scholarly journals A novel duplex qPCR assay for stepwise detection of multiple Perkinsea protistan infections of amphibian tissues

2021 ◽  
Vol 8 (3) ◽  
Author(s):  
Vanessa Smilansky ◽  
Aurelie Chambouvet ◽  
Mari Reeves ◽  
Thomas A. Richards ◽  
David S. Milner
Keyword(s):  
Disease Outbreaks ◽  
Small Subunit ◽  
Phylogenetic Group ◽  
Qpcr Assay ◽  
Rapid Screening ◽  
Rrna Gene ◽  
Rdna Sequences ◽  
Routine Surveillance ◽  
Asymptomatic Infections ◽  
Single Primer

Alveolate protists within the phylum Perkinsea have been found to infect amphibians across a broad taxonomic and geographic range. Phylogenetic analysis has suggested the existence of two clades of amphibian Perkinsea: a putatively non-pathogenic clade linked to asymptomatic infections of tadpoles in Africa, Europe and South America, and a putatively pathogenic clade linked to disease and mass mortality events of tadpoles in North America. Here, we describe the development of a duplex TaqMan qPCR assay to detect and discriminate between rDNA sequences from both clades of Perkinsea in amphibian tissues. The assay uses a single primer pair to target an 18S small subunit (SSU) ribosomal RNA (rRNA) gene region shared between the two clades, and two dual-labelled probes to target a region within this fragment that is diagnostic for each clade. This assay enables rapid screening for each of the two Perkinsea groups, allowing for detection, primarily of the phylogenetic group associated with disease outbreaks, and secondarily for the phylogenetic group with no current disease relationship identified. Incorporation of our novel qPCR assay into the routine surveillance of amphibian populations will allow for the assessment of the incidence of each protist clade, thereby providing an improved understanding of Perkinsea infection pervasiveness and a method to underpin future conservation planning.

scholarly journals Quantitative Measure of Small-Subunit rRNA Gene Sequences of the Kingdom Korarchaeota

1998 ◽  
Vol 64 (12) ◽  
pp. 5064-5066 ◽  
Author(s):  
Clifford F. Brunk ◽  
Nicole Eis
Keyword(s):  
Internal Standard ◽  
Pcr Amplification ◽  
Quantitative Measure ◽  
Pcr Primers ◽  
Small Subunit ◽  
Ssu Rdna ◽  
Rdna Sequence ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Rdna Sequences

ABSTRACT Comparative PCR amplification of small-subunit (SSU) rRNA gene (rDNA) sequences indicates substantial preferential PCR amplification of pJP27 sequences with korarchaeote-specific PCR primers. The coamplification of a modified SSU rDNA sequence can be used as an internal standard to determine the amount of a specific SSU rDNA sequence.

Morphological reports on two species of Dexiotricha (Ciliophora, Scuticociliatia), with a note on the phylogenetic position of the genus

2014 ◽  
Vol 64 (Pt_2) ◽  
pp. 680-688 ◽  
Author(s):  
Xinpeng Fan ◽  
Saleh A. Al-Farraj ◽  
Feng Gao ◽  
Fukang Gu
Keyword(s):  
Phylogenetic Analyses ◽  
Eastern China ◽  
Small Subunit ◽  
Ssu Rdna ◽  
Contractile Vacuole ◽  
Phylogenetic Position ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
North West ◽  
Rdna Sequences

Two Dexiotricha species (Dexiotricha elliptica nov. comb. and Dexiotricha cf. granulosa), respectively isolated from soil north-west of Riyadh, Saudi Arabia, and freshwater in Shanghai, eastern China, were investigated using standard methods. The species Loxocephalus ellipticus Kahl, 1931 is reclassified here in the genus Dexiotricha and was characterized mainly by constantly showing 16 somatic kineties, three post-oral kineties with the middle one shortened, a contractile vacuole located subcaudally with an excretory pore near the posterior end of somatic kinety 2 and single caudal cilia. A Dexiotricha granulosa-like organism having a subcaudally located contractile vacuole and fewer somatic kineties was designated D. cf. granulosa. The small-subunit rRNA gene (SSU rDNA) sequences of these two species were characterized and their phylogenetic positions based on SSU rDNA sequences were revealed by means of Bayesian inference and maximum-likelihood analysis. Phylogenetic analyses confirmed Dexiotricha as a monophyletic genus and supported its assignment to the order Loxocephalida. However, its family assignment remains unsupported.

scholarly journals Kinetic Bias in Estimates of Coastal Picoplankton Community Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR Amplicon Length Heterogeneity

1998 ◽  
Vol 64 (11) ◽  
pp. 4522-4529 ◽  
Author(s):  
Marcelino Suzuki ◽  
Michael S. Rappé ◽  
Stephen J. Giovannoni
Keyword(s):  
Clone Library ◽  
Fluorescence Emission ◽  
Small Subunit ◽  
Ssu Rdna ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Length Variation ◽  
Small Subunit Rrna ◽  
Gene Frequencies ◽  
Rdna Sequences

ABSTRACT Marine bacterioplankton diversity was examined by quantifying natural length variation in the 5′ domain of small-subunit (SSU) rRNA genes (rDNA) amplified by PCR from a DNA sample from the Oregon coast. This new technique, length heterogeneity analysis by PCR (LH-PCR), determines the relative proportions of amplicons originating from different organisms by measuring the fluorescence emission of a labeled primer used in the amplification reaction. Relationships between the sizes of amplicons and gene phylogeny were predicted by an analysis of 366 SSU rDNA sequences from cultivated marine bacteria and from bacterial genes cloned directly from environmental samples. LH-PCR was used to compare the distribution of bacterioplankton SSU rDNAs from a coastal water sample with that of an SSU rDNA clone library prepared from the same sample and also to examine the distribution of genes in the PCR products from which the clone library was prepared. The analysis revealed that the relative frequencies of genes amplified from natural communities are highly reproducible for replicate sets of PCRs but that a bias possibly caused by the reannealing kinetics of product molecules can skew gene frequencies when PCR product concentrations exceed threshold values.

Analysis of primary structure loops from Hairpins 35 and 48 of the Nematoda SSU rRNA gene provides further evidence that the genera Tripylina Brzeski, 1963, Trischistoma Cobb, 1913 and Rhabdolaimus de Man, 1880 are members of Enoplida

Zootaxa ◽  
2012 ◽  
Vol 3208 (1) ◽  
pp. 41 ◽  
Author(s):  
ZENGQI ZHAO ◽  
DONGMEI LI ◽  
THOMAS R. BUCKLEY
Keyword(s):  
Primary Structure ◽  
Phylogenetic Analyses ◽  
Small Subunit ◽  
Ssu Rdna ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Rdna Sequences ◽  
Evolutionarily Conserved ◽  
De Man ◽  
Nucleotide Transition

A comparison of small subunit (SSU) rDNA sequences has revealed that a rare nucleotide transition A→G and a trans-version G→Y occurred simultaneously in the evolutionarily conserved loops of Hairpins 35 and 48 of the SSU rRNA geneof Tripylina Brzeski 1963, Trischistoma Cobb 1913 and Rhabdolaimus de Man 1880. The same pair of substitutions oc-curred in all Enoplida analysed but not in other nematodes. This evidence from the primary structure of Hairpins 35 and48 indicates that Tripylina, Trischistoma and Rhabdolaimus are members of Enoplida, supporting previous phylogenetic analyses.

scholarly journals Axial Differences in Community Structure ofCrenarchaeota and Euryarchaeota in the Highly Compartmentalized Gut of the Soil-Feeding TermiteCubitermes orthognathus

2001 ◽  
Vol 67 (10) ◽  
pp. 4880-4890 ◽  
Author(s):  
Michael W. Friedrich ◽  
Dirk Schmitt-Wagner ◽  
Tillmann Lueders ◽  
Andreas Brune
Keyword(s):  
Community Structure ◽  
Archaeal Community ◽  
Small Subunit ◽  
Ssu Rdna ◽  
Ecological Niches ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Rdna Sequences ◽  
Electron Sink ◽  
Archaeal Communities

ABSTRACT Methanogenesis represents an important electron sink reaction in the hindgut of soil-feeding termites. This is the first comprehensive analysis of the archaeal community structure within the highly compartmentalized intestinal tract of a humivorous insect, combining clonal analysis and terminal restriction fragment (T-RF) length polymorphism (T-RFLP) fingerprinting of the archaeal communities in the different gut compartments of Cubitermes orthognathus. We found that the morphological and physicochemical heterogeneity of the gut is reflected in a large phylogenetic diversity and pronounced axial differences in the composition of the archaeal gut microbiota, notably among those clones or ribotypes that could be assigned to methanogenic taxa. Comparative analysis of the relative frequencies of different archaeal lineages among the small-subunit rRNA gene (SSU rDNA) clones and their corresponding T-RF indicated that the archaeal community in the anterior, extremely alkaline hindgut compartment (P1) consists mainly of members of theMethanosarcinaceae, whereasMethanobacteriaceae andMethanomicrobiales predominate in the subsequent, more posterior compartments (P3/4a and P4b). The relative abundance ofThermoplasmales increased towards the rectum (P5). SSU rDNA sequences representing Crenarchaeota, which have not yet been reported to occur in the intestinal tracts of arthropods, were detected in all gut sections. We discuss how the spatial distribution of methanogenic populations may be linked to axial heterogeneity in the physicochemical gut conditions and to functional adaptations to their respective ecological niches.

scholarly journals Molecular Differentiation of Fusarium solani f. sp. glycines from Other F. solani Based on Mitochondrial Small Subunit rDNA Sequences

2000 ◽  
Vol 90 (5) ◽  
pp. 491-497 ◽  
Author(s):  
Shuxian Li ◽  
Yan Kit Tam ◽  
Glen L. Hartman
Keyword(s):  
Genetic Distance ◽  
Fusarium Solani ◽  
Sequence Data ◽  
Cladistic Analysis ◽  
Sequence Divergence ◽  
Small Subunit ◽  
Rrna Gene ◽  
Rdna Sequences ◽  
Nucleotide Insertions ◽  
Host Affiliation

Fusarium solani is a soilborne plant pathogen that infects many different hosts. Within the species, there is some specialization, and a number of forma specialis have been described based on host affiliation. One of these, F. solani f. sp. glycines, infects soybean and causes sudden death syndrome. To differentiate between F. solani f. sp. glycines and other F. solani isolates, a partial sequence of the mitochondrial small subunit (mtSSU) rRNA gene was amplified by polymerase chain reaction and sequenced from 14 F. solani f. sp. glycines and 24 F. solani isolates from various plant hosts. All F. solani f. sp. glycines isolates had identical sequences. A single, unique insertion of cytosine occurred in all F. solaniisolates but not in any of the F. solani f. sp. glycines isolates. Two major lineages, distinguished by sequence divergence and the presence or absence of multiple insertions, occurred in F. solani isolates. Cladistic analysis produced a single most-parsimonious tree with three major clades. The first clade contained all F. solani f. sp. glycines isolates. A second clade grouped together all of the F. solani isolates that had only a single nucleotide insertion difference from the first clade. Genetic distance between these two clades was 0.016. A third clade was formed by five F. solaniisolates that had multiple insertions. Isolates in the third clade had a genetic distance of 0.040 from the first and second clades. Based on the sequence data, it is likely that F. solani f. sp. glycineshas a shorter evolutionary history than other F. solaniisolates that have either single or multiple nucleotide insertions. The differences in nucleotide insertions in part of the mtSSU rRNA gene between F. solani f. sp. glycinesand other F. solani isolates provide a direct and reliable way to distinguish isolates of F. solani.

scholarly journals Targeted Next-Generation Sequencing and Informatics as an Effective Tool to Establish the Composition of Bovine Piroplasm Populations in Endemic Regions

2020 ◽  
Vol 9 (1) ◽  
pp. 21
Author(s):  
Abdul Ghafar ◽  
Anson V. Koehler ◽  
Ross S. Hall ◽  
Charles G. Gauci ◽  
Robin B. Gasser ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Water Buffalo ◽  
Hypervariable Region ◽  
Small Subunit ◽  
Rrna Gene ◽  
Next Generation ◽  
Small Subunit Rrna ◽  
Tropical Theileriosis ◽  
Ecological Zones ◽  
Generation Sequencing

Protists of the genera Babesia and Theileria (piroplasms) cause some of the most prevalent and debilitating diseases for bovines worldwide. In this study, we established and used a next-generation sequencing-informatic approach to explore the composition of Babesia and Theileria populations in cattle and water buffalo in a country (Pakistan) endemic for these pathogens. We collected individual blood samples from cattle (n = 212) and water buffalo (n = 154), extracted genomic DNAs, PCR-amplified the V4 hypervariable region of 18S small subunit rRNA gene from piroplasms, sequenced amplicons using Illumina technology, and then analysed data using bioinformatic platforms. The results revealed piroplasms in 68.9% (252/366) samples, with overall occurrence being markedly higher in cattle (85.8%) than in water buffaloes (45.5%). Babesia (B.) occultans and Theileria (T.) lestoquardi-like species were recorded for the first time in Pakistan, and, overall, T. annulata was most commonly detected (65.8%) followed by B. bovis (7.1%), B. bigemina (4.4%), and T. orientalis (0.5%), with the genetic variability within B. bovis being pronounced. The occurrence and composition of piroplasm species varied markedly across different agro-ecological zones. The high detection of T. annulata in asymptomatic animals suggested a relatively high level of endemic stability of tropical theileriosis in the bovine population.

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

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.

scholarly journals Differences in Microbial Activity and Microbial Populations of Peat Associated with Suppression of Damping-Off Disease Caused by Pythium sylvaticum

2006 ◽  
Vol 72 (10) ◽  
pp. 6452-6460 ◽  
Author(s):  
Paul J. Hunter ◽  
Geoff M. Petch ◽  
Leo A. Calvo-Bado ◽  
Tim R. Pettitt ◽  
Nick R. Parsons ◽  
...  
Keyword(s):  
Microbial Activity ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Small Subunit ◽  
Disease Suppression ◽  
Rrna Gene ◽  
Damping Off ◽  
Small Subunit Rrna ◽  
Pythium Sylvaticum ◽  
Gradient Gel Electrophoresis ◽  
Microbial Groups

ABSTRACT The microbiological characteristics associated with disease-suppressive peats are unclear. We used a bioassay for Pythium sylvaticum-induced damping-off of cress seedlings to identify conducive and suppressive peats. Microbial activity in unconditioned peats was negatively correlated with the counts of P. sylvaticum at the end of the bioassay. Denaturing gradient gel electrophoresis (DGGE) profiling and clone library analyses of small-subunit rRNA gene sequences from two suppressive and two conducive peats differed in the bacterial profiles generated and the diversity of sequence populations. There were also significant differences between bacterial sequence populations from suppressive and conducive peats. The frequencies of a number of microbial groups, including the Rhizobium-Agrobacterium group (specifically sequences similar to those for the genera Ochrobactrum and Zoogloea) and the Acidobacteria, increased specifically in the suppressive peats, although no single bacterial group was associated with disease suppression. Fungal DGGE profiles varied little over the course of the bioassay; however, two bands associated specifically with suppressive samples were detected. Sequences from these bands corresponded to Basidiomycete yeast genera. Although the DGGE profiles were similar, fungal sequence diversity also increased during the bioassay. Sequences highly similar to those of Cryptococcus increased in relative abundance during the bioassay, particularly in the suppressive samples. This study highlights the importance of using complementary approaches to molecular profiling of complex populations and provides the first report that basidiomycetous yeasts may be associated with the suppression of Pythium-induced diseases in peats.

Detection of Kobe-typeBabesia microtiassociated with Japanese human babesiosis in field rodents in central Taiwan and southeastern mainland China

Parasitology ◽  
2008 ◽  
Vol 135 (6) ◽  
pp. 691-699 ◽  
Author(s):  
A. SAITO-ITO ◽  
N. TAKADA ◽  
F. ISHIGURO ◽  
H. FUJITA ◽  
Y. YANO ◽  
...  
Keyword(s):  
Mainland China ◽  
Small Subunit ◽  
Human Infection ◽  
Babesia Microti ◽  
Rrna Gene ◽  
Microscopical Examination ◽  
Small Subunit Rrna ◽  
Central Taiwan ◽  
Field Rodents ◽  
Human Babesiosis

SUMMARYField rodent surveys forBabesiainfection were performed from 2002 to 2005 in the vicinities of human babesiosis occurrences in Taiwan and mainland China.Babesia microtiwas identified by microscopical examination and/or PCR in 1Rattus coxingaand 1Crocidura horsfieldiiin central Taiwan and in 13Niviventer confucianusand 1Apodemus agrariusin Zhejiang and Fujian Provinces of southeastern China. Of 15B. microtisamples detected by PCR, all except 1 were shown to be the Kobe-type, the aetiological small subunit rRNA gene-type of the first Japanese patient; the exception was also a Kobe-related type. The Kobe-type had been found in rodents only in a few places including the human infection occurrence place in Japan. The internal transcribed spacer 1 to 2 sequences of the Taiwanese and Chinese Kobe-types were very similar to each other but considerably different (approx. 94% pairwise identities) from that of the Japanese Kobe-type. A Taiwanese Kobe-type strain was serologically differentiated from the Kobe strain originating from the Japanese first patient. The distribution of the Kobe-type in the vicinities of human babesiosis occurrences in Taiwan and China as well as in Japan is suggestive of involvement of the Kobe-type in Asian human babesiosis.

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