Molecular Characterization of Large Deletions in the von Hippel-Lindau (VHL) Gene by Quantitative Real-Time PCR

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

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Detection and Characterization of Circulating Tumor Cells by Quantitative Real-Time PCR

Methods in Molecular Biology - Quantitative Real-Time PCR ◽

10.1007/978-1-4939-9833-3_11 ◽

2019 ◽

pp. 139-151 ◽

Cited By ~ 1

Author(s):

Francesca Salvianti ◽

Filomena Costanza ◽

Gemma Sonnati ◽

Pamela Pinzani

Keyword(s):

Real Time ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Real Time Pcr ◽

Quantitative Real Time Pcr

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Rapid and reliable detection of α-globin copy number variations by quantitative real-time PCR

BMC Hematology ◽

10.1186/2052-1839-14-4 ◽

2014 ◽

Vol 14 (1) ◽

Cited By ~ 21

Author(s):

Runa M Grimholt ◽

Petter Urdal ◽

Olav Klingenberg ◽

Armin P Piehler

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Globin Gene ◽

Copy Number Variations ◽

Globin Genes ◽

Quantitative Real Time Pcr ◽

Human Genetic Disease ◽

Large Deletions ◽

Wide Range

Abstract Background Alpha-thalassemia is the most common human genetic disease worldwide. Copy number variations in the form of deletions of α-globin genes lead to α-thalassemia while duplications of α-globin genes can cause a severe phenotype in β-thalassemia carriers due to accentuation of globin chain imbalance. It is important to have simple and reliable methods to identify unknown or rare deletions and duplications in cases in which thalassemia is suspected but cannot be confirmed by multiplex gap-PCR. Here we describe a copy number variation assay to detect deletions and duplications in the α-globin gene cluster (HBA-CNV). Results Quantitative real-time PCR was performed using four TaqMan® assays which specifically amplify target sequences representing both the α-globin genes, the –α3.7 deletion and the HS-40 region. The copy number for each target was determined by the 2-ΔΔCq method. To validate our method, we compared the HBA-CNV method with traditional gap-PCR in 108 samples from patients referred to our laboratory for hemoglobinopathy evaluation. To determine the robustness of the four assays, we analyzed samples with and without deletions diluted to obtain different DNA concentrations. The HBA-CNV method identified the correct copy numbers in all 108 samples. All four assays showed the correct copy number within a wide range of DNA concentrations (3.2-100 ng/μL), showing that it is a robust and reliable method. By using the method in routine diagnostics of hemoglobinopathies we have also identified several deletions and duplications that are not detected with conventional gap-PCR. Conclusions HBA-CNV is able to detect all known large deletions and duplications affecting the α-globin genes, providing a flexible and simple workflow with rapid and reliable results.

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Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

Applied and Environmental Microbiology ◽

10.1128/aem.04137-13 ◽

2014 ◽

Vol 80 (10) ◽

pp. 3086-3094 ◽

Cited By ~ 115

Author(s):

Hyatt C. Green ◽

Richard A. Haugland ◽

Manju Varma ◽

Hana T. Millen ◽

Mark A. Borchardt ◽

...

Keyword(s):

Real Time ◽

Surface Waters ◽

Real Time Pcr ◽

Fecal Pollution ◽

Qpcr Assay ◽

Taqman Assay ◽

Reference Database ◽

Quantitative Real Time Pcr ◽

Ambient Surface

ABSTRACTQuantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genusBacteroidesare among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

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