Quantification of the copy number of nor-1, a gene of the aflatoxin biosynthetic pathway by real-time PCR, and its correlation to the cfu of Aspergillus flavus in foods

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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Quantification of the Transcripts Encoding Different Forms of AChE in Various Cell Types: Real-Time PCR Coupled with Standards in Revealing the Copy Number

Journal of Molecular Neuroscience ◽

10.1007/s12031-013-0210-6 ◽

2014 ◽

Vol 53 (3) ◽

pp. 461-468 ◽

Cited By ~ 9

Author(s):

Cathy W.C. Bi ◽

Wilson K.W. Luk ◽

María-Letizia Campanari ◽

Yuen H. Liu ◽

Li Xu ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Cell Types

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Photo-Induced Electron Transfer Real-Time PCR for Detection ofPlasmodium falciparum plasmepsin 2Gene Copy Number

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00317-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 1

Author(s):

Samaly Santos Souza ◽

Mariangela L'Episcopia ◽

Carlo Severini ◽

Venkatachalam Udhayakumar ◽

Naomi W. Lucchi

Keyword(s):

Electron Transfer ◽

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Gene Copy ◽

Content Type ◽

Copy Numbers ◽

Ofplasmodium Falciparum ◽

Photo Induced Electron Transfer ◽

Gene Copy Numbers

ABSTRACTPiperaquine is an important partner drug used in artemisinin-based combination therapies (ACTs). An increase in theplasmepsin 2and3gene copy numbers has been associated with decreased susceptibility ofPlasmodium falciparumto piperaquine in Cambodia. Here, we developed a photo-induced electron transfer real-time PCR (PET-PCR) assay to quantify the copy number of theP. falciparumplasmepsin 2gene (PfPM2) that can be used in countries whereP. falciparumis endemic to enhance molecular surveillance.

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Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillus flavus and A. parasiticus in Hazelnut

Toxins ◽

10.3390/toxins12120757 ◽

2020 ◽

Vol 12 (12) ◽

pp. 757

Author(s):

Sara Franco Ortega ◽

Ilenia Siciliano ◽

Simona Prencipe ◽

Maria Lodovica Gullino ◽

Davide Spadaro

Keyword(s):

Food Safety ◽

Real Time ◽

Aspergillus Flavus ◽

Real Time Pcr ◽

Limit Of Detection ◽

Screening Method ◽

Lamp Assay ◽

High Sensitivity ◽

Isothermal Amplification

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100–999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.

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