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.

Real-Time PCR for Quantitative Detection of Bovine Tissues in Food and Feed

2008 ◽  
Vol 71 (3) ◽  
pp. 564-572 ◽  
Author(s):  
IRENE MARTÍN ◽  
TERESA GARCÍA ◽  
VIOLETA FAJARDO ◽  
MARÍA ROJAS ◽  
PABLO E. HERNÁNDEZ ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Detection System ◽  
Total Content ◽  
18S Rrna Gene ◽  
Universal Primers ◽  
Quantitative Detection ◽  
12S Rrna ◽  
Rrna Gene

A real-time PCR approach with the SYBR Green detection system has been developed for the quantitative detection of bovine tissues in food and feedstuffs. The method combines the use of bovine-specific primers, which amplify an 84-bp fragment of the mitochondrial 12S rRNA gene, and universal primers, which amplify a 140-bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. The 18S rRNA primers are used as endogenous controls for the total content of PCR-amplifiable DNA in the sample. The specificity of the primers was tested against 18 animal species, including mammals, birds, and fish, as well as 6 plant species. Analysis of experimental bovine tissues–oats mixtures demonstrated the suitability of the assay for the detection of bovine DNA in mixtures containing as low as 0.1% of bovine tissues. The performance of the method is not affected by severe heat treatment (up to 133°C for 20 min at 300 kPa). The reported PCR assay could be very useful for detecting bovine-derived ingredients in raw and heat-treated food and feedstuffs.

Quantitative Detection and Differentiation of Free-Living Amoeba Species Using SYBR Green–Based Real-Time PCR Melting Curve Analysis

2006 ◽  
Vol 53 (6) ◽  
pp. 506-509 ◽  
Author(s):  
Jonas Behets ◽  
Priscilla Declerck ◽  
Yasmine Delaedt ◽  
Lieve Verelst ◽  
Frans Ollevier
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Curve Analysis ◽  
Sybr Green ◽  
Quantitative Detection ◽  
Melting Curve Analysis ◽  
Free Living ◽  
Free Living Amoeba

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

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 Real-Time PCR for Quantitative Detection of Chamois (Rupicapra rupicapra) and Pyrenean Ibex (Capra pyrenaica) in MeatMixtures

2008 ◽  
Vol 91 (1) ◽  
pp. 103-111 ◽  
Author(s):  
Violeta Fajardo ◽  
Isabel Gonzlez ◽  
Irene Martn ◽  
Mara Rojas ◽  
Pablo E Hernndez ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Quantitative Polymerase Chain Reaction ◽  
Universal Primers ◽  
Quantitative Detection ◽  
12S Rrna ◽  
Rrna Gene ◽  
Capra Pyrenaica ◽  
Loop Region

Abstract A real-time quantitative polymerase chain reaction (PCR) technique was developed for the quantification of chamois and pyrenean ibex DNAs in meat mixtures by using a SYBR green detection platform. Two species-specific systems and a eukaryotic endogenous system were combined in the real-time PCR approach to quantify the target species. In the specific systems, a 133 base pair (bp) fragment of the 12S rRNA gene was amplified from chamois DNA, and an 88 bp fragment from the D-loop region was amplified from pyrenean ibex DNA. In the endogenous system, universal primers amplified a 141 bp fragment on the nuclear 18S rRNA gene from eukaryotic DNA. The threshold cycle values obtained with the 18S rRNA primers were used to normalize those obtained from chamois- or pyrenean ibex-specific systems, serving as endogenous control for the total content of PCR-amplifiable DNA in the sample. Analysis of experimental raw and heat-treated binary mixtures of chamois and pyrenean ibex meat in a swine meat matrix demonstrated the suitability of the assay for the detection and quantification of the target DNAs in the range of 0.10.8, depending on the species and treatment of the meat samples.

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.  

scholarly journals Pooled sample testing for Bonamia ostreae: A tale of two SYBR Green real-time PCR assays

2017 ◽  
Vol 29 (5) ◽  
pp. 752-756 ◽  
Author(s):  
Henry S. Lane ◽  
J. Brian Jones ◽  
Wendy L. McDonald
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Pool Size ◽  
Rrna Gene ◽  
Sample Testing ◽  
Bonamia Ostreae ◽  
Pcr Assays ◽  
Pooled Sample

Pooled testing of samples is a common laboratory practice to increase efficiency and reduce expenses. We investigated the efficacy of 2 published SYBR Green real-time PCR assays when used to detect the haplosporidian parasite Bonamia ostreae in pooled samples of infected oyster tissue. Each PCR targets a different gene within the B. ostreae genome: the actin 1 gene or the 18S rRNA gene. Tissue homogenates (150 mg) of the New Zealand flat oyster Ostrea chilensis were spiked with ~1.5 × 103 purified B. ostreae cells to create experimental pools of 3, 5, and 10. Ten positive replicates of each pool size were assayed twice with each PCR and at 2 different amounts of DNA template. The PCR targeting the actin 1 gene was unable to reproducibly detect B. ostreae in any pool size. Conversely, the 18S rRNA gene PCR could reproducibly detect B. ostreae in pools of up to 5. Using a general linear model, there was a significant difference in the number of pools that correctly detected B. ostreae between each PCR ( p < 0.01) and each pool size ( p < 0.01). It is likely that the single copy actin 1 gene is more likely to be diluted and not detected by pooling than the multi-copy 18S rRNA gene. Our study highlights that validation data are necessary for pooled sample testing because detection efficacy may not be comparable to individual sample testing.

scholarly journals Detection of Protozoan Hosts for Legionella pneumophila in Engineered Water Systems by Using a Biofilm Batch Test

2010 ◽  
Vol 76 (21) ◽  
pp. 7144-7153 ◽  
Author(s):  
Rinske M. Valster ◽  
Bart A. Wullings ◽  
Dick van der Kooij
Keyword(s):  
Legionella Pneumophila ◽  
Biofilm Formation ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Batch Test ◽  
Free Living ◽  
Water Types ◽  
Operational Taxonomic Units ◽  
Hartmannella Vermiformis

ABSTRACT Legionella pneumophila proliferates in aquatic habitats within free-living protozoa, 17 species of which have been identified as hosts by using in vitro experiments. The present study aimed at identifying protozoan hosts for L. pneumophila by using a biofilm batch test (BBT). Samples (600 ml) collected from 21 engineered freshwater systems, with added polyethylene cylinders to promote biofilm formation, were inoculated with L. pneumophila and subsequently incubated at 37°C for 20 days. Growth of L. pneumophila was observed in 16 of 18 water types when the host protozoan Hartmannella vermiformis was added. Twelve of the tested water types supported growth of L. pneumophila or indigenous Legionella anisa without added H. vermiformis. In 12 of 19 BBT flasks H. vermiformis was indicated as a host, based on the ratio between maximum concentrations of L. pneumophila and H. vermiformis, determined with quantitative PCR (Q-PCR), and the composition of clone libraries of partial 18S rRNA gene fragments. Analyses of 609 eukaryotic clones from the BBTs revealed that 68 operational taxonomic units (OTUs) showed the highest similarity to free-living protozoa. Forty percent of the sequences clustering with protozoa showed ≥99.5% similarity to H. vermiformis. None of the other protozoa serving as hosts in in vitro studies were detected in the BBTs. In several tests with growth of L. pneumophila, the protozoa Diphylleia rotans, Echinamoeba thermarum, and Neoparamoeba sp. were identified as candidate hosts. In vitro studies are needed to confirm their role as hosts for L. pneumophila. Unidentified protozoa were implicated as hosts for uncultured Legionella spp. grown in BBT flasks at 15°C.

scholarly journals Implementation and Validation of the Roche Light Cycler 480 96-Well Plate Platform as a Real-Time PCR Assay for the Quantitative Detection of Cytomegalovirus (CMV) in Clinical Specimens Using the Luminex MultiCode ASRs System

2020 ◽  
Vol 8 (1) ◽  
pp. 14
Author(s):  
Shengwen Calvin Li ◽  
Kara J. Sparks ◽  
Leonard S. Sender
Keyword(s):  
Stem Cell ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Range ◽  
Measurement Range ◽  
Quantitative Detection ◽  
Clinical Settings ◽  
Stem Cell Therapies ◽  
Cell Therapies ◽  
Copy Numbers

Allogenic stem-cell therapies benefit patients in the treatment of multiple diseases; however, the side effects of stem-cell therapies (SCT) derived from the concomitant use of immune suppression agents often include triggering infection diseases. Thus, analysis is required to improve the detection of pathogen infections in SCT. We develop a polymerase chain reaction (PCR)-based methodology for the qualitative real-time DNA detection of cytomegalovirus (CMV), with reference to herpes simplex virus types 1 (HSVI), Epstein–Barr virus (EBV), and varicella-zoster virus (VZV) in blood, urine, solid tissues, and cerebrospinal fluid. This real-time PCR of 96-well plate format provides a rapid framework as required by the Food and Drug Administration (FDA) for clinical settings, including the processing of specimens, reagent handling, special safety precautions, quality control criteria and analytical accuracy, precisely reportable range (analyst measurement range), reference range, limit of detection (LOD), analytical specificity established by interference study, and analyte stability. Specifically, we determined the reportable range (analyst measurement range) with the following criteria: CMV copies ≥200 copies/mL; report copy/mL value; CMV copies ≤199 copies/mL; report detected but below quantitative range; CMV copies = 0 with report <200 copies/mL. That is, with reference range, copy numbers (CN) per milliliter (mL) of the LOD were determined by standard curves that correlated Ct value and calibrated standard DNA panels. The three repeats determined that the measuring range was 1E2~1E6 copies/mL. The standard curves show the slopes were within the range −2.99 to −3.65 with R2 ≥ 0.98. High copy (HC) controls were within 0.17–0.18 log differences of DNA copy numbers; (2) low copy (LC) controls were within 0.17–0.18 log differences; (3) LOD was within 0.14–0.15 log differences. As such, we set up a fast, simple, inexpensive, sensitive, and reliable molecular approach for the qualitative detection of CMV pathogens. Conclusion: This real-time PCR of the 96-well plate format provides a rapid framework as required by the FDA for clinical settings.

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.

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