scholarly journals Genotyping of Acantamoeba spp. from rhisophere in Hungary

2020 ◽  
Vol 67 (3) ◽  
pp. 171-175
Author(s):  
Erika Orosz ◽  
Katalin Posta
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Human Pathogens ◽  
Quantitative Real Time Pcr ◽  
Potential Health ◽  
Granulomatous Amoebic Encephalitis ◽  
Related Genotype ◽  
Biological Methods

The protista Acanthamoeba is a free-living amoeba existing in various environments. A number of species among protista are recognized as human pathogens, potentially causing Acanthamoeba keratitis (AK), granulomatous amoebic encephalitis (GAE), and chronic granulomatous lesions. In this study, 10 rhizosphere samples were collected from maize and alfalfa plants in experimental station at Institute of Genetics, Microbiology and Biotechnology, Szent István University. We detected Acanthamoeba based on the quantitative real-time PCR assay and sequence analysis of the 18S rRNA gene. All studied molecular biological methods are suitable for the detection of Acanthamoeba infection in humans. The quantitative real-time PCR-based methods are more sensitive, simple, and easy to perform; moreover, these are opening avenue to detect the effect of number of parasites on human disease. Acanthamoeba species were detected in five (5/10; 50%) samples. All Acanthamoeba strains belonged to T4 genotype, the main AK-related genotype worldwide. Our result confirmed Acanthamoeba strains in rhizosphere that should be considered as a potential health risk associated with human activities in the environment.

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

2004 ◽  
Vol 42 (12) ◽  
pp. 5636-5643 ◽  
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

Analysis of community structures in anaerobic processes using a quantitative real-time PCR method

2005 ◽  
Vol 52 (1-2) ◽  
pp. 85-91 ◽  
Author(s):  
Y. Yu ◽  
C. Lee ◽  
S. Hwang
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rrna Gene ◽  
Quantitative Real Time Pcr ◽  
Community Structures ◽  
Specific Primer ◽  
Anaerobic Processes ◽  
Promising Tool ◽  
Pcr Method ◽  
Group Specific Primer

The methanogenic community structures of four different anaerobic processes were characterized using a quantitative real-time PCR with group-specific primer and probe sets targeting the 16S rRNA gene (rDNA). The group specific primer and probe sets were developed and used to detect the orders Methanosarcinales, and the families Methanosarcinaceae and Methanosaetaceae. Two separate sets targeting the domains Archaea and Bacteria were also used. Each microbial population in different anaerobic processes was determined and the relative abundance in the system was compared with each other. Dominant methanogenic populations and the community structures in the processes were varied by hydraulic retention time and acetate concentration. This indicates that the real-time PCR method with the primer and probe sets is a promising tool to analyze community structures in anaerobic processes.

scholarly journals Development and Validation of a Real-Time PCR Method To Quantify Rumen Protozoa and Examination of Variability between Entodinium Populations in Sheep Offered a Hay-Based Diet

2006 ◽  
Vol 72 (1) ◽  
pp. 200-206 ◽  
Author(s):  
Lucy C. Skillman ◽  
Andrew F. Toovey ◽  
Andrew J. Williams ◽  
André-Denis G. Wright
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Advantages And Disadvantages ◽  
Pcr Efficiency ◽  
Rumen Protozoa ◽  
Pcr Method ◽  
Serial Dilutions

ABSTRACT PCR and real-time PCR primers for the 18S rRNA gene of rumen protozoa (Entodinium and Dasytricha spp.) were designed, and their specificities were tested against a range of rumen microbes and protozoal groups. External standards were prepared from DNA extracts of a rumen matrix containing known numbers and species of protozoa. The efficiency of PCR (ε) was calculated following amplification of serial dilutions of each standard and was used to calculate the numbers of protozoa in each sample collected; serial dilutions of DNA were used similarly to calculate PCR efficiency. Species of Entodinium, the most prevalent of the rumen protozoa, were enumerated in rumen samples collected from 100 1-year-old merino wethers by microscopy and real-time PCR. Both the counts developed by the real-time PCR method and microscopic counts were accurate and repeatable, with a strong correlation between them (R 2 = 0.8), particularly when the PCR efficiency was close to optimal (i.e., two copies per cycle). The advantages and disadvantages of each procedure are discussed. Entodinium represented on average 98% of the total protozoa, and populations within the same sheep were relatively stable, but greater variation occurred between different sheep (100 and 106 entodinia per gram of rumen contents). With this inherent variability, it was estimated that, to detect a statistically significant (P = 0.05) 20% change in Entodinium populations, 52 sheep per treatment group would be required.

scholarly journals Real-time PCR detection and speciation of Cryptosporidium infection using Scorpion probes

2006 ◽  
Vol 55 (9) ◽  
pp. 1217-1222 ◽  
Author(s):  
Suzanne E. Stroup ◽  
Shantanu Roy ◽  
John Mchele ◽  
Venance Maro ◽  
Simon Ntabaguzi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rflp Analysis ◽  
18S Rrna Gene ◽  
Human Infection ◽  
Rrna Gene ◽  
Dna Extraction Method ◽  
Stool Samples ◽  
Stool Specimens ◽  
Human Stool

At least eight species of Cryptosporidium can cause human infection and disease. A real-time PCR (qPCR) assay based on the 18S rRNA gene and utilizing a Scorpion probe was developed to detect all human-pathogenic Cryptosporidium without the usual need for nested amplification. Sensitivity of detection in stool samples was highest using a glass bead-based DNA extraction method (under 103 oocysts per stool sample). The assay was validated against 123 human stool specimens from Bangladesh and Tanzania, exhibited a sensitivity and specificity of >91 % versus microscopy, and detected an additional eight microscopy-negative infections. Cryptosporidium parvum-specific and Cryptosporidium meleagridis-specific Scorpion qPCR assays that provided 100 % accurate speciation compared with VspI RFLP analysis and sequencing were developed subsequently. These Scorpion probe qPCR assays are simpler to perform than existing nested PCR and RFLP methods for diagnosis and epidemiological investigation of cryptosporidiosis.

scholarly journals Sequencing of 18S rRNA gene of Bdelloid rotifers and design of the primers for real-time PCR

2019 ◽  
Vol 1 (3) ◽  
pp. 55-63
Author(s):  
Watcharapong Thakong ◽  
Kazuya Shimizu ◽  
Miwa Kodato ◽  
Norio Iwami ◽  
Niwooti Whangchai ◽  
...  
Keyword(s):  
Cell Suspension ◽  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Consensus Sequence ◽  
Treatment Plant ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Bdelloid Rotifer ◽  
Bdelloid Rotifers

Three individuals of Bdelloid rotifer (J1, J2 and J3) were isolated from a MBR system in Nagasaki University and one individual of rotifer (J4) in the original seed sludge collected from a wastewater treatment plant for the MBR was isolated. The four rotifer species were able to proliferate in toxic Microcystis cell suspension. The partial sequence of 18S rRNA gene of each isolated rotifer was determined using In-fusion cloning and searched by BLAST. The gene of the four rotifers J1, J2, J3 and J4 showed the same sequence, then the consensus sequence was in the branch of Bdelloid rotifers in the phylogenetic tree. Furthermore, a specific Bdelloid forward primer 55F and reverse primer 395R for real-time PCR was designed based on the consensus sequence for quantitative researches on the Bdelloid rotifers population. We succeeded to quantify the population of a Bdelloid rotifer cultured in toxic Microcystis cell suspension using the new designed primer pairs.

scholarly journals Quantitative Detection of the Free-Living Amoeba Hartmannella vermiformis in Surface Water by Using Real-Time PCR

2006 ◽  
Vol 72 (9) ◽  
pp. 5750-5756 ◽  
Author(s):  
Melanie W. Kuiper ◽  
Rinske M. Valster ◽  
Bart A. Wullings ◽  
Harry Boonstra ◽  
Hauke Smidt ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Quantitative Detection ◽  
Rrna Gene ◽  
Free Living ◽  
Copy Numbers ◽  
Free Living Amoeba ◽  
Hartmannella Vermiformis

ABSTRACT A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 � 10−1 and 1.14 � 104 cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 � 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (≥98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.

scholarly journals Rapid diagnosis of candidaemia by real-time PCR detection of Candida DNA in blood samples

2009 ◽  
Vol 58 (8) ◽  
pp. 1106-1111 ◽  
Author(s):  
Nele Wellinghausen ◽  
Dunja Siegel ◽  
Juliane Winter ◽  
Susanne Gebert
Keyword(s):  
Real Time ◽  
Antifungal Therapy ◽  
Real Time Pcr ◽  
Positive Culture ◽  
18S Rrna Gene ◽  
Rapid Diagnosis ◽  
Rrna Gene ◽  
Blood Samples ◽  
Specific Pcr ◽  
Patients At Risk

This study prospectively evaluated an 18S rRNA gene-targeted real-time PCR approach in comparison with standard blood culture (BC) diagnostics for rapid diagnosis of candidaemia in a large study population of 384 patients, including 902 whole blood samples from 468 infectious episodes (IEs) of 329 adults and 55 children with haematological malignancies and various forms of immunodeficiency, and intensive care unit patients. Seven out of eight BC-proven cases (87.5 %) of candidaemia and seven out of twelve BC-positive samples (58.3 %) were positive by the Candida-specific PCR. A positive PCR result was also obtained for 28/460 BC-negative samples from IEs, including 8 patients with culture-confirmed Candida infection at primary sterile body sites. Of the PCR-positive, culture-negative patients, more than 50 % received systemic antifungal therapy. In 432/460 BC-negative IEs, the Candida specific-PCR was negative, resulting in a negative predictive value of 99.8 %. In conclusion, the Candida specific-PCR approach facilitates rapid detection of Candida DNA in blood samples of patients at risk of candidaemia within a few hours. Although standard BC diagnostics appear to remain indispensable for the detection of all cases of candidaemia, this PCR assay allowed the detection of candidaemia at a mean of 3 days earlier than BC diagnostics. Thus, it enables earlier antifungal therapy for patients with suspected candidaemia and may prevent further complications.

scholarly journals  Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR

2012 ◽  
Vol 57 (No. 5) ◽  
pp. 224-232 ◽  
Author(s):  
M. Adamska ◽  
A. Leonska-Duniec ◽  
M. Sawczuk ◽  
A. Maciejewska ◽  
B. Skotarczak
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
High Concentration ◽  
Intestinal Protozoan ◽  
Wide Range ◽  
Stool Samples

Cryptosporidium parvum is a common intestinal protozoan parasite infecting humans and a wide range of animals, whose diagnostics present considerable difficulties. These arise from the exceptionally robust nature of the oocyst’s walls, which necessitates more stringent treatments for disruption and recovery of DNA for analysis using molecular methods. In the case of water, which is the major source of Cryptosporidium oocysts, investigations concern the detection of the presence of the oocysts. Their concentration in water is very low, and moreover, many substances that may have significance as inhibitors of DNA amplification, are present in environmental water and stool. We have carried out trials in order to assess the effectiveness of recovery of C. parvum oocysts, from spiked environmental and distilled water samples, filtrated and concentrated with the use of special laboratory equipment. Inactivation of inhibitors was carried out with use of bovine serum albumin (BSA) in PCR mixes at ten different concentrations. DNA extraction was carried out from stool samples spiked with C. parvum oocysts, concentrated using two methods, and unconcentrated. Nested PCR and a TaqMan nested real time PCR assay, targeting the 18S rRNA gene, was used to detect C. parvum DNA in spiked water and additionally in spiked stool samples. The obtained results showed that losses of C. parvum oocysts occur during the filtration and concentration of spiked water samples. The addition of small amounts of BSA (5–20 ng/µl) to PCR and TaqMan PCR mixes increases the sensitivity of both methods, but a high concentration of BSA (100 ng/µl and above) has an inhibiting effect on the polymerase reaction. The extraction of DNA from C. parvum oocysts from spiked stool samples preceded by concentration with PBS, ether and Percoll resulted in a higher copy number of the 18S rRNA gene.  

Sign in / Sign up

Export Citation Format

Share Document