A new semi-nested PCR protocol to amplify large 18S rRNA gene fragments for PCR-DGGE analysis of soil fungal communities

2006 ◽  
Vol 65 (1) ◽  
pp. 63-75 ◽  
Author(s):  
Miruna Oros-Sichler ◽  
Newton C.M. Gomes ◽  
Gabriele Neuber ◽  
Kornelia Smalla
2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Jasem Saki ◽  
Masoud Foroutan-Rad ◽  
Reza Asadpouri

Background. Rodents could act as reservoir forCryptosporidiumspp. speciallyC. parvum, a zoonotic agent responsible for human infections. Since there is no information aboutCryptosporidiuminfection in rodents of Ahvaz city, southwest of Iran, hence, this survey was performed to determine the prevalence and molecular characterization ofCryptosporidiumspp. in this region.Materials and Methods. One hundred rodents were trapped from different regions of Ahvaz city. Intestine contents and fecal specimens of rodents were studied using both microscopy examination to identify oocyst and nested-polymerase chain reaction (PCR) technique for 18s rRNA gene detection. Eventually restriction fragment length polymorphism (RFLP) method usingSspIandVspIrestriction enzymes was carried out to genotype the species and then obtained results were sequenced.Results. Three out of 100 samples were diagnosed as positive and overall prevalence ofCryptosporidiumspp. was 3% using both modified Ziehl-Neelsen staining under light microscope and nested-PCR (830 bp) methods. Afterwards, PCR-RFLP was performed on positive samples andC. parvumpattern was identified. Finally PCR-RFLP findings were sequenced and presence ofC. parvumwas confirmed again.Conclusions. Our study showed rodents could be potential reservoir forC. parvum. So an integrated program for control and combat with them should be adopted and continued.


Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1061
Author(s):  
Olusesan Adeyemi Adelabu ◽  
Benson Chuks Iweriebor ◽  
Anthony Ifeanyi Okoh ◽  
Larry Chikwelu Obi

Importation of tick-infected animals and the uncontrollable migration of birds and wild animals across borders can lead to geographical expansion and redistribution of ticks and pathogen vectors, thus leading to the emergence and re-emergence of tick-borne diseases in humans and animals. Comparatively, little is known about the occurrence of piroplasms in ixodid ticks in the Eastern Cape, South Africa, thus necessitating this study, which is aimed at detecting piroplasms (Theileria and Babesia) from feeding tick samples collected from cattle, sheep, and goats in selected sites in the Eastern Cape, South Africa. A total of 1200 feeding ixodid ticks collected from farm animals at selected homesteads were first subjected to molecular identification using mitochondrial 12S ribosomal RNA (rRNA) gene by PCR and were further tested for the presence of piroplasms through amplification of the 18S rRNA gene via nested-PCR followed by sequencing of the PCR products. The results indicated that 853 (71.1%) corresponded to the genus Rhipicephalus, 335 (27.9%) corresponded to genus Amblyomma, and 12 (1%) corresponded to genus Haemaphysalis. Amblyomma hebraeum and Rhipicephalus appendiculatus were the most common identified ticks from this study. The 18S rRNA nested-PCR revealed that 44 (3.7%) samples were confirmed positive for Theileria. A homology search for the generated sequences revealed a high percentage identity of 98–98.9% similarity to T. buffeli, T. orientalis, and T. sergenti in the GenBank. Based on the results obtained herein, we conclude that there is a big diversity of Theileria species; therefore, we suggest that this research should cover more geographical areas in order to reveal the true prevalence of this pathogen in the studied area because this will be a great step in the possible prevention of an outbreak that could have devastating effects on livestock production and human health in both the studied areas and South Africa at large.


2017 ◽  
Vol 7 (9) ◽  
pp. 548-552 ◽  
Author(s):  
Mohd Aiman Barudin ◽  
◽  
Muhammad Lokman Md Isa ◽  
Noratikah Othman ◽  
Afzan Mat Yusof ◽  
...  

2015 ◽  
Vol 5 (3) ◽  
pp. 202-207 ◽  
Author(s):  
AMAM Zonaed Siddiki ◽  
Sohana Akter Mina ◽  
Zinat Farzana ◽  
Bibi Ayesa ◽  
Rasel Das ◽  
...  

2018 ◽  
Vol 84 (21) ◽  
Author(s):  
Yingbo Duan ◽  
Ningdong Xie ◽  
Zhiquan Song ◽  
Christopher S. Ward ◽  
Cheuk-Man Yung ◽  
...  

ABSTRACTThere is a growing awareness of the ecological and biogeochemical importance of fungi in coastal marine systems. While highly diverse fungi have been discovered in these marine systems, still, little is known about their seasonality and associated drivers in coastal waters. Here, we examined fungal communities over 3 years of weekly sampling at a dynamic, temperate coastal site (Pivers Island Coastal Observatory [PICO], Beaufort, NC, USA). Fungal 18S rRNA gene abundance, operational taxonomic unit (OTU) richness, and Shannon's diversity index values exhibited prominent seasonality. Fungal 18S rRNA gene copies peaked in abundance during the summer and fall, with positive correlations with chlorophylla, SiO4, and oxygen saturation. Diversity (measured using internal transcribed spacer [ITS] libraries) was highest during winter and lowest during summer; it was linked to temperature, pH, chlorophylla, insolation, salinity, and dissolved inorganic carbon (DIC). Fungal communities derived from ITS libraries were dominated throughout the year byAscomycota, with contributions fromBasidiomycota,Chytridiomycota, andMucoromycotina, and their seasonal patterns linked to water temperature, light, and the carbonate system. Network analysis revealed that while cooccurrence and exclusion existed within fungus networks, exclusion dominated the fungus-and-phytoplankton network, in contrast with reported pathogenic and nutritional interactions between marine phytoplankton and fungi. Compared with the seasonality of bacterial communities in the same samples, the timing, extent, and associated environmental variables for fungi community are unique. These results highlight the fungal seasonal dynamics in coastal water and improve our understanding of the ecology of planktonic fungi.IMPORTANCECoastal fungal dynamics were long assumed to be due to terrestrial inputs; here, a high-resolution time series reveals strong, repeating annual patterns linked toin situenvironmental conditions, arguing for a resident coastal fungal community shaped by environmental factors. These seasonal patterns do, however, differ from those observed in the bacterioplankton at the same site; e.g., fungal diversity peaks in winter, whereas bacterial diversity maxima occur in the spring and fall. While the dynamics of these communities are linked to water temperature and insolation, fungi are also influenced by the carbonate system (pH and DIC). As both fungi and heterotrophic bacteria are thought to be key organic-material metabolizers, differences in their environmental drivers may offer clues as to which group dominates secondary production at this dynamic site. Overall, this study suggests the unique ecological roles of mycoplankton and their potentially broad niche complementarities to other microbial groups in the coastal ocean.


2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Claire Y. T. Wang ◽  
Emma L. Ballard ◽  
Zuleima Pava ◽  
Louise Marquart ◽  
Jane Gaydon ◽  
...  

Abstract Background Volunteer infection studies have become a standard model for evaluating drug efficacy against Plasmodium infections. Molecular techniques such as qPCR are used in these studies due to their ability to provide robust and accurate estimates of parasitaemia at increased sensitivity compared to microscopy. The validity and reliability of assays need to be ensured when used to evaluate the efficacy of candidate drugs in clinical trials. Methods A previously described 18S rRNA gene qPCR assay for quantifying Plasmodium falciparum in blood samples was evaluated. Assay performance characteristics including analytical sensitivity, reportable range, precision, accuracy and specificity were assessed using experimental data and data compiled from phase 1 volunteer infection studies conducted between 2013 and 2019. Guidelines for validation of laboratory-developed molecular assays were followed. Results The reportable range was 1.50 to 6.50 log10 parasites/mL with a limit of detection of 2.045 log10 parasites/mL of whole blood based on a parasite diluted standard series over this range. The assay was highly reproducible with minimal intra-assay (SD = 0.456 quantification cycle (Cq) units [0.137 log10 parasites/mL] over 21 replicates) and inter-assay (SD = 0.604 Cq units [0.182 log10 parasites/mL] over 786 qPCR runs) variability. Through an external quality assurance program, the QIMR assay was shown to generate accurate results (quantitative bias + 0.019 log10 parasites/mL against nominal values). Specificity was 100% after assessing 164 parasite-free human blood samples. Conclusions The 18S rRNA gene qPCR assay is specific and highly reproducible and can provide reliable and accurate parasite quantification. The assay is considered fit for use in evaluating drug efficacy in malaria clinical trials.


Polar Biology ◽  
2021 ◽  
Author(s):  
Eleanor E. Jackson ◽  
Ian Hawes ◽  
Anne D. Jungblut

AbstractThe undulating ice of the McMurdo Ice Shelf, Southern Victoria Land, supports one of the largest networks of ice-based, multiyear meltwater pond habitats in Antarctica, where microbial mats are abundant and contribute most of the biomass and biodiversity. We used 16S rRNA and 18S rRNA gene high-throughput sequencing to compare variance of the community structure in microbial mats within and between ponds with different salinities and pH. Proteobacteria and Cyanobacteria were the most abundant phyla, and composition at OTU level was highly specific for the meltwater ponds with strong community sorting along the salinity gradient. Our study provides the first detailed evaluation of eukaryote communities for the McMurdo Ice Shelf using the 18S rRNA gene. They were dominated by Ochrophyta, Chlorophyta and Ciliophora, consistent with previous microscopic analyses, but many OTUs belonging to less well-described heterotrophic protists from Antarctic ice shelves were also identified including Amoebozoa, Rhizaria and Labyrinthulea. Comparison of 16S and 18S rRNA gene communities showed that the Eukaryotes had lower richness and greater similarity between ponds in comparison with Bacteria and Archaea communities on the McMurdo Ice shelf. While there was a weak correlation between community dissimilarity and geographic distance, the congruity of microbial assemblages within ponds, especially for Bacteria and Archaea, implies strong habitat filtering in ice shelf meltwater pond ecosystems, especially due to salinity. These findings help to understand processes that are important in sustaining biodiversity and the impact of climate change on ice-based aquatic habitats in Antarctica.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tsuyoshi Watanabe ◽  
Satoshi Nagai ◽  
Yoko Kawakami ◽  
Taiga Asakura ◽  
Jun Kikuchi ◽  
...  

AbstractEel larvae apparently feed on marine snow, but many aspects of their feeding ecology remain unknown. The eukaryotic 18S rRNA gene sequence compositions in the gut contents of four taxa of anguilliform eel larvae were compared with the sequence compositions of vertically sampled seawater particulate organic matter (POM) in the oligotrophic western North Pacific Ocean. Both gut contents and POM were mainly composed of dinoflagellates as well as other phytoplankton (cryptophytes and diatoms) and zooplankton (ciliophoran and copepod) sequences. Gut contents also contained cryptophyte and ciliophoran genera and a few other taxa. Dinoflagellates (family Gymnodiniaceae) may be an important food source and these phytoplankton were predominant in gut contents and POM as evidenced by DNA analysis and phytoplankton cell counting. The compositions of the gut contents were not specific to the species of eel larvae or the different sampling areas, and they were most similar to POM at the chlorophyll maximum in the upper part of the thermocline (mean depth: 112 m). Our results are consistent with eel larvae feeding on marine snow at a low trophic level, and feeding may frequently occur in the chlorophyll maximum in the western North Pacific.


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