Sequencing and Analysis of Fungal rRNA Operons for Development of Broad-Range Fungal PCR Assays

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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Diversity of cultured photosynthetic flagellates in the North East Pacific and Arctic Oceans in summer

Biogeosciences Discussions ◽

10.5194/bgd-9-6219-2012 ◽

2012 ◽

Vol 9 (6) ◽

pp. 6219-6259 ◽

Cited By ~ 3

Author(s):

S. Balzano ◽

P. Gourvil ◽

R. Siano ◽

M. Chanoine ◽

D. Marie ◽

...

Keyword(s):

18S Rrna ◽

Gene Sequencing ◽

18S Rrna Gene ◽

Rrna Gene ◽

28S Rrna ◽

28S Rrna Gene ◽

North East ◽

The North ◽

Green Algal ◽

Rrna Gene Sequencing

Abstract. During the MALINA cruise (summer 2009) an extensive effort was undertaken to isolate phytoplankton strains from the North East (NE) Pacific Ocean, the Bering Strait, and the Beaufort Sea. Strains were isolated by flow cytometry sorting (FCS) and pipetting before or after phytoplankton enrichment of seawater samples. Strains were isolated both onboard and back in the laboratory and cultured at 4 °C under light/dark conditions. Overall, we isolated and characterised by light microscopy and 18S rRNA gene sequencing 104 strains of photosynthetic flagellates which grouped into 21 genotypes (defined by 99.5% 18S rRNA gene sequence similarity) mainly affiliated to Chlorophyta and Heterokontophyta. The taxon most frequently isolated was an Arctic ecotype of the green algal genus Micromonas (Arctic Micromonas) which was almost the only phytoplankter recovered within picoplankton (≤ 2 μm) size range. Strains of Arctic Micromonas as well as three unidentified strains related to the same genus were identified in further details by sequencing the Internal Transcribed Spacer (ITS) region of the rRNA operon. The MALINA Micromonas strains share identical 18S rRNA and ITS sequences suggesting high genetic homogeneity within Arctic Micromonas. The unidentified strains form a genotype likely belonging to a new genus within the family Mamiellaceae to which Micromonas belongs. Other green algae genotypes from the genera Nephroselmis, Chlamydomonas, Pyramimonas were also isolated whereas Heterokontophyta included Pelagophyceae, Dictyochophyceae and Chrysophyceae. Dictyochophyceae included Pedinellales which could not be identified to the genus level whereas Chrysophyceae comprised Dinobryon faculiferum. Moreover, we isolated Rhodomonas sp. as well as a few Haptophyta and dinoflagellates. We identified the dinoflagellate Woloszynskia cincta by Scanning Electron Microscopy (SEM) and 28S rRNA gene sequencing. Our morphological analyses show that this species possess the diagnostic features of the genus Biecheleria, and the 28S rRNA gene topology corroborates this affiliation. We thus propose the transfer of W. cincta to the genus Biecheleria and its recombination as Biecheleria cincta.

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Finding Sarcocystis spp. on the Tioman Island: 28S rRNA gene next-generation sequencing reveals nine new Sarcocystis species

Journal of Water and Health ◽

10.2166/wh.2019.124 ◽

2019 ◽

Vol 17 (3) ◽

pp. 416-427

Author(s):

Florence C. H. Lee

Keyword(s):

Next Generation Sequencing ◽

Water Samples ◽

18S Rrna ◽

18S Rrna Gene ◽

Rrna Gene ◽

28S Rrna ◽

Sarcocystis Species ◽

Next Generation ◽

28S Rrna Gene ◽

Generation Sequencing

Abstract The Tioman Island of Malaysia experienced acute muscular sarcocystosis outbreaks from 2011 to 2014. So far, a previous study based on the 18S rRNA gene sequencing has reported S. singaporensis, S. nesbitti and Sarcocystis sp. YLL-2013 in water samples acquired from the island, thus confirming the waterborne nature of this emerging parasitic disease. This study aimed to improve the detection methods for Sarcocystis, in order to have a clearer picture of the true diversity of Sarcocystis species in Tioman. A new primer set (28S R7F–28S R8 Deg R) was designed to amplify the 28S rRNA gene of Sarcocystis. Subsequently, Sarcocystidae was detected in 65.6% (21/32) of water samples and 28% (7/25) of soil samples acquired between 2014 and 2015 from Tioman. Next-generation sequencing (NGS) on 18 of the positive samples was then performed using amplicons generated from the same primer set. This yielded 53 potentially unique Sarcocystidae sequences (290 bp), of which nine of the most abundant, prevalent and unique sequences were named herein. In contrast, NGS of the 18S rRNA gene V9 hypervariable region of 10 selected samples detected only two Sarcocystis species (160 bp). S. mantioni was the most ubiquitous sequence found in this study.

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Morphological and molecular characterization of Mermisnigrescens Dujardin, (Nematoda: Mermithidae) parasitizing the introduced European earwig (Dermaptera: Forficulidae) in New Zealand

Journal of Helminthology ◽

10.1017/s0022149x14000017 ◽

2014 ◽

Vol 89 (3) ◽

pp. 267-276 ◽

Cited By ~ 7

Author(s):

B. Presswell ◽

S. Evans ◽

R. Poulin ◽

F. Jorge

Keyword(s):

New Zealand ◽

18S Rrna ◽

Sequence Data ◽

18S Rrna Gene ◽

Parasitic Nematodes ◽

Rrna Gene ◽

28S Rrna ◽

Forficula Auricularia ◽

Adult Females ◽

European Earwig

AbstractParasitic nematodes of the family Mermithidae were found to be infecting the introduced European earwig Forficula auricularia (Dermaptera: Forficulidae) in Dunedin, South Island, New Zealand. Adult females were later collected from various garden plants while depositing eggs. These mermithid specimens were identified morphologically as Mermis nigrescens Dujardin, 1842. A genetic distance of 0.7% between these specimens and a M. nigrescens isolate from Canada (18S rRNA gene), suggests that they have diverged genetically, but there are currently no available comparable sequences for the European M. nigrescens. Two additional nuclear fragments were also amplified, the 28S rRNA and the ribosomal DNA first internal transcribed spacer (ITS1), providing a basis for future studies. Bearing in mind the morphological similarity with other reported M. nigrescens and the lack of sequence data from other parts of the world, we retain the name M.nigrescens, and suggest that the species may be found to represent a complex of cryptic species when more worldwide data are available. Herein, we present a brief description of the post-parasitic worms and adult females, along with an inferred phylogeny using 18S rRNA gene sequences.

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Molecular characterisation of two known species of Paratylenchus Micoletzky, 1922 from Iran with notes on the validity of Paratylenchus audriellus Brown, 1959

Nematology ◽

10.1163/15685411-00002979 ◽

2016 ◽

Vol 18 (5) ◽

pp. 591-604 ◽

Cited By ~ 4

Author(s):

Mehrab Esmaeili ◽

Ramin Heydari ◽

Pablo Castillo ◽

Mozhgan Ziaie Bidhendi ◽

Juan E. Palomares-Rius

Keyword(s):

18S Rrna ◽

First Record ◽

18S Rrna Gene ◽

Rrna Gene ◽

28S Rrna ◽

Molecular Characterisation ◽

Female Tail ◽

Valid Taxon ◽

Two Populations ◽

Partial 18S

During a survey on pin nematodes in western Iran, two populations of Paratylenchus audriellus and Paratylenchus tenuicaudatus were collected and subsequently analysed morphologically and molecularly. Paratylenchus audriellus is characterised by the long stylet (48-61 μm) and the typical female tail with a characteristic claw-like process with sharply pointed terminus. To our knowledge, the Iranian population of P. tenuicaudatus is the first record from Iran. The molecular characterisation of P. audriellus nematodes using the D2-D3 of 28S rRNA and the partial 18S rRNA gene sequences revealed that this species is clearly separated from P. straeleni and should be considered as a valid taxon.

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Detection of Four Plasmodium Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays

Journal of Clinical Microbiology ◽

10.1128/jcm.42.12.5636-5643.2004 ◽

2004 ◽

Vol 42 (12) ◽

pp. 5636-5643 ◽

Cited By ~ 215

Author(s):

M. Rougemont ◽

M. Van Saanen ◽

R. Sahli ◽

H. P. Hinrikson ◽

J. Bille ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

18S Rrna ◽

Plasmodium Species ◽

18S Rrna Gene ◽

Rrna Gene ◽

Pcr Assays ◽

Species Specific

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

BMC Ophthalmology ◽

10.1186/1471-2415-8-7 ◽

2008 ◽

Vol 8 (1) ◽

Cited By ~ 31

Author(s):

Zunaina Embong ◽

Wan Hazabbah Wan Hitam ◽

Chan Yean Yean ◽

Nur Haslindawaty Abdul Rashid ◽

Balqis Kamarudin ◽

...

Keyword(s):

Fungal Pathogens ◽

18S Rrna ◽

18S Rrna Gene ◽

Rrna Gene ◽

Microbial Keratitis ◽

Specific Detection

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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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Morphological and molecular characterisation of Xiphinema ifacolum Luc, 1961 (Nematoda: Longidoridae) from Sri Lanka

Nematology ◽

10.1163/15685411-00003187 ◽

2018 ◽

Vol 20 (10) ◽

pp. 925-937 ◽

Cited By ~ 1

Author(s):

Solomia Susulovska ◽

Carolina Cantalapiedra-Navarrete ◽

Andrij Susulovsky ◽

Pablo Castillo ◽

Antonio Archidona-Yuste

Keyword(s):

Sri Lanka ◽

18S Rrna ◽

Mitochondrial Gene ◽

Molecular Data ◽

18S Rrna Gene ◽

Rrna Gene ◽

28S Rrna ◽

Molecular Characterisation ◽

Group 4 ◽

Sao Tome And Principe

Summary Females and juveniles from a population of Xiphinema ifacolum from Sri Lanka are described based on morphology, morphometrics and molecular analyses. Morphologically, females and juveniles from Sri Lanka are similar to original descriptions and other reports from Brazil, Cameroon, Liberia, and São Tomé and Príncipe. The identity of the species was also confirmed by 18S rRNA gene sequences deposited in NCBI from Brazil (AY297826). Integrative diagnosis was completed with molecular data using D2-D3 expansion segments of 28S rRNA, ITS1 region, partial 18S-rRNA and the partial mitochondrial gene cytochrome c oxidase subunit 1 (coxI). This is the third molecular characterisation for a species of the X. non-americanum Group 4, after X. oleae and X. tica. The use of different ribosomal and mitochondrial markers in this study, particularly, D2-D3, ITS1 and partial coxI, provided a precise and unequivocal tool for the identification of X. ifacolum and contributes to a better knowledge of the diversity within Xiphinema. Morphospecies Group 4 appears to be a paraphyletic group within the X. non-americanum assemblage.

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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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A reappraisal of the monophyly of the genus Pseudomonocelis Meixner, 1943 (Platyhelminthes: Proseriata), with the description of a new species from the Mediterranean

Zootaxa ◽

10.11646/zootaxa.3011.1.6 ◽

2011 ◽

Vol 3011 (1) ◽

pp. 59 ◽

Cited By ~ 10

Author(s):

MARCO CASU ◽

PIERO COSSU ◽

DARIA SANNA ◽

TIZIANA LAI ◽

FABIO SCARPA ◽

...

Keyword(s):

18S Rrna ◽

Copulatory Organ ◽

18S Rrna Gene ◽

Rrna Gene ◽

28S Rrna ◽

East African ◽

Statistical Support ◽

Variable Domains ◽

The Mediterranean ◽

A New Species

Pseudomonocelis paupercula nov. sp. is described from brackish-water habitats of the Mediterranean. It is distinguished from other members of the genus by the copulatory organ provided with a stylet, combined with lack of vagina and presence of a muscular organ close to the female pore. Its phylogenetic relationships have been investigated sequencing complete 18S rRNA gene and partial 28S rRNA gene, spanning variable domains D1-D6. Both BI and ML suggest a sistertaxon relationships of P. paupercula nov. sp. with the east African P. cf cavernicola. However, statistical support is low. Conversely, MP indicates P. paupercula nov. sp. as sister-taxon to all the remaining Pseudomonocelis and Minona ilenae. Overall, results of the combined analysis do not support the monophyly of the genus Pseudomonocelis. The need for wider molecular and taxonomic sampling is stressed.

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