Specific detection of fungal pathogens by 18S rRNA gene PCR in microbial keratitis

ABSTRACT rRNA genes are attractive targets for developing PCR assays targeting human fungal pathogens. Most studies have focused on the 18S rRNA gene, internal transcribed spacers, and the 5′ end of the 28S rRNA gene. An approximately 2,900-bp region of the 28S rRNA gene remains largely unexplored because sequences of many medically relevant fungi are either unavailable or undefined in genomic databases. The internal transcribed spacers and 28S rRNA gene of nine medically and phylogenetically important fungi were sequenced. In addition, 42 sequences from this region were acquired from public databases, resulting in an alignment of 51 fungal sequences spanning 30 fungal genera. For the nearly 3,950-bp region from the 3′ end of 18S rRNA gene to the 3′ end of the 28S rRNA gene, 27 broad-range PCR primers were designed such that their sequence homology with the human rRNA gene was minimal. All 62 possible amplicons in the size range from 75 to 400 bp from 27 primers were screened using fungal genomic DNA from 26 species spanning 14 genera. Eleven of the 62 amplicons did not cross-react with 1 μg/PCR human DNA but simultaneously amplified 10 fg of fungal DNA. Phylogenetic distance matrices were calculated for regions covered by these 11 amplicons based on 51 fungi. Two of these 11 amplicons successfully amplified 30 fg of fungal DNA from 25 of 26 fungi and provided the most phylogenetic information for species identification based on the distance matrices. These PCR assays hold promise for detection and identification of fungal pathogens in human tissues.

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Design of primer pairs for species-specific diagnosis of Leishmania (Leishmania) infantum chagasi using PCR

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612012000300024 ◽

2012 ◽

Vol 21 (3) ◽

pp. 304-307 ◽

Cited By ~ 4

Author(s):

Osvaldo José da Silveira Neto ◽

Sabrina Castilho Duarte ◽

Hérika Xavier da Costa ◽

Guido Fontgalland Coelho Linhares

Keyword(s):

18S Rrna ◽

18S Rrna Gene ◽

Reference Sequence ◽

Rrna Gene ◽

Analytical Sensitivity ◽

Specific Detection ◽

Pcr Products ◽

Specific Amplification ◽

Pcr Assays ◽

Species Specific

The objective of this study was to design and evaluate new primers for species-specific detection of L. infantum chagasi using PCR. Two combinations of primer pairs were established with the aim of obtaining specific amplification products from the L. infantum chagasi 18S rRNA gene. The combinations of the primer pairs and the respective sizes of the PCR products, based on the U422465 GenBank reference sequence of L. infantum chagasi, were: LCS1/LCS3 (259 bp) and LCS2/LCS3 (820 bp). It was concluded that the new PCR assays optimized using the primer pairs LCS1/LCS3 and LCS2/LCS3 were effective for specific detection of L. infantum chagasi, with analytical sensitivity to detect 1 pg/µL of DNA.

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Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS)

Microbiome ◽

10.1186/s40168-021-01180-0 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Kevin Xu Zhong ◽

Anna Cho ◽

Christoph M. Deeg ◽

Amy M. Chan ◽

Curtis A. Suttle

Keyword(s):

Fungal Pathogens ◽

18S Rrna ◽

Amplicon Sequencing ◽

18S Rrna Gene ◽

Model Organisms ◽

Rrna Gene ◽

Gene Sequences ◽

Oyster Spat ◽

16S Rna ◽

Eukaryotic Microbes

Abstract Background The microbiome affects the health of plants and animals, including humans, and has many biological, ecological, and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbes this approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped by contaminating host rRNA gene sequences. Results To overcome this problem, we developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS), a high-resolution and efficient approach for characterizing eukaryotic microbiomes. CCSAS uses taxon-specific single-guide RNA (sgRNA) to direct Cas9 to cut 18S rRNA gene sequences of the host, while leaving protistan and fungal sequences intact. We validated the specificity of the sgRNA on ten model organisms and an artificially constructed (mock) community of nine protistan and fungal pathogens. The results showed that > 96.5% of host rRNA gene amplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used to assess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community of eukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than other methods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that were not detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryotic microbiome in complex communities. To facilitate the application of CCSAS, we designed taxon-specific sgRNA for ~16,000 metazoan and plant taxa, making CCSAS widely available for characterizing eukaryotic microbiomes that have largely been neglected. Conclusion CCSAS provides a high-through-put and cost-effective approach for resolving the eukaryotic microbiome of metazoa and plants with minimal contamination from host 18S rRNA gene sequences.

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0591 - 16S and 18S rRNA gene metabarcoding show comparable responses of sediment communities to eutrophication

10.26226/morressier.5b5199bfb1b87b000ecef7ad ◽

2018 ◽

Author(s):

Jesse Harrison ◽

Myrsini Chronopoulou

Keyword(s):

18S Rrna ◽

18S Rrna Gene ◽

Rrna Gene

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Analytical validation of a real-time hydrolysis probe PCR assay for quantifying Plasmodium falciparum parasites in experimentally infected human adults

Malaria Journal ◽

10.1186/s12936-021-03717-y ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Claire Y. T. Wang ◽

Emma L. Ballard ◽

Zuleima Pava ◽

Louise Marquart ◽

Jane Gaydon ◽

...

Keyword(s):

Clinical Trials ◽

Plasmodium Falciparum ◽

18S Rrna ◽

Drug Efficacy ◽

18S Rrna Gene ◽

Molecular Techniques ◽

Qpcr Assay ◽

Rrna Gene ◽

Analytical Sensitivity ◽

Blood Samples

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.

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16S rRNA gene and 18S rRNA gene diversity in microbial mat communities in meltwater ponds on the McMurdo Ice Shelf, Antarctica

Polar Biology ◽

10.1007/s00300-021-02843-2 ◽

2021 ◽

Author(s):

Eleanor E. Jackson ◽

Ian Hawes ◽

Anne D. Jungblut

Keyword(s):

16S Rrna ◽

18S Rrna ◽

High Throughput Sequencing ◽

Microbial Mats ◽

Gene Diversity ◽

Salinity Gradient ◽

18S Rrna Gene ◽

Rrna Gene ◽

Ice Shelf ◽

The Impact

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.

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18S rRNA gene sequences of leptocephalus gut contents, particulate organic matter, and biological oceanographic conditions in the western North Pacific

Scientific Reports ◽

10.1038/s41598-021-84532-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tsuyoshi Watanabe ◽

Satoshi Nagai ◽

Yoko Kawakami ◽

Taiga Asakura ◽

Jun Kikuchi ◽

...

Keyword(s):

Organic Matter ◽

Particulate Organic Matter ◽

North Pacific ◽

Western North Pacific ◽

18S Rrna ◽

18S Rrna Gene ◽

Gut Contents ◽

Rrna Gene ◽

Marine Snow ◽

Chlorophyll Maximum

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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Molecular Characterization of Ciliate Diversity in Stream Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.01438-07 ◽

2008 ◽

Vol 74 (6) ◽

pp. 1740-1747 ◽

Cited By ~ 52

Author(s):

Andrew Dopheide ◽

Gavin Lear ◽

Rebecca Stott ◽

Gillian Lewis

Keyword(s):

18S Rrna ◽

Pcr Primers ◽

18S Rrna Gene ◽

Sequence Diversity ◽

Rrna Genes ◽

Rrna Gene ◽

Sequencing Analysis ◽

Specific Pcr ◽

Stream Biofilms

ABSTRACT Free-living protozoa are thought to be of fundamental importance in aquatic ecosystems, but there is limited understanding of their diversity and ecological role, particularly in surface-associated communities such as biofilms. Existing eukaryote-specific PCR primers were used to survey 18S rRNA gene sequence diversity in stream biofilms but poorly revealed protozoan diversity, demonstrating a need for protozoan-targeted primers. Group-specific PCR primers targeting 18S rRNA genes of the protozoan phylum Ciliophora were therefore designed and tested using DNA extracted from cultured protozoan isolates. The two most reliable primer combinations were applied to stream biofilm DNA, followed by cloning and sequencing analysis. Of 44 clones derived from primer set 384F/1147R, 86% were of probable ciliate origin, as were 25% of 44 clones detected by primer set 121F/1147R. A further 29% of 121F/1147R-detected clones matched sequences from the closely related phylum Apicomplexa. The highly ciliate-specific primer set 384F/1147R was subsequently used in PCRs on biofilm DNA from four streams exhibiting different levels of human impact, revealing differences in ciliate sequence diversity in samples from each site. Of a total of 240 clones, 73% were of probable ciliate origin; 54 different putative ciliate sequences were detected from throughout seven taxonomic ciliate classes. Sequences from Oligohymenophorea were most commonly detected in all samples, followed by either Spirotrichea or Phyllopharyngea. Restriction fragment length polymorphism profile-based analysis of clones suggested a potentially higher level of diversity than did sequencing. Nevertheless, newly designed PCR primers 384F/1147R were considered to provide an effective molecular basis for characterization of ciliate diversity in stream biofilms.

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PHYLOGENETIC RELATIONSHIPS OF THE FRESHWATER ALGA BOLDIA ERYTHROSIPHON (COMPSOPOGONALES, RHODOPHYTA) BASED ON 18S rRNA GENE SEQUENCES

Journal of Phycology ◽

10.1046/j.1529-8817.1998.340555.x ◽

1998 ◽

Vol 34 (3) ◽

pp. 555-557 ◽

Cited By ~ 3

Author(s):

Raymond W. Holton ◽

Stella A. Boele-Bos ◽

Wytze T. Stam

Keyword(s):

Phylogenetic Relationships ◽

18S Rrna ◽

18S Rrna Gene ◽

Freshwater Alga ◽

Rrna Gene ◽

Gene Sequences

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Crassicauda boopis in a fin whale (Balaenoptera physalus) ship-struck in the eastern North Atlantic Ocean

Parasitology Open ◽

10.1017/pao.2017.10 ◽

2017 ◽

Vol 3 ◽

Cited By ~ 1

Author(s):

LAETITIA LEMPEREUR ◽

MORGAN DELOBELLE ◽

MARJAN DOOM ◽

JAN HAELTERS ◽

ETIENNE LEVY ◽

...

Keyword(s):

North Atlantic ◽

18S Rrna ◽

North Atlantic Ocean ◽

18S Rrna Gene ◽

The Body ◽

Rrna Gene ◽

Balaenoptera Physalus ◽

Fin Whale ◽

Eastern North Atlantic ◽

The Right

SUMMARY On 9 November 2015, a juvenile male fin whale of 11·60 m length was observed on the bulb of a merchant vessel in the Channel Terneuzen – Ghent (The Netherlands – Belgium). A severe parasitosis was present in the right heart ventricle and caudal caval vein. Parasites were identified as Crassicauda boopis based on macroscopic and microscopic observations. The sequence of the 18S rRNA gene obtained from the parasite samples was 100% similar to the sequence of the 18S rRNA gene from Crassicauda magna available on GenBank. While adults of C. boopis and C. magna are morphologically distinct and found at different locations in the body, the molecular analysis of the 18S rRNA gene seems insufficient for reliable species identification. Although numerous C. boopis were found, the cause of death was identified as due to the collision with the ship, as suggested by the presence of a large haematoma, and the absence of evidence of renal failure. The young age of this whale and the absence of severe chronic reaction may suggest that the infestation was not yet at an advanced chronic stage.

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