A Real-Time PCR Protocol to Determine the Number of Amelogenin (X–Y) Gene Copies From Forensic DNA Samples

2004 ◽  
pp. 031-044 ◽  
Author(s):  
Antonio Alonso ◽  
Pablo Martín
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Forensic Dna ◽  
Gene Copies

scholarly journals Modeling of Phenoxy Acid Herbicide Mineralization and Growth of Microbial Degraders in 15 Soils Monitored by Quantitative Real-Time PCR of the FunctionaltfdAGene

2012 ◽  
Vol 78 (15) ◽  
pp. 5305-5312 ◽  
Author(s):  
Jacob Bælum ◽  
Emmanuel Prestat ◽  
Maude M. David ◽  
Bjarne W. Strobel ◽  
Carsten S. Jacobsen
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Kinetic Models ◽  
Exponential Model ◽  
Dichlorophenoxyacetic Acid ◽  
Order Kinetic ◽  
Clear Trend ◽  
Gene Copies ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
Kinetics Of

ABSTRACTMineralization potentials, rates, and kinetics of the three phenoxy acid (PA) herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), and 2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP), were investigated and compared in 15 soils collected from five continents. The mineralization patterns were fitted by zero/linear or exponential growth forms of the three-half-order models and by logarithmic (log), first-order, or zero-order kinetic models. Prior and subsequent to the mineralization event,tfdAgenes were quantified using real-time PCR to estimate the genetic potential for degrading PA in the soils. In 25 of the 45 mineralization scenarios, ∼60% mineralization was observed within 118 days. Elevated concentrations oftfdAin the range 1 × 105to 5 × 107gene copies g−1of soil were observed in soils where mineralization could be described by using growth-linked kinetic models. A clear trend was observed that the mineralization rates of the three PAs occurred in the order 2,4-D > MCPA > MCPP, and a correlation was observed between rapid mineralization and soils exposed to PA previously. Finally, for 2,4-D mineralization, all seven mineralization patterns which were best fitted by the exponential model yielded a highertfdAgene potential after mineralization had occurred than the three mineralization patterns best fitted by the Lin model.

scholarly journals Detection of Bifidobacterium animalis subsp. lactis (Bb12) in the Intestine after Feeding of Sows and Their Piglets

2008 ◽  
Vol 74 (20) ◽  
pp. 6338-6347 ◽  
Author(s):  
Gloria Solano-Aguilar ◽  
Harry Dawson ◽  
Marta Restrepo ◽  
Kate Andrews ◽  
Bryan Vinyard ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Protein Biosynthesis ◽  
Bifidobacterium Longum ◽  
Elongation Factor ◽  
Single Copy ◽  
Bifidobacterium Animalis ◽  
Gene Copies ◽  
Intestinal Contents ◽  
High Level

ABSTRACT A real-time PCR method has been developed to distinguish Bifidobacterium animalis subspecies in the gastrointestinal tracts of pigs. Identification of a highly conserved single-copy tuf gene encoding the elongation factor Tu involved in bacterial protein biosynthesis was used as a marker to differentiate homologous Bifidobacterium animalis subsp. lactis (strain Bb12) from Bifidobacterium animalis subsp. animalis, as well as Bifidobacterium suis, Bifidobacterium breve, Bifidobacterium longum, several species of Lactobacillus, and Enterococcus faecium. Real-time PCR detection of serially diluted DNA extracted from a pure culture of Bb12 was linear for bacterial numbers ranging from 10 to 10,000 tuf gene copies per PCR (r 2 = 0.99). Relative differences in Bb12 bacterial numbers in pigs fed daily with Bb12 were determined after detection of Bb12 tuf gene copies in DNA extracted from the intestinal contents. Piglets treated with Bb12 immediately after birth maintained a high level of Bb12 in their large intestines with continuous daily administration of Bb12. Piglets born to Bb12-treated sows during the last third of their gestation and also treated with Bb12 at birth (T/T group) had a higher number of Bb12 organisms per gram of intestinal contents compared to placebo-treated piglets born to placebo-treated sows (C/C group), Bb12-treated sows (T/C group), or piglets born to placebo sows but treated with Bb12 immediately after birth (C/T group). In addition, there was a significant increase in gene expression for Toll-like receptor 9 (TLR9) in piglets from the T/T group, with no change in TLR2 and TLR4. These findings suggest that the tuf gene represents a specific and functional marker for detecting Bifidobacterium animalis subsp. lactis strain Bb12 within the microbiota of the intestine.

The utility and application of real-time PCR and FISH in the detection of single-copy gene targets inEscherichia coliO157:H7 andSalmonellaTyphimurium

2010 ◽  
Vol 56 (3) ◽  
pp. 254-262 ◽  
Author(s):  
Merriam Haffar ◽  
Kimberley A. Gilbride
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Receptor Gene ◽  
Detection Limits ◽  
Single Copy ◽  
Target Cells ◽  
Single Copy Gene ◽  
Gene Copies ◽  
Copy Gene

The ultimate specificity in molecular-based assays for pathogen detection relies on the design of the primers and probes. Their ability to hybridize to DNA sequences found only in pathogens can be realized by designing primers and probes that are complementary to pathogen-specific virulence genes. This study evaluates the detection and enumeration strengths of real-time PCR (qPCR) and fluorescent in situ hybridization (FISH) for selected waterborne pathogens and their ultimate applicability within a monitoring framework. Detection limits calculated in the qPCR assay were 150 tir (intimin protein receptor) gene copies for Escherichia coli O157:H7 and 2 × 103invA (inner membrane invasive protein) gene copies for Salmonella enterica serovar Typhimurium. Detection limits were, however, at least 100-fold less sensitive in wastewater extracts, partly because of the inhibitory effect of the wastewater itself. Fluorescent signals from hybridized whole target cells were below the detection limit of the FISH assay. While this research demonstrates the potential detection strength of qPCR, it highlights the need for strong dependable primer and probe sets among PCR and FISH methodologies as well as the need for further signal amplification with DNA-targeted FISH for single-copy gene targets within environmental samples.

scholarly journals Monitoring the Production of Aflatoxin B1 in Wheat by Measuring the Concentration of nor-1 mRNA

2003 ◽  
Vol 69 (2) ◽  
pp. 1154-1158 ◽  
Author(s):  
Zsuzsanna Mayer ◽  
Paul Färber ◽  
Rolf Geisen
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Biosynthetic Pathway ◽  
Fungal Growth ◽  
Reverse Transcription Pcr ◽  
Gene Copies ◽  
Aflatoxin Concentration ◽  
Probe System ◽  
Aflatoxin B ◽  
First Time

ABSTRACT A real-time reverse transcription-PCR system has been used to monitor the expression of an aflatoxin biosynthetic gene of Aspergillus flavus in wheat. Therefore, total RNA was isolated from infected wheat samples, reverse transcribed and subjected to real-time PCR. In parallel all samples were analyzed by high-pressure liquid chromatography for aflatoxin B1 production. The primer-probe system of the real-time PCR was targeted against nor-1, a gene of the aflatoxin biosynthetic pathway. By application of this method the nor-1 transcription was quantified during the course of incubation. After 4 days of incubation nor-1 mRNA could be detected for the first time. The amount of nor-1 mRNA increased rapidly, and the maximum was achieved after 6 days. Then, starting very slowly, the mRNA was degraded until day 8, and this was followed by a very fast degradation, reaching nondetectable levels at days 9 and 10. First traces of aflatoxin B1could be detected between the 5th and 6th day of incubation. The aflatoxin concentration reached its maximum after 9 days of incubation and remained constant for the whole period of observation. To ensure that differences in the nor-1 mRNA concentration were due to different expression levels, the expression of the constitutively expressed β-tubulin gene (benA56) has also been monitored. The expression of benA56 remained constant during the whole incubation time. As a parameter for fungal growth, the number of nor-1 gene copies was determined during the course of incubation. The numbers of nor-1 gene copies increased at the beginning of the incubation and reached a plateau at day 5. They correlate well with the viable counts albeit at a higher level.

scholarly journals Comparative detection and enumeration strengths of quantitative real-time PCR & FISH for waterborne bacterial pathogens in municipal wastewater

2021 ◽  
Author(s):  
Merriam Haffar
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Municipal Wastewater ◽  
Bacterial Pathogens ◽  
Virulence Gene ◽  
Target Cells ◽  
Rt Pcr ◽  
Hybridization Probes ◽  
Gene Copies ◽  
Pure Cultures

This study comparatively evaluates the detection and enumeration strengths of Real-Time PCR (RT PCR) and FISH, for selected bacterial pathogens in municipal wastewater. Both assays were performed using three primer and probe sets complementary to the same chromosomal virulence gene sequences. Primer & probe specificity was confirmed with DNA & fixed cells from pure bacterial cultures as well as seeded wastewater samples. Detection limits calculated for the RT PCR assay were 25 to 3030 tir gene copies for Escherichia coli O157:H7 and 3 x 10⁴ to 293 x10⁷ invA gene copies for Salmonella enterica, using pure cultures and seeded wasewater samples, respectively. In spite of the confirmed specificity of the DNA hybridization probes with target nucleic acids, fluorescent signals from hybridized whole target cells were below the detection limit of the FISH assay, and consequently were not quantified. This research demonstrates both the utility of RT PCR in detecting bacterial pathogens and the need for further optimization with DNA-targeted FISH, using environmental samples.

scholarly journals Quantitative Real-Time PCR Detection of Toxic Nodularia Cyanobacteria in the Baltic Sea

2007 ◽  
Vol 73 (7) ◽  
pp. 2173-2179 ◽  
Author(s):  
Kerttu Koskenniemi ◽  
Christina Lyra ◽  
Pirjo Rajaniemi-Wacklin ◽  
Jouni Jokela ◽  
Kaarina Sivonen
Keyword(s):  
Baltic Sea ◽  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Gene Copy ◽  
Quantitative Real Time Pcr ◽  
The Baltic Sea ◽  
Gene Copies ◽  
Qpcr Method ◽  
The Baltic

ABSTRACT A specific quantitative real-time PCR (qPCR) method was developed for the quantification of hepatotoxin nodularin-producing Nodularia, one of the main bloom-forming cyanobacteria in the Baltic Sea. Specific PCR primers were designed for subunit F of the nodularin synthetase gene (ndaF), which encodes the NdaF subunit of the nodularin synthetase gene complex needed for nodularin production. The qPCR method was applied to water samples (a total of 120 samples) collected from the Baltic Sea in July 2004. As few as 30 ndaF gene copies ml−1 of seawater could be detected, and thus, the method was very sensitive. The ndaF gene copy numbers and nodularin concentrations were shown to correlate in the Baltic seawater, indicating the constant production of nodularin by Nodularia. This qPCR method for the ndaF gene can be used for detailed studies of Nodularia blooms and their formation. ndaF gene copies and nodularin were detected mostly in the surface water but also in deeper water layers (down to 30 m). Toxic Nodularia blooms are not only horizontally but also vertically widely distributed, and thus, the Baltic fauna is extensively exposed to nodularin.

scholarly journals Lean Breed Landrace Pigs Harbor Fecal Methanogens at Higher Diversity and Density than Obese Breed Erhualian Pigs

Archaea ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Yu-heng Luo ◽  
Yong Su ◽  
André-Denis G. Wright ◽  
Ling-li Zhang ◽  
Hauke Smidt ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Rrna Gene ◽  
Pig Breeds ◽  
Operational Taxonomic Units ◽  
Gene Copies ◽  
Gene Libraries ◽  
Landrace Pig

The diversity of fecal methanogens of Erhualian (obese type) and Landrace (lean type) pigs was examined using separate 16S rRNA gene libraries for each breed. A total of 763 clones were analyzed; 381 from the Erhualian library and 382 from the Landrace library were identified belonging to the genus Methanobrevibacter. Others were identified belonging to the genus Methanosphaera. The two libraries showed significant differences in diversity (P<0.05) and composition (P<0.0001). Only two operational taxonomic units (OTUs) were found in both libraries, whereas six OTUs were found only in the Erhualian library and 23 OTUs were found only in the Landrace library. Real-time PCR showed that the abundance of fecal methanogens in Landrace pigs was significantly higher than that in Erhualian pigs (P<0.05). Results showed that the Landrace pig (lean) harbored a greater diversity and higher numbers of methanogenmcrAgene copies than the Erhualian pig (obese). These differences may be related to the fatness or leanness in these two pig breeds. The results provide new leads for further investigations on the fat storage of pigs or even humans.

scholarly journals Real-Time PCR Method Combined with a Matrix Lysis Procedure for the Quantification of Listeria monocytogenes in Meat Products

Foods ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 735
Author(s):  
Mirian Labrador ◽  
Carlota Giménez-Rota ◽  
Carmen Rota
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Foodborne Pathogen ◽  
Meat Products ◽  
Reference Method ◽  
Meat Industry ◽  
Gene Copies ◽  
Pcr Method ◽  
Meat Samples

In this study a real-time PCR method has been developed for the specific quantification of the foodborne pathogen Listeria monocytogenes on meat products through the gene hlyA. The PCR was combined with a matrix lysis that allowed the obtaining of the microorganisms without sample dilution and the elimination the PCR inhibitors from dry-cured ham. The qPCR method calibration curve had an efficiency of 100.4%, limits of detection and quantification were 30.1 ± 6.2 CFU/g which is under the legal limit of L. monocytogenes in ready-to-eat products, and an analytical variability <0.25 log hlyA gene copies/reaction. The analysis was performed simultaneously with the reference method ISO 11290-2. The comparison of the qPCR-matrix lysis results with the reference method showed an excellent correspondence, with a relative accuracy between 95.83–105.20%. Finally, the method was applied to commercial derived meat samples and the pathogen was quantified in one of the commercial samples assayed in 69.1 ± 13.9 CFU/g while the reference method did not quantify it. The optimized qPCR showed higher precision and sensitivity than the reference method at low concentrations of the microorganism in a shorter time. Therefore, qPCR-matrix lysis shows a potential application in the meat industry for L. monocytogenes routine control.

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