scholarly journals Quantitative Detection of Methanotrophs in Soil by Novel pmoA-Targeted Real-Time PCR Assays

2003 ◽  
Vol 69 (5) ◽  
pp. 2423-2429 ◽  
Author(s):  
Steffen Kolb ◽  
Claudia Knief ◽  
Stephan Stubner ◽  
Ralf Conrad
Keyword(s):  
Community Structure ◽  
Real Time ◽  
Real Time Pcr ◽  
Pcr Analysis ◽  
Quantitative Detection ◽  
Methanotrophic Bacteria ◽  
Methylococcus Capsulatus ◽  
Α Subunit ◽  
Target Molecules ◽  
Pcr Assays

ABSTRACT Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S ribosomal DNA and pmoA (α subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the α and γ subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. Detection limits were between 101 and 102 target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album, and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 ± 1.4) × 106 pmoA molecules g−1 for all methanotrophs. The Methylosinus group was predominant (2.7 × 106 ± 1.1 × 106 target molecules g−1). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 × 106 ± 0.9 × 106 target molecules g of soil−1). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 × 104 target molecules g of soil−1. Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the community structure of methanotrophs in nature.

scholarly journals Quantitative Detection of Legionella pneumophila in Water Samples by Immunomagnetic Purification and Real-Time PCR Amplification of the dotA Gene

2005 ◽  
Vol 71 (7) ◽  
pp. 3433-3441 ◽  
Author(s):  
M. A. Yáñez ◽  
C. Carrasco-Serrano ◽  
V. M. Barberá ◽  
V. Catalán
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Legionella Pneumophila ◽  
Water Samples ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Immunomagnetic Separation ◽  
Pcr Analysis ◽  
Quantitative Detection ◽  
Specific Sequence

ABSTRACT A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.

scholarly journals Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting thegroELGene

2012 ◽  
Vol 78 (8) ◽  
pp. 2613-2622 ◽  
Author(s):  
Jana Junick ◽  
Michael Blaut
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Housekeeping Gene ◽  
Cell Number ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Bifidobacterium Adolescentis ◽  
Fecal Samples ◽  
Content Type ◽  
Pcr Assays

ABSTRACTQuantitative real-time PCR assays targeting thegroELgene for the specific enumeration of 12 human fecalBifidobacteriumspecies were developed. The housekeeping genegroEL(HSP60in eukaryotes) was used as a discriminative marker for the differentiation ofBifidobacterium adolescentis,B. angulatum,B. animalis,B. bifidum,B. breve,B. catenulatum,B. dentium,B. gallicum,B. longum,B. pseudocatenulatum,B. pseudolongum, andB. thermophilum. The bifidobacterial chromosome contains a single copy of thegroELgene, allowing the determination of the cell number by quantification of thegroELcopy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a givenBifidobacteriumspecies. Independent of theBifidobacteriumspecies tested, the proportion ofgroELcopies recovered from fecal samples spiked with 5 to 9 log10cells/g feces was approximately 50%. The quantification limit was 5 to 6 log10groELcopies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3Bifidobacteriumspecies andB. longumfrequently detected. The predominant species in infant and adult fecal samples wereB. breveandB. adolescentis, respectively. It was possible to distinguishB. catenulatumandB. pseudocatenulatum. We conclude that thegroELgene is a suitable molecular marker for the specific and accurate quantification of human fecalBifidobacteriumspecies by real-time PCR.

Comparison of Real-Time Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay for Sensitive and Quantitative Detection of Hazelnuts in Nut Pastes

2018 ◽  
Vol 101 (6) ◽  
pp. 1864-1867 ◽  
Author(s):  
Ľubica Piknová ◽  
Veronika Janská ◽  
Tomáš Kuchta ◽  
Peter Siekel
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Sandwich Elisa ◽  
Pcr Analysis ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Wide Range ◽  
Order Of Magnitude ◽  
Polymerase Chain

Abstract Background: Hazelnuts, being a frequent agent of allergenic reactions, need to be detected in food products. Thus, it is necessary to develop and further investigate appropriate methods for detection. Objective: The aim of the study was to compare the analysis of nut pastes (peanut paste spiked with different amounts of hazelnut paste) as a model of contamination of confectionery. Methods: Real-time PCR and sandwich ELISA (RidaScreen Hazelnut Fast Kit) were used. Results: For real-time PCR, LOQ of 2 mg/kg and a quantification range from 2 to 10 000 mg/kg were determined. For ELISA, LOQ of 1 mg/kg and a quantification range from 1 to 100 mg/kg were determined. Conclusions: The comparison shows that the methods had comparable sensitivity with LOQs in the same order of magnitude. Although ELISA was slightly more sensitive, it required dilution of samples at higher concentrations of the analyte because of its narrow quantification range. Results of this study suggest that real-time PCR and ELISA are both suitable methods for the analysis of nut pastes over a wide range of concentrations. Achieved results could be useful for control as well as for technological purposes. Highlights: Real-time PCR analysis of peanut paste spiked with different amounts of hazelnut paste as a model is proposed. Sandwich ELISA analysis of peanut paste spiked with different amounts of hazelnut paste as a model is proposed. The analytical parameters of real-time PCR and ELISA methods are compared.

Real-time PCR assays for the quantitative detection of Kazachstania servazzii and Candida sake related to undesirable white colony on kimchi

Food Control ◽  
2021 ◽  
Vol 125 ◽  
pp. 107984
Author(s):  
Mi-Ju Kim ◽  
Sung-gi Min ◽  
So Won Shin ◽  
Jiyong Shin ◽  
Hae-Yeong Kim
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Detection ◽  
Pcr Assays ◽  
Candida Sake

scholarly journals A Field Investigation of Bacillus anthracis Contamination of U.S. Department of Agriculture and Other Washington, D.C., Buildings during the Anthrax Attack of October 2001

2003 ◽  
Vol 69 (1) ◽  
pp. 593-599 ◽  
Author(s):  
James A. Higgins ◽  
Mary Cooper ◽  
Linda Schroeder-Tucker ◽  
Scott Black ◽  
David Miller ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Real Time ◽  
Metropolitan Area ◽  
Real Time Pcr ◽  
Lethal Factor ◽  
Field Investigation ◽  
Pcr Analysis ◽  
Air Samples ◽  
Freeze Dried ◽  
Pcr Assays

ABSTRACT In response to a bioterrorism attack in the Washington, D.C., area in October 2001, a mobile laboratory (ML) was set up in the city to conduct rapid molecular tests on environmental samples for the presence of Bacillus anthracis spores and to route samples for further culture analysis. The ML contained class I laminar-flow hoods, a portable autoclave, two portable real-time PCR devices (Ruggedized Advanced Pathogen Identification Device [RAPID]), and miscellaneous supplies and equipment to process samples. Envelopes and swab and air samples collected from 30 locations in the metropolitan area once every three days were subjected to visual examination and DNA extraction, followed by real-time PCR using freeze-dried, fluorescent-probe-based reagents. Surface swabs and air samples were also cultured for B. anthracis at the National Veterinary Service Laboratory (NVSL) in Ames, Iowa. From 24 October 2001 to 15 September 2002, 2,092 pieces of mail were examined, 405 real-time PCR assays were performed (comprising 4,639 samples), and at the NVSL 6,275 samples were subjected to over 18,000 platings. None of the PCR assays on DNA extracted from swab and air samples were positive, but viable spores were cultured from surface swabs taken from six locations in the metropolitan area in October, November, and December 2001 and February, March, and May 2002. DNA extracted from these suspected B. anthracis colonies was positive by real-time and conventional PCRs for the lethal factor, pXO1, and for capA and vrr genes; sequence analysis of the latter amplicons indicated >99% homology with the Ames, vollum, B6273-93, C93022281, and W-21 strains of B. anthracis, suggesting they arose from cross-contamination during the attack through the mail. The RAPID-based PCR analysis provided fast confirmation of suspect colonies from an overnight incubation on agar plates.

scholarly journals Multicenter Comparison of Different Real-Time PCR Assays for Quantitative Detection of Epstein-Barr Virus

2007 ◽  
Vol 46 (1) ◽  
pp. 157-163 ◽  
Author(s):  
R. T. Hayden ◽  
K. M. Hokanson ◽  
S. B. Pounds ◽  
M. J Bankowski ◽  
S. W. Belzer ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Epstein Barr Virus ◽  
Quantitative Detection ◽  
Barr Virus ◽  
Pcr Assays ◽  
Epstein Barr

scholarly journals Evaluation of Molecular Serotyping Assays for Shigella flexneri Directly on Stool Samples

2020 ◽  
Author(s):  
Jie Liu ◽  
Suporn Pholwat ◽  
Jixian Zhang ◽  
Mami Taniuchi ◽  
Rashidul Haque ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vaccine Development ◽  
Shigella Flexneri ◽  
Detection Algorithm ◽  
Quantitative Detection ◽  
Stool Samples ◽  
Pcr Assays ◽  
Validation Set ◽  
Serotype Identification

Shigella flexneri is prevalent worldwide and is the most common Shigella species in many countries. At least 19 S. flexneri serotypes exist, and serotype information is important for epidemiologic and vaccine development purposes. We evaluated the performance of real time PCR (PCR) assays for O-antigen modification genes to identify the major serotypes on isolates and direct stool samples. The assays were formulated into two multiplex panels: one panel included gtrII, gtrV, gtrX, oac, and wzx6 to identify S. flexneri serotype 2a, 2b, 3a, 5a, 5b, 6, and X, the other panel included ipaH, gtrI, gtrIc, gtrIV, to confirm Shigella detection and further identify S. flexneri serotype 1a, 1b, 1d, 3b, 4a, 4b, 7a, and 7b. We first evaluated 283 Shigella isolates and PCR serotyping demonstrated 97.0% (95% confidence interval 93.0% - 99.0%) sensitivity and 99.9% (99.9%-100%) specificity compared to conventional serotyping. The assays were then utilized on direct stool specimens. A quantitative detection algorithm was developed with a validation set of 174 Shigella culture positive stool samples, and further tested with a derivation set of 164 samples. The PCR serotyping on stool achieved 93% (89%-96%) sensitivity and 99% (99%-100%) specificity compared to serotyping. Most discrepancies were genotypic-phenotypic discordance, not genotypic failure. These real-time PCR assays provide an efficient and novel tool for S. flexneri serotype identification.

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