatures

ABSTRACTThis large-scale study compared incubation temperatures (37°C versus 42°C) to study the detection of thermophilicCampylobacterspecies, includingCampylobacter jejuni,C. coli, andC. lari, in various surface water samples and bird fecal droppings around Hamilton Harbor, Lake Ontario. The putative culture isolates obtained from incubation temperatures of 37 and 42°C were confirmed byCampylobactergenus- and species-specific triplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA gene internal transcribed spacer (ITS) region. A total of 759 water, wastewater, and bird fecal dropping samples were tested. Positive amplification reactions for the genusCampylobacterwere found for 454 (60%) samples incubated at 37°C, compared to 258 (34%) samples incubated at 42°C.C. jejuni(16%) andC. lari(12%) were detected significantly more frequently at the 42°C incubation temperature than at 37°C (8% and 5%, respectively). In contrast, significantly higher rates ofC. coli(14%) and otherCampylobacterspp. (36%) were detected at the 37°C incubation temperature than at 42°C (8% and 7%, respectively). These results were consistent across surface water, wastewater, and bird fecal dropping samples. At times,Campylobacterspp. were recovered and detected at 37°C (3% forC. jejuni, 10% forC. coli, and 3% forC. lari) when the same samples incubated at 42°C were negative. A significantly higher rate of otherCampylobacterspp. was detected only at 37°C (32%) than only at 42°C (3%). These results indicate that incubation temperature can significantly influence the culturability and detection of thermophilic and other fastidiousCampylobacterspp. and that a comprehensive characterization of theCampylobacterspp. in surface water, wastewaters, or bird fecal droppings will require incubation at both 37 and 42°C.

scholarly journals Authentication of the Herbal Medicine Angelicae Dahuricae Radix Using an ITS Sequence-Based Multiplex SCAR Assay

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2134 ◽  
Author(s):  
Pureum Noh ◽  
Wook Kim ◽  
Sungyu Yang ◽  
Inkyu Park ◽  
Byeong Moon
Keyword(s):  
Herbal Medicines ◽  
Morphological Characteristics ◽  
Pcr Amplification ◽  
Its Region ◽  
Sequencing Analysis ◽  
Accurate Identification ◽  
Republic Of China ◽  
Sequence Characterized Amplified Region ◽  
Angelicae Dahuricae Radix ◽  
Species Specific

The accurate identification of plant species is of great concern for the quality control of herbal medicines. The Korean Pharmacopoeia and the Pharmacopoeia of the People’s Republic of China define Angelicae Dahuricae Radix (Baek-Ji in Korean and Bai-zhi in Chinese) as the dried roots of Angelica dahurica or A. dahurica var. formosana belonging to the family Apiaceae. Discrimination among Angelica species on the basis of morphological characteristics is difficult due to their extremely polymorphic traits and controversial taxonomic history. Furthermore, dried roots processed for medicinal applications are indistinguishable using conventional methods. DNA barcoding is a useful and reliable method for the identification of species. In this study, we sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes in A. dahurica, A. dahurica var. formosana, and the related species A. anomala and A. japonica. Using these sequences, we designed species-specific primers, and developed and optimized a multiplex sequence-characterized amplified region (SCAR) assay that can simply and rapidly identify respective species, and verify the contamination of adulterant depending on the polymerase chain reaction (PCR) amplification without sequencing analysis in a single PCR reaction. This assay successfully identified commercial samples of Angelicae Dahuricae Radix collected from Korean and Chinese herbal markets, and distinguished them from adulterants. This multiplex SCAR assay shows a great potential in reducing the time and cost involved in the identification of genuine Angelicae Dahuricae Radix and adulterant contamination.

scholarly journals Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin

2015 ◽  
Vol 54 (1) ◽  
pp. 208-211 ◽  
Author(s):  
Patrick McGann ◽  
Sarah Chahine ◽  
Darius Okafor ◽  
Ana C. Ong ◽  
Rosslyn Maybank ◽  
...  
Keyword(s):  
16S Rrna ◽  
High Specificity ◽  
Susceptibility Test ◽  
Methyltransferase Activity ◽  
Content Type ◽  
Phenotypic Methods ◽  
Disk Susceptibility ◽  
Standard Disk

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity inEnterobacteriaceaewith high specificity using the standard disk susceptibility test.

scholarly journals Loop-Mediated Isothermal Amplification (LAMP) Method for the Rapid Identification of Dendrobium officinale

2018 ◽  
Author(s):  
Lu Yang ◽  
Hua Zhou ◽  
Huili Lai ◽  
Fei Fu ◽  
Wenru Wu
Keyword(s):  
Color Change ◽  
Dna Barcode ◽  
Its Region ◽  
Rapid Identification ◽  
Isothermal Amplification ◽  
Dendrobium Officinale ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Species Specific

Background: Dendrobium officinale is not only an ornamental plant, but also a valuable medicinal herb that is both effective and widely used in traditional Chinese medicine. However, distinguishing D. officinale from other Dendrobium species is usually a difficult task that need much time and complex technologies due to their very similar external morphologies. The aim of this study is to develop a fast, even on-spot approach to identify D. officinale. Methods: We used DNA barcode-based loop-mediated isothermal amplification (LAMP) method with species-specific LAMP primers targeting the internal transcribed spacer (ITS) region of the rDNA of D. officinale. LAMP reaction time and temperature were optimized and the specificity and sensitivity of LAMP species-specific primers were assessed. Results: This technique showed a high specificity and sensitivity to amplify the genomic DNA of D. officinale and allowed for rapid amplification (within 40 min) of the ITS region under a constant and mild temperature range of 65 °C without using thermocyclers. Besides, by using SYBR® Green I dye as the color developing agent, the color change was easily observed with naked eye. Reaction mixture containing DNA of D. officinale changed from orange to green, while the other Dendrobium species and the negative control retained original orange color. The specificity of this LAMP-based method was confirmed by testing 17 samples of D. officinale and 32 adulterant samples from other Dendrobium species. Conclusions: This LAMP-based rapid identification method does not require expensive equipment or specialized techniques and can be used in field surveys for accurate and fast on site identification.

scholarly journals Application of molecular genetic methods for identification of wood-decaying fungi in wood constructions

2008 ◽  
Vol 56 (2) ◽  
pp. 281-284 ◽  
Author(s):  
Elena Bobeková ◽  
Michal Tomšovský ◽  
Petr Horáček
Keyword(s):  
Dna Isolation ◽  
Molecular Genetic ◽  
Its Region ◽  
Dry Rot ◽  
Wood Structures ◽  
Specific Pcr ◽  
Fungal Dna ◽  
Serpula Lacrymans ◽  
Species Specific ◽  
Soil Substrates

The aim of the paper is to evaluate the utilization of molecular biology methods for detection of wood decaying fungi directly from decomposed wood using a commercial DNA extraction kit developed for soil substrates (PowerSoil™ DNA isolation kit). The experiment based on dry rot fungus (Serpula lacrymans) detection from inoculated wooden pieces under laboratory conditions was followed by field detection of wood-decaying fungi from wood structures on building constructions. Fungal DNA was identified using the PCR–based methods including species-specific PCR and sequencing of amplified ITS region of ribosomal DNA.

Development of a species-specific PCR to detect the cereal cyst nematode, Heterodera latipons

Nematology ◽  
2013 ◽  
Vol 15 (6) ◽  
pp. 709-717 ◽  
Author(s):  
Fateh Toumi ◽  
Fateh Toumi ◽  
Lieven Waeyenberge ◽  
Fateh Toumi ◽  
Lieven Waeyenberge ◽  
...  
Keyword(s):  
Morphological Characteristics ◽  
Molecular Techniques ◽  
Actin Gene ◽  
Specific Primers ◽  
Second Stage ◽  
Specific Pcr ◽  
Pcr Products ◽  
Heterodera Latipons ◽  
Species Specific ◽  
Very High

Several Heterodera species can reduce the yield of wheat and barley, among which H. avenae, H. filipjevi and H. latipons are economically the most important. Their identification, based on morphological characteristics, is not straightforward but can be made easier using molecular techniques. In this study, we developed species-specific primers for the detection of H. latipons. The actin gene of eight Heterodera species was partially sequenced and, after purifying and sequencing the PCR products, all sequences were aligned to find unique sites. The alignment showed moderate to very high similarities between the species. However, a small fragment of the actin gene was suitable for the construction of a potentially useful species-specific primer for H. latipons. The optimised PCR was subsequently tested with several populations of 14 Heterodera species and a single population of Punctodera punctata. Heterodera latipons was represented by 16 populations originating from six different countries. The primer set (Hlat-act), designed using AlleleID 7.73, was shown to be very specific. To test its sensitivity further, the PCR was conducted on DNA extracted from five second-stage juveniles (J2) of H. latipons mixed with five or 100 J2 belonging to H. avenae. The PCR was able to detect up to 1:10 dilution of the DNA obtained from five J2. The results showed that a specific and sensitive H. latipons species-specific PCR was constructed.

scholarly journals Interactions between Head Blight Pathogens: Consequences for Disease Development and Toxin Production in Wheat Spikes

2014 ◽  
Vol 81 (3) ◽  
pp. 957-965 ◽  
Author(s):  
Dorothée Siou ◽  
Sandrine Gélisse ◽  
Valérie Laval ◽  
Sonia Elbelt ◽  
Cédric Repinçay ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Field Experiments ◽  
Field Studies ◽  
Toxin Production ◽  
Head Blight ◽  
Complex Species ◽  
Content Type ◽  
Significant Yield ◽  
Fungal Dna ◽  
Species Specific

ABSTRACTHead blight (HB) is one of the most damaging diseases on wheat, inducing significant yield losses and toxin accumulation in grains. Fungal pathogens responsible for HB include the genusMicrodochium, with two species, and the toxin producer genusFusarium, with several species. Field studies and surveys show that two or more species can coexist within a same field and coinfect the same plant or the same spike. In the current study, we investigated how the concomitant presence ofF. graminearumand another of the HB complex species influences the spike colonization and the toxin production by the fungi. To study these interactions, 17 well-characterized isolates representing five species were inoculated alone or in pairs on wheat spikes in greenhouse and field experiments. The fungal DNA in the grains was estimated by quantitative PCR and toxin contents (deoxynivalenol and nivalenol) by ultraperformance liquid chromatography-UV detection-tandem mass spectrometry. The responses of the different isolates to the presence of a competitor were variable and isolate specific more than species specific. The development of the most aggressive isolates was either unchanged or a slightly increased, while the development of the less aggressive isolates was reduced. The main outcome of the study was that no trend of increased toxin production was observed in coinoculations compared to single inoculations. On the contrary, the amount of toxin produced was often lower than expected in coinoculations. We thus conclude against the hypothesis that the co-occurrence of several HB-causing species in the same field might aggravate the risk linked to fusarium toxins in wheat production.

scholarly journals Evaluation of Daphnid Grazing on Microscopic Zoosporic Fungi by Using Comparative Threshold Cycle Quantitative PCR

2016 ◽  
Vol 82 (13) ◽  
pp. 3868-3874 ◽  
Author(s):  
Michelle A. Maier ◽  
Kimiko Uchii ◽  
Tawnya D. Peterson ◽  
Maiko Kagami
Keyword(s):  
Organic Matter ◽  
Food Web ◽  
Food Webs ◽  
Quantitative Pcr ◽  
Morphological Characteristics ◽  
Life Cycles ◽  
Molecular Techniques ◽  
Aquatic Environments ◽  
Content Type ◽  
Zoosporic Fungi

ABSTRACTLethal parasitism of large phytoplankton by chytrids (microscopic zoosporic fungi) may play an important role in organic matter and nutrient cycling in aquatic environments by shunting carbon away from hosts and into much smaller zoospores, which are more readily consumed by zooplankton. This pathway provides a mechanism to more efficiently retain carbon within food webs and reduce export losses. However, challenges in accurate identification and quantification of chytrids have prevented a robust assessment of the relative importance of parasitism for carbon and energy flows within aquatic systems. The use of molecular techniques has greatly advanced our ability to detect small, nondescript microorganisms in aquatic environments in recent years, including chytrids. We used quantitative PCR (qPCR) to quantify the consumption of zoospores byDaphniain laboratory experiments using a culture-based comparative threshold cycle (CT) method. We successfully quantified the reduction of zoospores in water samples duringDaphniagrazing and confirmed the presence of chytrid DNA inside the daphnid gut. We demonstrate that comparativeCTqPCR is a robust and effective method to quantify zoospores and evaluate zoospore grazing by zooplankton and will aid in better understanding how chytrids contribute to organic matter cycling and trophic energy transfer within food webs.IMPORTANCEThe study of aquatic fungi is often complicated by the fact that they possess complex life cycles that include a variety of morphological forms. Studies that rely on morphological characteristics to quantify the abundances of all stages of the fungal life cycle face the challenge of correctly identifying and enumerating the nondescript zoospores. These zoospores, however, provide an important trophic link between large colonial phytoplankton and zooplankton: that is, once the carbon is liberated from phytoplankton into the parasitic zoospores, the latter are consumed by zooplankton and carbon is retained in the aquatic food web rather than exported from the system. This study provides a tool to quantify zoospores and evaluate the consumption of zoospores by zooplankton in order to further our understanding of their role in food web dynamics.

scholarly journals Biological efficiency and nutritional composition of Pleurotus djamor cultivated on bagasse of Agave salmiana

2021 ◽  
Author(s):  
Brianda Susana Velázquez de Lucio ◽  
Edna María Hernández Domínguez ◽  
Xochitl Tovar Jiménez ◽  
Laura Sofia Castillo Ortega ◽  
Gerardo Díaz Godínez ◽  
...  
Keyword(s):  
Production Rate ◽  
Morphological Characteristics ◽  
Its Region ◽  
Nutritional Composition ◽  
Molecular Techniques ◽  
Biological Efficiency ◽  
Fruiting Bodies ◽  
Suitable Alternative ◽  
Pleurotus Djamor ◽  
Nitrogen Concentrations

Abstract The objective of the present study was to use molecular techniques to identify a wild mushroom isolated from A. salmiana, and then evaluate its biological efficiency, production rate, and nutritional and morphological characteristics when grown on A. salmiana bagasse with various concentrations of urea as a source of nitrogen. Two types of inoculum were employed: in grain (WG) and pellet (WP) form. The substrate was supplemented with total nitrogen concentrations (TN) of 0.77, 0.95, 1.14, 1.32, and 1.5% to evaluate its effect on the biological efficiency (BE), production rate (PR), and morphological and nutritional characteristics of the fruiting bodies. The molecular analysis of the ITS region permitted the amplification of a product of 750 pb. The mushroom was identified as Pleurotus djamor. After supplementing the substrate with urea, a BE of 70% was obtained in the sample inoculated with WG at 1.32% TN. Observations found that the TN concentration of 1.5% produced malformations in the fruiting bodies. The analysis of the sporocarps indicated a raw protein content (RP) of 15–26% and that the mushroom’s nutritional composition changed according to the inoculum utilized and the percentage of nitrogen in the substrate. This is the first report on the isolation of P. djamor on A. salmiana as an atypical substrate, and so represents an opportunity for further study and commercialization. To its chemical composition and high availability, A. salmiana bagasse is a suitable alternative substrate for cultivating edible mushrooms, specifically P. djamor.

scholarly journals PCR-Based Strategy To Detect and Identify Species of Phaeoacremonium Causing Grapevine Diseases

2007 ◽  
Vol 73 (9) ◽  
pp. 2911-2918 ◽  
Author(s):  
Angeles Aroca ◽  
Rosa Raposo
Keyword(s):  
Morphological Characteristics ◽  
Pcr Amplification ◽  
Its Region ◽  
Restriction Enzymes ◽  
Effective Measure ◽  
Petri Disease ◽  
Specific Pcr ◽  
Plant Nursery ◽  
Species Specific ◽  
Single Amplicon

ABSTRACT Species of Phaeoacremonium (especially Phaeoacremonium aleophilum) are associated with two severe diseases in grapevines, Petri disease in young plants and Esca disease in adult plants. Phaeoacremonium species grow slowly on culture medium, and it is difficult to identify these species on the basis of morphological characteristics. Primers Pm1 and Pm2 were designed in the ribosomal DNA internal transcribed spacer (ITS) regions ITS1 and ITS2, respectively. They yielded a single amplicon of 415 bp for nine species of Phaeoacremonium that may occur in grapevines. A nested PCR (using general fungal primers ITS1F/ITS4 in the primary reaction) was developed to detect Phaeoacremonium directly in grapevine wood. Molecular detection was more sensitive than the traditional method of culturing in growth medium was. Identification of Phaeoacremonium species was achieved by digesting the PCR-amplified fragment with the restriction enzymes BssKI, EcoO109I, and HhaI. It was possible to distinguish these species by their restriction fragment length polymorphism patterns, except for Phaeoacremonium viticola and Phaeoacremonium angustius, which had 100% similarity in their ITS region sequences. A species-specific PCR amplification of the partial β-tubulin gene using the primer pair Pbr4_1/T1 and Pbr8/T1 was necessary to differentiate P. angustius from P. viticola, respectively. An easy and fast protocol was developed to detect and identify species of Phaeoacremonium in a few hours. Primers defined here can be used in a plant nursery sanitation program to produce plants free of Phaeoacremonium spp. Use of healthy grapevine plants in new plantations is the most effective measure to manage Petri disease.

Sign in / Sign up

Export Citation Format

Share Document