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

ABSTRACT Invasive aspergillosis caused by triazole-resistant strains of Aspergillus fumigatus is a growing public health concern, as is the occurrence of mixed infections with triazole-resistant and -susceptible A. fumigatus strains. Therefore, it is crucial to develop robust methods to identify triazole-resistant strains of A. fumigatus, even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus. In this work, we developed a robust, highly selective, and broad-range allele-specific TaqMan real-time PCR platform consisting of 7 simultaneous assays that detect TR34 (a 34-bp tandem repeat in the promoter region), TR46, G54W (a change of G to W at position 54), G54R, L98H, Y121F, and M220I mutations in the cyp51A gene of A. fumigatus. The method is based on the widely used TaqMan real-time PCR technology and combines allele-specific PCR with a blocking reagent (minor groove binder [MGB] oligonucleotide blocker) to suppress amplification of the wild-type cyp51A alleles. We used this method to detect triazole-resistant clinical strains of A. fumigatus with a variety of cyp51A gene mutations, as well as the triazole-resistant strains in mixtures of triazole-resistant and -susceptible strains of A. fumigatus. The method had high efficiency and sensitivity (300 fg/well, corresponding to about 100 CFU per reaction mixture volume). It could promptly detect triazole resistance in a panel of 30 clinical strains of A. fumigatus within about 6 h. It could also detect cyp51A-associated resistance alleles, even in mixtures containing only 1% triazole-resistant A. fumigatus strains. These results suggest that this method is robustly able to detect cyp51A-associated resistance alleles even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus and that it should have important clinical applications.

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Single-copy detection of somatic variants from solid and liquid biopsy

Scientific Reports ◽

10.1038/s41598-021-85545-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ana-Luisa Silva ◽

Paulina Klaudyna Powalowska ◽

Magdalena Stolarek ◽

Eleanor Ruth Gray ◽

Rebecca Natalie Palmer ◽

...

Keyword(s):

Real Time ◽

Single Molecule ◽

Real Time Pcr ◽

Clinical Decision Making ◽

High Sensitivity ◽

Clinical Decision ◽

Single Copy ◽

Turnaround Time ◽

Copy Detection ◽

Allele Specific

AbstractAccurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

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Real-Time PCR Determination of Escherichia coli Genomic DNA Contamination in Plasmid Preparations

Analytical Biochemistry ◽

10.1006/abio.2001.5480 ◽

2002 ◽

Vol 301 (1) ◽

pp. 151-153 ◽

Cited By ~ 10

Author(s):

Adrián Vilalta ◽

Vanessa Whitlow ◽

Terrie Martin

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Real Time Pcr ◽

Genomic Dna ◽

Dna Contamination

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Combining allele-specific fluorescent probes and restriction assay in real-time PCR to achieve SNP scoring beyond allele ratios of 1:1000

BioTechniques ◽

10.2144/000112719 ◽

2008 ◽

Vol 44 (2) ◽

pp. 193-199 ◽

Cited By ~ 12

Author(s):

Jonci N. Wolff ◽

Neil J. Gemmell

Keyword(s):

Real Time ◽

Fluorescent Probes ◽

Real Time Pcr ◽

Allele Specific

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Allele-Specific Real-Time PCR System for Detection of Subpopulations of Genotype 1a and 1b Hepatitis C NS5B Y448H Mutant Viruses in Clinical Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.00274-11 ◽

2011 ◽

Vol 49 (9) ◽

pp. 3168-3174 ◽

Cited By ~ 6

Author(s):

Andrew S. Bae ◽

Karin S. Ku ◽

Michael D. Miller ◽

Hongmei Mo ◽

Evguenia S. Svarovskaia

Keyword(s):

Hepatitis C ◽

Real Time ◽

Real Time Pcr ◽

Clinical Samples ◽

Genotype 1A ◽

Allele Specific

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Rapid, specific and quantitative polymerase chain reaction (PCR) detection of pathogenic protozoa Entamoeba histolytica for drinking water supply

Water Science & Technology Water Supply ◽

10.2166/ws.2011.057 ◽

2011 ◽

Vol 11 (4) ◽

pp. 418-425 ◽

Cited By ~ 1

Author(s):

S. W. Lam ◽

H. B. Zhang ◽

L. Yu ◽

C. H. Woo ◽

K. N. Tiew ◽

...

Keyword(s):

Real Time ◽

Water Samples ◽

Real Time Pcr ◽

Genomic Dna ◽

Tap Water ◽

Pcr Detection ◽

Raw Water ◽

Conventional Pcr ◽

Polymerase Chain ◽

Species Specific

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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