scholarly journals Development and Application of a Most-Probable-Number–PCR Assay To Quantify Flagellate Populations in Soil Samples

2001 ◽  
Vol 67 (4) ◽  
pp. 1613-1618 ◽  
Author(s):  
Line Fredslund ◽  
Flemming Ekelund ◽  
Carsten Suhr Jacobsen ◽  
Kaare Johnsen
Keyword(s):  
Dna Sequences ◽  
Sequence Data ◽  
Small Subunit ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Heterotrophic Flagellates ◽  
Small Subunit Rrna ◽  
Pcr Assay ◽  
Probable Number ◽  
Soil Protozoa

ABSTRACT This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.

The Relationships among Microorganisms in Paddy Soil of Rice Varieties at Sanamchaikate, Chachoengsao

2017 ◽  
Vol 866 ◽  
pp. 144-147
Author(s):  
Duongruitai Nicomrat ◽  
Paisan Kanthang ◽  
Siriphatrc Chamutpong
Keyword(s):  
Paddy Soil ◽  
Small Subunit ◽  
Organic Fertilizers ◽  
Rrna Genes ◽  
Most Probable Number ◽  
Small Subunit Rrna ◽  
Rice Varieties ◽  
Probable Number ◽  
Jasmine Rice ◽  
Ph Range

The research was conducted to understand the diversity of microbial communities in the rice cultivars KDM 105 in the rice fields at Sanamchaikate, Chachoengsao Province. The culturing bacterial community in paddy soil before planting, during the planting and sowing of rice, and after rice collection as well as isolation of free nitrogen fixing bacteria under aerobic and anaerobic conditions were identified by molecular comparision of 16S small subunit rRNA genes as well as species diversity and their richness by Most Probable Number (MPN) method. Culturable bacterial isolates in the soil around the roots of rice varieties were determined for their physical appearances on the solid culture (Plate culturing method) and the microscopic observation under light microscope. It was found that bacteria in the paddy soil complemented with organic fertilizers and no pesticide application for over five years had a pH range from 5.2 to 5.5 cultivated jasmine rice, 8-9 log Units of free N2-fixing bacteria near the roots compared with those in other area having 4-5 log Units. Most of them were identified to be Pseudomonas sp. Microbacterium sp. Bacillus sp. Stenotrophomonas sp. and Burkholderia sp., by homology comparison of 16S rDNA gene at 98, 97, 99, 99.5, and 99%, respectively. This research revealed the recognizable complex and change in soil bacteria presented in paddy ecosystem. In any critical change of to the soil, the study of microbial diversity, compositions and their richness can be further useful for indicating proper soil management.

The molecular evidence of Babesia microti infection in small mammals collected in Slovenia

Parasitology ◽  
2003 ◽  
Vol 126 (2) ◽  
pp. 113-117 ◽  
Author(s):  
D. DUH ◽  
M. PETROVEC ◽  
T. TRILAR ◽  
T. AVSIC-ZUPANC
Keyword(s):  
Small Mammals ◽  
Small Subunit ◽  
Clethrionomys Glareolus ◽  
Babesia Microti ◽  
Molecular Evidence ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Pcr Assay ◽  
Small Subunit Rrna Gene ◽  
Yellow Necked Mouse

In Europe, the zoonotic cycle of Babesia microti has not been determined so far. Recently, B. microti was detected in Ixodes ricinus ticks in Slovenia by using molecular methods. In order to investigate the mammalian hosts of B. microti in Slovenia we collected 261 small mammals representing 11 species. They were tested for the presence of babesial parasites with a PCR assay based on the nuclear small subunit rRNA gene (nss-rDNA). The bank vole (Clethrionomys glareolus) and yellow-necked mouse (Apodemus flavicollis) were infected with B. microti. The prevalence rate was 15·9% for C. glareolus and 11·8% for A. flavicollis. Nucleotide sequences of amplified portions of B. microti nss-rDNA from C. glareolus and A. flavicollis were indistinguishable from each other and identical with those previously described in I. ricinus ticks collected in Slovenia. The results of this study represent molecular evidence of B. microti in small mammals in Europe.

scholarly journals Morphology, morphogenesis and molecular phylogeny of an oxytrichid ciliate, Rubrioxytricha haematoplasma (Blatterer & Foissner, 1990) Berger, 1999 (Ciliophora, Hypotricha)

2015 ◽  
Vol 65 (Pt_1) ◽  
pp. 309-320 ◽  
Author(s):  
Wenping Chen ◽  
Xumiao Chen ◽  
Lifang Li ◽  
Alan Warren ◽  
Xiaofeng Lin
Keyword(s):  
Gene Sequence ◽  
Staining Method ◽  
Sequence Data ◽  
Small Subunit ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Small Subunit Rrna ◽  
Small Subunit Rrna Gene ◽  
Single Mass ◽  
The Right

The morphology and morphogenesis of an oxytrichid ciliate, Rubrioxytricha haematoplasma (Blatterer & Foissner, 1990) Berger, 1999, collected from brackish and marine waters in China, were investigated using live observation and the protargol staining method. The main features of the morphogenetic process are: (i) the parental adoral zone of membranelles is retained completely in the proter and the anlage of undulating membranes originates from dedifferentiation of the old structures; (ii) three frontal, four frontoventral, one buccal, five ventral and five transverse cirri are derived from the anlagen of the undulating membranes and the five streaks of frontal-ventral-transverse anlagen in the pattern of 1 : 3 : 3 : 3 : 4 : 4 from left to right; (iii) the morphogenesis of the dorsal kineties is simpler than the Oxytricha pattern, i.e. without fragmentation of the dorsal kinety 3 anlagen; (iv) the single caudal cirrus originates from the dorsal kinety 3 anlage on the right side; (v) the two macronuclear nodules fuse into a single mass during the mid-stage of morphogenesis. These features correspond well with Rubrioxytricha indica, indicating that the morphogenetic pattern of Rubrioxytricha is stable. Phylogenetic analysis based on small-subunit rRNA gene sequence data supports the monophyly of the genus Rubrioxytricha, which is nested within the non-Stylonychinae clade.

Schistonchus hirtus n. sp. (Nematoda: Aphelenchoididae), an associate of Ficus hirta in China

Nematology ◽  
2010 ◽  
Vol 12 (4) ◽  
pp. 543-556 ◽  
Author(s):  
Yongsan Zeng ◽  
Weimin Ye ◽  
Robin M. Giblin-Davis ◽  
Changhui Li ◽  
Zhijian Du ◽  
...  
Keyword(s):  
Sequence Data ◽  
Large Subunit ◽  
Morphological Characters ◽  
Small Subunit ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Wasp Species ◽  
Dna Sequence Data ◽  
Ficus Hirta ◽  
Large Subunit Rrna

Abstract A nematode recovered from syconia of Ficus hirta from Guangzhou, P. R. China, during a survey of nematode biodiversity from 2007 to 2009, is described herein as Schistonchus hirtus n. sp. and is differentiated by a combination of morphological characters, including excretory pore (EP) located near the metacorpus, a short post-uterine sac (PUS) (0.5 vulval body diam. (VBD) long), rose thorn-shaped spicules, amoeboid sperm, absence of gubernaculum, three pairs of subventral papillae on the male tail, host-Ficus and host-wasp species and DNA sequence data. Morphologically, S. hirtus n. sp. is close to S. centerae, S. altermacrophylla, S. aureus, S. laevigatus and S. virens based upon the length of the PUS (about 0.5 VBD long). However, the relative position of the EP in S. hirtus n. sp. is very different from these species (near metacorpus vs near head). With regard to the EP character, S. hirtus n. sp. is very similar to S. macrophylla, S. guangzhouensis and S. caprifici where the EP is at metacorpus level. However, S. hirtus n. sp. differs from S. macrophylla and S. guangzhouensis by possessing a shorter PUS and smaller spicules, and differs from S. caprifici by a shorter female stylet and smaller spicules. Schistonchus hirtus n. sp. was easily differentiated from other sequenced species by the proportion of parsimony informative changes in the partial small subunit rRNA gene (SSU) and D2/D3 expansion segments of the large subunit rRNA gene (LSU). Phylogenetic analysis with SSU sequences suggests that S. hirtus n. sp. is in a highly supported monophyletic clade with Aphelenchoides and Laimaphelenchus and is polyphyletic to other sequenced Schistonchus species. With LSU sequence data, it forms a clade with S. caprifici and they appear polyphyletic relative to S. guangzhouensis, S. centerae, S. aureus, S. laevigatus and S. virens.

Taxonomy, morphology and molecular systematics of three oligotrich ciliates, including a description of Apostrombidium parakielum spec. nov. (Ciliophora, Oligotrichia)

2013 ◽  
Vol 63 (Pt_3) ◽  
pp. 1179-1191 ◽  
Author(s):  
Wen Song ◽  
Jiamei Li ◽  
Weiwei Liu ◽  
Jiamei Jiang ◽  
Khaled A. S. Al- Rasheid ◽  
...  
Keyword(s):  
Sequence Data ◽  
Novel Species ◽  
Small Subunit ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
The Novel ◽  
Small Subunit Rrna ◽  
Ssu Rrna Gene ◽  
Single Clade ◽  
Ssu Rrna Gene Sequence

Three oligotrich ciliates, Apostrombidium parakielum spec. nov., Novistrombidium apsheronicum (Alekperov & Asadullayeva, 1997) Agatha, 2003 and Novistrombidium testaceum (Anigstein, 1914) Song & Bradbury, 1998 were collected from the coastal waters of China and their morphology and small-subunit rRNA (SSU rRNA) gene sequences were studied. The novel species can be recognized by the combination of its obconical body shape, 14–16 anterior and 6–8 ventral membranelles, somatic kinety in three parts and conspicuously long dorsal cilia. Based on the data obtained for this novel species, an improved diagnosis of the genus Apostrombidium is supplied. Descriptions of the population of N. apsheronicum and N. testaceum collected in this study are also provided and compared with the existing descriptions. In addition, the phylogenetic positions of these three species are inferred from their SSU rRNA gene sequence data. The results indicate that the genus Apostrombidium, the systematics of which has not previously been discussed using molecular information, clusters with Varistrombidium kielum and Omegastrombidium elegans, whereas N. testaceum and N. apsheronicum form a single clade.

Redescriptions of three trachelocercid ciliates (Protista, Ciliophora, Karyorelictea), with notes on their phylogeny based on small subunit rRNA gene sequences

2013 ◽  
Vol 63 (Pt_9) ◽  
pp. 3506-3514 ◽  
Author(s):  
Ying Yan ◽  
Yuan Xu ◽  
Zhenzhen Yi ◽  
Alan Warren
Keyword(s):  
Sequence Data ◽  
Phylogenetic Analyses ◽  
Small Subunit ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Small Subunit Rrna ◽  
Small Subunit Rrna Gene ◽  
Ssu Rrna Gene Sequence ◽  
First Time

Three trachelocercid ciliates, Kovalevaia sulcata (Kovaleva, 1966) Foissner, 1997, Trachelocerca sagitta (Müller, 1786) Ehrenberg, 1840 and Trachelocerca ditis (Wright, 1982) Foissner, 1996, isolated from two coastal habitats at Qingdao, China, were investigated using live observation and silver impregnation methods. Data on their infraciliature and morphology are supplied. The small subunit rRNA (SSU rRNA) genes of K. sulcata and Trachelocerca sagitta were sequenced for the first time. Phylogenetic analyses based on SSU rRNA gene sequence data indicate that both organisms, and the previously sequenced Trachelocerca ditis, are located within the trachelocercid assemblage and that K. sulcata is sister to an unidentified taxon forming a clade that is basal to the core trachelocercids.

scholarly journals Panfungal PCR Assay for Detection of Fungal Infection in Human Blood Specimens

1998 ◽  
Vol 36 (5) ◽  
pp. 1169-1175 ◽  
Author(s):  
Jo-Anne Van Burik ◽  
David Myerson ◽  
Randall W. Schreckhise ◽  
Raleigh A. Bowden
Keyword(s):  
Fungal Infection ◽  
Whole Blood ◽  
Limit Of Detection ◽  
Fungal Infections ◽  
Small Subunit ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Pcr Assay ◽  
Blood Specimens ◽  
Panfungal Pcr

A novel panfungal PCR assay which detects the small-subunit rRNA gene sequence of the two major fungal organism groups was used to test whole-blood specimens obtained from a series of blood or bone marrow transplant recipients. The 580-bp PCR product was identified after amplification by panfungal primers and hybridization to a 245-bp digoxigenin-labeled probe. The lower limit of detection of the assay was approximately four organisms per milliliter of blood. Multiple whole-blood specimens from five patients without fungal infection or colonization had negative PCR results. Specimens from 11 infected patients had positive PCR results. Blood from three patients with pulmonary aspergillosis had positive PCR results: one patient’s blood specimen obtained in the week prior to the diagnosis of infection by a positive bronchoalveolar lavage fluid culture result was positive by PCR, and blood specimens obtained from two patients 1 to 2 days after lung biopsy and which were sterile by culture were positive by PCR. The blood of four patients with candidemia, three patients with mixed fungal infections, and one patient with fusariosis also had positive PCR signals. The panfungal PCR assay can detect multiple fungal genera and may be used as an adjunct to conventional methods for the detection of fungal infection or for describing the natural history of fungal infection. Further studies are needed to define the sensitivity and specificity of this assay for the diagnosis of fungal infection prior to the existence of other clinical or laboratory indications of invasive fungal infection.

scholarly journals Seasonal Shedding of Multiple Cryptosporidium Genotypes in California Ground Squirrels (Spermophilus beecheyi)

2004 ◽  
Vol 70 (11) ◽  
pp. 6748-6752 ◽  
Author(s):  
Edward R. Atwill ◽  
Ralph Phillips ◽  
Maria Das Gra�as C. Pereira ◽  
Xunde Li ◽  
Brenda McCowan
Keyword(s):  
Dna Sequences ◽  
Small Subunit ◽  
Ground Squirrels ◽  
Rrna Gene ◽  
Spermophilus Beecheyi ◽  
Small Subunit Rrna ◽  
Fecal Shedding ◽  
Link Type ◽  
The Mean ◽  
California Ground Squirrels

ABSTRACT Twelve percent of 853 California ground squirrels (Spermophilus beecheyi) from six different geographic locations in Kern County, Calif., were found to be shedding on average 44,482 oocysts g of feces−1. The mean annual environmental loading rate of Cryptosporidium oocysts was 57,882 oocysts squirrel−1 day−1, with seasonal patterns of fecal shedding ranging from <10,000 oocysts squirrel−1 day−1 in fall, winter, and spring to levels of 2 � 105 oocysts squirrel−1 day−1 in summer. Juveniles were about twice as likely as adult squirrels to be infected and shed higher concentrations of oocysts than adults did, with particularly high levels of infection and shedding being found among juvenile male squirrels. Based on DNA sequencing of a portion of the 18S small-subunit rRNA gene, there existed three genotypes of Cryptosporidium species in these populations of squirrels (Sbey03a, Sbey03b, and Sbey03c; accession numbers AY462231 to AY462233 , respectively). These unique DNA sequences were most closely related (96 to 97% homology) to porcine C. parvum (AF115377) and C. wrairi (AF115378). Inoculating BALB/c neonatal mice with up to 10,000 Sbey03b or Sbey03c fresh oocysts from different infected hosts did not produce detectable levels of infection, suggesting that this common genotype shed by California ground squirrels is not infectious for mice and may constitute a new species of Cryptosporidium.

Description of Schistonchus microcarpus n. sp. (Nematoda: Aphelenchoididae), an associate of Ficus microcarpa in China

Nematology ◽  
2011 ◽  
Vol 13 (2) ◽  
pp. 221-233 ◽  
Author(s):  
Yongsan Zeng ◽  
Weimin Ye ◽  
Robin Giblin-Davis ◽  
Changhui Li ◽  
Shinian Zhang ◽  
...  
Keyword(s):  
Excretory Pore ◽  
Sequence Data ◽  
Large Subunit ◽  
Small Subunit ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Male Tail ◽  
Mtdna Coi ◽  
Ficus Microcarpa ◽  
Tail Tip

AbstractSchistonchus microcarpus n. sp. was recovered from the syconia of Ficus microcarpa from Shenzhen and Guangzhou, Guangdong Province, China, during a survey of nematode biodiversity from 2007 to 2009. It is characterised by possessing the combined characters of a short post-uterine sac (PUS) (3-11 μm or <0.4 vulval body diam. (VBD) long), excretory pore located just posterior to the head but anterior to the conus level of the stylet, prominent amphids, three pairs of subventral papillae on the male tail (one pair adcloacal, one pair halfway between cloaca and tail terminus, and one pair near tail tip), unique recurved and sickleshaped spicules with finely rounded tip with cucullus, amoeboid sperm, and rounded male tail tip with or without mucron. Schistonchus microcarpus n. sp. is morphologically differentiated from all other described species in this genus by the possession of a spicule with a cucullus on the tip. Schistonchus microcarpus n. sp. was easily differentiated from other sequenced species by the partial small subunit rRNA gene (SSU) and D3 expansion segments of the large subunit rRNA gene (LSU). Phylogenetic analysis with partial SSU sequences suggests that S. microcarpus n. sp. is in a highly supported monophyletic clade with sequenced Schistonchus species except for S. hirtus. Based upon inferences using D3 LSU sequence data, it forms a clade with an undescribed species of Schistonchus ex F. benjamini from Australia and is part of a larger clade of Schistonchus that mostly share the character of an anteriorly placed excretory pore. Sequences of partial mtDNA COI (590 bp) from males of S. microcarpus n. sp. with and without a mucronate tail tip were identical, proving that these two morphotypes are conspecific.

scholarly journals Single-cell genomics unveils a canonical origin of the diverse mitochondrial genomes of euglenozoans

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Kristína Záhonová ◽  
Gordon Lax ◽  
Savar D. Sinha ◽  
Guy Leonard ◽  
Thomas A. Richards ◽  
...  
Keyword(s):  
Single Cell ◽  
Small Subunit ◽  
Rrna Genes ◽  
Mitochondrial Genomes ◽  
Trna Genes ◽  
Rrna Gene ◽  
Heterotrophic Flagellates ◽  
Small Subunit Rrna ◽  
Single Chromosome ◽  
Linear Chromosomes

Abstract Background The supergroup Euglenozoa unites heterotrophic flagellates from three major clades, kinetoplastids, diplonemids, and euglenids, each of which exhibits extremely divergent mitochondrial characteristics. Mitochondrial genomes (mtDNAs) of euglenids comprise multiple linear chromosomes carrying single genes, whereas mitochondrial chromosomes are circular non-catenated in diplonemids, but circular and catenated in kinetoplastids. In diplonemids and kinetoplastids, mitochondrial mRNAs require extensive and diverse editing and/or trans-splicing to produce mature transcripts. All known euglenozoan mtDNAs exhibit extremely short mitochondrial small (rns) and large (rnl) subunit rRNA genes, and absence of tRNA genes. How these features evolved from an ancestral bacteria-like circular mitochondrial genome remains unanswered. Results We sequenced and assembled 20 euglenozoan single-cell amplified genomes (SAGs). In our phylogenetic and phylogenomic analyses, three SAGs were placed within kinetoplastids, 14 within diplonemids, one (EU2) within euglenids, and two SAGs with nearly identical small subunit rRNA gene (18S) sequences (EU17/18) branched as either a basal lineage of euglenids, or as a sister to all euglenozoans. Near-complete mitochondrial genomes were identified in EU2 and EU17/18. Surprisingly, both EU2 and EU17/18 mitochondrial contigs contained multiple genes and one tRNA gene. Furthermore, EU17/18 mtDNA possessed several features unique among euglenozoans including full-length rns and rnl genes, six mitoribosomal genes, and nad11, all likely on a single chromosome. Conclusions Our data strongly suggest that EU17/18 is an early-branching euglenozoan with numerous ancestral mitochondrial features. Collectively these data contribute to untangling the early evolution of euglenozoan mitochondria.

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