102. Method Validation for Quantification of Recombinant Adeno-Associated Virus 5 in Mouse Genomic DNA Using Real-Time PCR

In this study, a quantitative species-specific polymerase chain reaction (PCR) method to rapidly detect E. histolytica in water is developed. First, the specificity of E. histolytica PCR detection was verified by using species-specific primers of 16S-like rRNA genes to clearly differentiate it from the closely related amoebae species E. dispar and E. moshkovskii. The sensitivity of this method was subsequently determined using purified E. histolytica genomic DNA and culture cells as PCR reaction templates. Results indicated that conventional PCR visualized on 1% agarose gel was able to detect as low as 0.02 pg genomic DNA and 5 cells, while real-time PCR could detect 0.01 pg genomic DNA and 2 cells of E. histolytica. The protocols for E. histolytica PCR detection in real water samples were then optimized by spiking E. histolytica cells into tap water and reservoir raw water samples. A two-round centrifugation treatment to concentrate amoeba cells directly as a PCR template was the most effective way to detect E. histolytica in spiked tap water samples, while DNA extraction after concentrating amoeba cells was required for spiked reservoir raw water samples. The detection limit of 50 E. histolytica cells in 100 ml tap water was achieved in 2 h from sample collection to real-time PCR data readout. With these established protocols, 78 tap water samples, 11 reservoir raw water samples and 4 feed water samples from Singapore water supply systems were analyzed by both conventional PCR and real-time PCR methods. No E. histolytica cell was detected in tested samples.

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Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate

Applied and Environmental Microbiology ◽

10.1128/aem.00779-07 ◽

2007 ◽

Vol 73 (20) ◽

pp. 6557-6565 ◽

Cited By ~ 38

Author(s):

Pascal E. Saikaly ◽

Morton A. Barlaz ◽

Francis L. de los Reyes

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Genomic Dna ◽

Dynamic Range ◽

Limit Of Detection ◽

Fate And Transport ◽

Biological Warfare ◽

Quantitative Real Time Pcr ◽

Pcr Assays ◽

Detection And Quantification

ABSTRACT Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R 2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R 2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.

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Analysis of Polymorphisms in Genes Differentially Expressed in the Enamel of Mice with Different Genetic Susceptibilities to Dental Fluorosis

Caries Research ◽

10.1159/000491554 ◽

2018 ◽

Vol 53 (2) ◽

pp. 228-233 ◽

Cited By ~ 4

Author(s):

Senda Charone ◽

Erika Calvano Küchler ◽

Aline de Lima Leite ◽

Mileni Silva Fernandes ◽

Vinicius Taioqui Pelá ◽

...

Keyword(s):

Drinking Water ◽

Real Time ◽

Dna Extraction ◽

Genetic Polymorphisms ◽

Clinical Evaluation ◽

Real Time Pcr ◽

Genomic Dna ◽

Dental Fluorosis ◽

Clinical Aspects ◽

Genomic Dna Extraction

Genes expressed during amelogenesis are candidates to increase the risk of dental fluorosis (DF). Thus, this study aimed to evaluate the association between polymorphisms in enamel development genes and susceptibility to DF in mice. Mice of both sexes, representing strains 129P3/J (n = 20; resistant to DF) and A/J (n = 20; susceptible to DF), were divided into 2 groups. Each strain received a diet with a low concentration of fluoride (F) and drinking water containing 0 or 50 mg/L of F for 6 weeks. Clinical evaluation and analysis of Vickers enamel microhardness of the incisors were performed. Livers were collected for genomic DNA extraction. Seventeen genetic polymorphisms in Amelx, Ambn, Ambn, Col14a1, Col1a1, Col5a2, Enam, Fam20a, Fam83h, Foxo1, Klk4, Mmp20, Serpinf1, Serpinh1, Smad3, Tuft1, and Wdr72 were genotyped by real-time PCR using Taqman chemistry. Overrepresentation of alleles and genotypes in DF was evaluated using the χ2 test with an alpha of 5%. The clinical aspects of the enamel and the surface enamel microhardness confirmed the DF condition. In the polymorphisms rs29569969, rs13482592, and rs13480057 in Ambn, Col14a1, and Mmp20, respectively, genotype and allele distributions were statistically significantly different between A/J and 129P3/J strains (p < 0.05). In conclusion, polymorphisms in Ambn, Col14a1, and Mmp20 are associated with the susceptibility to DF.

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Multiplex Real-Time PCR Detection of Fumonisin-Producing and Trichothecene-Producing Groups of Fusarium Species

Journal of Food Protection ◽

10.4315/0362-028x-67.3.536 ◽

2004 ◽

Vol 67 (3) ◽

pp. 536-543 ◽

Cited By ~ 58

Author(s):

B. H. BLUHM ◽

M. A. COUSIN ◽

C. P. WOLOSHUK

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Genomic Dna ◽

Fusarium Species ◽

Fusarium Spp ◽

Pcr Assay ◽

Traditional Methods ◽

Microbiological Methods ◽

Pcr Products ◽

Its Pcr

Some species of Fusarium can produce mycotoxins during food processing procedures that facilitate fungal growth, such as the malting of barley. The objectives of this study were to develop a 5′ fluorogenic (Taqman) real-time PCR assay for group-specific detection of trichothecene- and fumonisin-producing Fusarium spp. and to identify Fusarium graminearum and Fusarium verticillioides in field-collected barley and corn samples. Primers and probes were designed from genes involved in mycotoxin biosynthesis (TRI6 and FUM1), and for a genus-specific internal positive control, primers and a probe were designed from Fusarium rDNA sequences. Real-time PCR conditions were optimized for amplification of the three products in a single reaction format. The specificity of the assay was confirmed by testing 9 Fusarium spp. and 33 non- Fusarium fungal species. With serial dilutions of purified genomic DNA from F. verticillioides, F. graminearum, or both as the template, the detection limit of the assay was 5 pg of genomic DNA per reaction. The three products were detectable over four orders of magnitude of template concentration (5 pg to 5 ng of genomic DNA per reaction); at 50 ng template per reaction, only the TRI6 and FUM1 PCR products were detected. Barley and corn samples were evaluated for the presence of Fusarium spp. with traditional microbiological methods and with the real-time PCR assay. The 20 barley samples and 1 corn sample that contained F. graminearum by traditional methods of analysis tested positive for the TRI6 and internal transcribedspacer (ITS) PCR products. The five corn samples that tested positive for F. verticillioides by traditional methods also were positive for the FUM1 and ITS PCR products. These results indicate that the described multiplex real-time PCR assay provides sensitive and accurate differential detection of fumonisin- and trichothecene-producing groups of Fusarium spp. in complex matrices.

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Reliability of non-invasively acquired human genomic DNA as a substrate for real-time PCR-assisted analysis of genetic polymorphisms

Archives of Toxicology ◽

10.1007/s00204-004-0554-3 ◽

2004 ◽

Vol 78 (7) ◽

Cited By ~ 7

Author(s):

T. Neuhaus ◽

G. Geisen ◽

H.M. Bolt ◽

V. Janzen ◽

A. Kraemer ◽

...

Keyword(s):

Real Time ◽

Genetic Polymorphisms ◽

Real Time Pcr ◽

Genomic Dna ◽

Human Genomic ◽

Assisted Analysis ◽

Human Genomic Dna

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BRCA1 gene mutations frequency estimation by allele-specific real-time PCR of pooled genomic DNA samples

The Breast ◽

10.1016/j.breast.2012.12.007 ◽

2013 ◽

Vol 22 (4) ◽

pp. 532-536 ◽

Cited By ~ 4

Author(s):

Maksim S. Anisimenko ◽

Dmitriy V. Mitrofanov ◽

Olga B. Chasovnikova ◽

Mikhail I. Voevoda ◽

Sergey P. Kovalenko

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Genomic Dna ◽

Frequency Estimation ◽

Gene Mutations ◽

Brca1 Gene ◽

Allele Specific

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Multiple Displacement Amplification Enables Large Scale Clonal Analysis after Retroviral Gene Therapy.

Blood ◽

10.1182/blood.v108.11.5469.5469 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5469-5469

Author(s):

Stephanie Bleier ◽

Patrick Maier ◽

Frederik Wenz ◽

W. Jens Zeller ◽

Stephanie Laufs ◽

...

Keyword(s):

Gene Therapy ◽

Real Time ◽

Real Time Pcr ◽

Genomic Dna ◽

Large Scale ◽

Integration Site ◽

Multiple Displacement Amplification ◽

Quantitative Real Time Pcr ◽

Integration Site Analysis ◽

Reliable Quantification

Abstract Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole genome amplification named multiple displacement amplification (MDA) with respect to the even and accurate representation of retrovirally transduced genomic DNA. We were able to show that MDA is a suitable method to subsequently specify engraftment efficiencies by quantitative real-time PCR as the retroviral integrations are amplified the same way and by the same probability as all other parts of the genome. We validated the method by analyzing a dilution series containing retrovirally transduced DNA and untransduced background DNA and retroviral integrations found in primary material from a retroviral transplantation model by quantitative real-time PCR. The representation of the portion of retroviral DNA in the amplified samples was 0.9-fold (range 0.2 – 2.1-fold) of the portion determined in the original genomic DNA. Furthermore, the succession of the combination of MDA and integration site analysis by ligation-mediated PCR showed an increase in the sensitivity of the method as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show that MDA enables large scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow up analysis in gene therapy studies even from smallest amounts of starting material.

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Polymorphisms in Nonamelogenin Enamel Matrix Genes Are Associated with Dental Fluorosis

Caries Research ◽

10.1159/000479826 ◽

2017 ◽

Vol 52 (1-2) ◽

pp. 1-6 ◽

Cited By ~ 14

Author(s):

Erika Calvano Küchler ◽

Carolina Dea Bruzamolin ◽

Marjorie Ayumi Omori ◽

Marcelo C. Costa ◽

Leonardo Santos Antunes ◽

...

Keyword(s):

Real Time ◽

Genetic Polymorphisms ◽

Real Time Pcr ◽

Genomic Dna ◽

Dental Fluorosis ◽

Permanent Teeth ◽

Enamel Matrix ◽

Water Supplies ◽

Allele Distribution

The aim of this study was to evaluate whether genetic polymorphisms in AMELX, AMBN, ENAM, TFIP11, and TUFT1 genes are associated with dental fluorosis (DF). A total of 1,017 children from 2 Brazilian cohorts were evaluated. These populations lived in cities with fluoridation of public water supplies. DF was assessed in erupted permanent teeth using the modified Dean index. The polymorphisms rs946252, rs12640848, rs4694075, rs5997096, and rs4970957 were analyzed by real-time PCR from genomic DNA. Associations between DF, genotype, and allele distribution were evaluated using the χ2 test, with an alpha of 5%. The polymorphisms rs4694075, rs5997096, and rs4970957 in AMBN, TFIP11, and TUFT1 were associated with DF (p < 0.05). In conclusion, enamel matrix genes are associated with DF.

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Methods for DNA quantification yield similar relative but different absolute values

Bulletin of Russian State Medical University ◽

10.24075/brsmu.2019.043 ◽

2019 ◽

pp. 25-31

Author(s):

O. P. Balanovsky ◽

ZhA Kagazezheva ◽

M. V. Olkova

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Genomic Dna ◽

Correlation Coefficients ◽

Dna Quantification ◽

Sample Storage ◽

Excellent Correlation ◽

The Real ◽

Absolute Concentrations

DNA quantification is a routine yet important procedure that determines the efficacy of long-term sample storage and further manipulations with the sample. There are a few well-established methods for measuring DNA concentrations. However, it still not fully clear how concordant their results are. The aim of this work was to measure DNA concentrations in a set of samples using different quantification methods and to compare the obtained values. In 2 independent experiments, a total of 100 genomic DNA samples were analyzed using 3 different DNA quantification methods, including spectrophotometry (NanoDrop), fluorometry (Qubit) and real-time PCR (Quantifiler). The obtained relative concentrations demonstrated an excellent correlation (the correlation coefficients were as high as 0.98 to 0.99). However, the absolute concentrations showed a considerable variation and even a twofold difference. Spectrophotometry yielded the highest concentrations, whereas fluorometry yielded the lowest. The real-time PCR results were intermediate. The differences were more pronounced for the samples with low DNA concentrations. We recommend that such differences should be accounted for when estimating DNA concentrations using an arsenal of different quantification methods.

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