scholarly journals Phylogeny of the Genus Synchytrium and the Development of TaqMan PCR Assay for Sensitive Detection of Synchytrium endobioticum in Soil

2014 ◽  
Vol 104 (4) ◽  
pp. 422-432 ◽  
Author(s):  
Donna S. Smith ◽  
Hélène Rocheleau ◽  
Julie T. Chapados ◽  
Cathryn Abbott ◽  
Sharon Ribero ◽  
...  
Keyword(s):  
Soil Extract ◽  
Small Subunit ◽  
High Rate ◽  
Confirmatory Test ◽  
Taqman Assay ◽  
Economic Damage ◽  
Soil Extracts ◽  
Synchytrium Endobioticum ◽  
Negative Results ◽  
Rdna Sequences

Potato wart, caused by the fungal pathogen Synchytrium endobioticum, is a serious disease with the potential to cause significant economic damage. The small subunit (SSU) and internal transcribed spacer (ITS) ribosomal DNA (rDNA) were sequenced for several Synchytrium spp., showing a high rate of variability for both of these markers among the different species and monophyly of the genus within phylum Chytridiomycota. The intergenic nontranscribed spacer (IGS) of rDNA was sequenced for different pathotypes and showed no intraspecific variation within S. endobioticum, similar to the other rDNA markers from this study. To facilitate screening for the pathogen in soil, three TaqMan polymerase chain reaction (PCR) assays were developed from SSU, ITS, and IGS rDNA sequences to detect S. endobioticum sporangia in the chloroform-flotation fraction of sieved soil extracts. In the screening portion of the method, a first TaqMan assay targeting the SSU rDNA was developed with positive results that were further confirmed with amplicon melt analysis. A synthetic reaction control cloned into a plasmid was incorporated into the procedure, facilitating the validation of negative results. The presence of the reaction control did not adversely affect the efficiency of the SSU target amplification. A second TaqMan assay targeting the ITS-1 region was developed as a confirmatory test. There was 100% accordance between the SSU and ITS-1 TaqMan assays. Utilizing these two assays in tandem achieved good specificity for S. endobioticum, generating negative results with the cloned SSU and ITS-1 regions from all 14 other Synchytrium spp. considered. Spike recovery experiments indicated that these assays, targeting the SSU and ITS-1 rDNA regions, developed from a phylogeny dataset of the genus, could reliably detect a single sporangium in the chloroform flotation fraction of a soil extract. Good correlation between microscopic detection of sporangia and PCR results in both positive and negative soil samples was dually demonstrated for both the SSU and ITS-1 assays.

scholarly journals The Effectiveness of Soil Extracts from Selangor Peat Swamp and Pristine Forest Soils on the Growth of Green Microalgae sp.

Forests ◽  
2022 ◽  
Vol 13 (1) ◽  
pp. 79
Author(s):  
Nor Suhaila Yaacob ◽  
Mohd Fadzli Ahmad ◽  
Ashvini Sivam ◽  
Emi Fazlina Hashim ◽  
Maegala Nallapan Maniyam ◽  
...  
Keyword(s):  
Extraction Method ◽  
Mineral Soil ◽  
Extraction Methods ◽  
Soil Extract ◽  
High Rate ◽  
Growth Promoter ◽  
Forest Reserve ◽  
Soil Extracts ◽  
Peat Swamp ◽  
Pristine Forest

Microalgae are widely utilized in commercial industries. The addition of a modified artificial medium (soil extract) could enhance their growth. Soil extract collected from the Raja Musa peat swamp and mineral soil from the Ayer Hitam Forest Reserve (AHFR), Selangor, Malaysia, were treated using various extraction methods. Carteria radiosa PHG2-A01, Neochloris conjuncta, and Nephrochlamys subsolitaria were grown in microplates at 25 °C, light intensity 33.75 µmol photons m−2s−1 for 9 days. N. conjuncta dominated the growth in 121 °C twice extraction method AFHR samples, with 47.17% increment. The highest concentrations of ammonia and nitrate were detected in the medium with soil extract treated with 121 °C twice extraction method, yielding the concentrations of 2 mg NL−1 and 35 mg NL−1 for ammonia and nitrate of RM soil and 2 mg NL−1 and 2.85 mg NL−1 for the AH soil. These extracts are proved successful as a microalgal growth stimulant, increasing revenue and the need for enriched medium. The high rate of nutrient recovery has the potential to serve as a growth promoter for microalgae.

scholarly journals Results of the Toxoplasmosis Screening in 9311 Pregnant Women in a Tertiary Center in Turkey

2020 ◽  
Vol 25 (3) ◽  
pp. 332-338
Author(s):  
Özgür Koçak ◽  
Özgür Kan
Keyword(s):  
Pregnant Women ◽  
Acute Infection ◽  
Congenital Toxoplasmosis ◽  
Tertiary Hospital ◽  
High Rate ◽  
Central Anatolia ◽  
Confirmatory Test ◽  
Serological Examination ◽  
Tertiary Center ◽  
Avidity Test

Introduction: Toxoplasma gondii is an important parasite that can cause permanent sequelae to the fetus when infected during pregnancy in humans. Although the frequency of this parasite varies widely between countries, it is known that it is common in our country. The aim of this study was to determine the seroprevalence of toxoplasma in pregnant women admitted to a tertiary hospital in central Anatolia and to evaluate the pregnancy outcomes together with seroprevalence. Materials and Methods: A total of 9311 patients admitted to a tertiary hospital between January 2016 and December 2018 were included into the study. After serological examination, avidity test was performed in cases suggestive of acute infection. Amniocentesis was recommended to be performed by Polimeraze Chain Reaction (PCR) in patients with low avidity. Results: The frequency of Toxoplasma immunoglobulin (Ig) G and M seropositivity rates were 20.3% and 0.28%, respectively. Low avidity was found in approximately 27% of the patients with IgM positivity, and only 15.4% had low avidity by confirmatory test. One patient could not be reached during follow-up. PCR was performed in 4 patients whose low avidity value was confirmed by re-tests and all of their PCR results were reported negative. No cases of congenital toxoplasmosis were detected during the 3 years in our clinic. Conclusion: The inclusion of toxoplasma in routine screening programme is still controversial and differs between countries. Screening in areas with a high rate of toxoplasma, such as in our country, may be rational. If infection is detected, treatment may be recommended because it may reduce the transmission to the fetus.

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.

scholarly journals Chloroform extraction of trace nutrients as oxinates from soil extracts for spectrographic analysis

1960 ◽  
Vol 32 (1) ◽  
pp. 223-228
Author(s):  
Osmo Mäkitie
Keyword(s):  
Trace Metals ◽  
Chloroform Solution ◽  
Soil Extract ◽  
Hydrogen Ion ◽  
Ion Concentration ◽  
Spectrographic Analysis ◽  
Hydrogen Ion Concentration ◽  
Soil Extracts ◽  
Copper Manganese ◽  
The Common

The experiments show that under these conditions the common trace nutrients, cobalt, copper, manganese, molybdenum and zinc are sufficiently completely extracted as chelates by shaking the soil extract with oxine-chloroform solution. The hydrogen ion concentration of the extract and the concentration of oxine in chloroform have decisive effects on the extractability. Using the reported and discussed procedure it is possible to separate the common trace metals from the major soil extract constituents, especially for spectrographic analysis.

Low Rate of False-Positive Results with Use of A Rapid HIV Test

2002 ◽  
Vol 23 (6) ◽  
pp. 335-337 ◽  
Author(s):  
Cassandra D. Salgado ◽  
Heidi L. Flanagan ◽  
Doris M. Haverstick ◽  
Barry M. Farr
Keyword(s):  
Enzyme Immunoassay ◽  
False Positive ◽  
Diagnostic System ◽  
Rapid Test ◽  
Occupational Exposures ◽  
High Rate ◽  
Negative Results ◽  
Hiv Test ◽  
Rapid Hiv Test ◽  
Positive Results

Background:Occupational exposure to human immunodeficiency virus (HIV) is an important threat to healthcare workers. Centers for Disease Control and Prevention guidelines recommend prompt institution of prophylaxis. This requires (1) immediate prophylaxis after exposure, pending test results that may take more than 24 hours in many hospitals; or (2) performance of a rapid test. The Single Use Diagnostic System (SUDS)® HIV-1 Test is used to screen rapidly for antibodies to HIV type 1 in plasma or serum, with a reported sensitivity of more than 99.9%. We used this test from January 1999 until September 2000, when it was withdrawn from the market following reports claiming a high rate of false-positive results.Methods:We reviewed the results of postexposure HIV testing during 21 months.Results:A total of 884 SUDS tests were performed on source patients after occupational exposures (883 negative results, 1 reactive result). The results of repeat SUDS testing on the reactive specimen were also reactive, but the results of enzyme immunoassay and Western blot testing were negative. A new specimen from the same patient showed a negative result on SUDS testing. This suggested a specificity of 99.9%. In the 4 months after SUDS testing was suspended, there was 1 false-positive result on enzyme immunoassay for 1 of 132 source patients (presumed specificity, 99.2%).Conclusion:Use of the SUDS test facilitated rapid and accurate evaluation of source specimens, obviating unnecessary prophylaxis.

Eimeria species: studies using rRNA and rDNA probes

Parasitology ◽  
1990 ◽  
Vol 101 (1) ◽  
pp. 1-6 ◽  
Author(s):  
J. Ellis ◽  
J. Bumstead
Keyword(s):  
Genomic Dna ◽  
Southern Hybridization ◽  
Eimeria Species ◽  
Small Subunit ◽  
Rrna Genes ◽  
Rdna Probe ◽  
Dna Library ◽  
Rdna Sequences ◽  
Genomic Dna Library

SUMMARYrRNA and a heterologous cloned rDNA probe have been used to detect the rRNA genes of Eimeria species which infe the chicken, and has allowed the isolation and preliminary characterization of cloned rDNA sequences from a genomic DNA library of Eimeria tenella. It is demonstrated that rRNA and rDNA probes can be used to identify individual Eimeria species by the restriction fragment patterns detected after Southern hybridization. In addition, studies have shown that the large and small subunit rRNAs are expressed throughout sporulation.

Whirling Disease: Reviews and Current Topics

2002 ◽  
Keyword(s):  
Life Cycle ◽  
Developmental Stages ◽  
Small Subunit ◽  
Fish Host ◽  
Complex Life Cycle ◽  
Developmental Cycle ◽  
Myxobolus Cerebralis ◽  
Rdna Sequences ◽  
Complex Development ◽  
The One

<em>ABSTRACT. Myxobolus cerebralis </em>possesses unique phenotypic and genotypic characteristics when compared with other histozoic parasites from the phylum Myxozoa. The parasite infects the cartilage and thereby induces a serious and potentially lethal disease in salmonid fish. Comparisons of the small subunit ribosomal DNA (ssu rDNA) sequences of <em>M. cerebralis </em>to other myxozoans demonstrate that the parasite has evolved separately from other <em>Myxobolus </em>spp. that may appear in cartilage or nervous tissues of the fish host. <em>Myxobolus cerebralis </em>has a complex life cycle involving two hosts and numerous developmental stages that may divide by mitosis, endogeny, or plasmotomy, and, at one stage, by meiosis. In the salmonid host, the parasite undergoes extensive migration from initial sites of attachment to the epidermis, through the nervous system, to reach cartilage, the site where sporogenesis occurs. During this migration, parasite numbers may increase by replication. Sporogenesis is initiated by autogamy, a process typical of pansporoblastic myxosporean development that involves the union of the one cell (pericyte) with another (sporogonic). Following this union, the sporogonic cell will give rise to all subsequent cells that differentiate into the lenticular shaped spore with a diameter of approximately 10 µm. This spore or myxospore is an environmentally resistant stage characterized by two hardened valves surrounding two polar capsules with coiled filaments and a binucleate sporoplasm cell. In the fish, these spores are found only in cartilage where they reside until released from fish that die or are consumed by other fish or fish-eating animals (e.g., birds). Spores reaching the aquatic sediments can be ingested and hatch in susceptible oligochaete hosts. The released sporoplasm invades and then resides between cells of the intestinal mucosa. In contrast to the parasite in the fish host, the parasite in the oligochaete undergoes the entire developmental cycle in this location. This developmental cycle involves merogony, gametogamy or the formation of haploid gametes, and sporogony. The actinosporean spores, formed at the culmination of this development, are released into the lumen of the intestine, prior to discharging into the aquatic environment. The mechanisms underlying the complex development of <em>M. cerebralis</em>, and its interactions with both hosts, are poorly understood. Recent advances, however, are providing insights into the factors that mediate certain phases of the infection. In this review, we consider known and recently obtained information on the taxonomy, development, and life cycle of the parasite.

scholarly journals A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans

2020 ◽  
Vol 59 (1) ◽  
pp. e01986-20
Author(s):  
Ibne Karim M. Ali ◽  
Shantanu Roy
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Small Subunit ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
Rdna Sequences ◽  
Appropriate Treatment

ABSTRACTThere are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.

Sign in / Sign up

Export Citation Format

Share Document