VP06.04: The novel screening method based on real‐time PCR for common fetal trisomies

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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Detection and identification of Acanthamoeba and other non-viral causes of infectious keratitis in corneal scrapings by real-time PCR and next-generation sequencing-based 16S-18S gene analysis

Journal of Clinical Microbiology ◽

10.1128/jcm.02224-20 ◽

2020 ◽

Author(s):

Dennis Back Holmgaard ◽

Celine Barnadas ◽

Seyed Hossein Mirbarati ◽

Lee O’Brien Andersen ◽

Henrik Vedel Nielsen ◽

...

Keyword(s):

Next Generation Sequencing ◽

Real Time ◽

Real Time Pcr ◽

Clinical Relevance ◽

Bacterial Species ◽

Screening Method ◽

Infectious Keratitis ◽

Next Generation ◽

Specificity And Sensitivity ◽

Generation Sequencing

Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for detection of Acanthamoeba. Two hundred DNAs extracted from corneal scrapings and screened by Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive using specific real-time PCR, 21 of which were positive using the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (n = 19) and T6 (n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for Acanthamoeba, exemplified by Pseudomonas aeruginosa (n = 11), Moraxella spp. (n = 6), Staphylococcus aureus (n = 2), Fusarium spp. (n = 4), and Candida albicans (n = 1). Conclusively, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotype was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for Acanthamoeba. NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting non-viral causes of IK, including Acanthamoeba.

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Analytical Performance of a Real-time PCR-based Assay for V600 Mutations in the BRAF Gene, Used as the Companion Diagnostic Test for the Novel BRAF Inhibitor Vemurafenib in Metastatic Melanoma

Diagnostic Molecular Pathology ◽

10.1097/pdm.0b013e31823b216f ◽

2012 ◽

Vol 21 (1) ◽

pp. 1-8 ◽

Cited By ~ 122

Author(s):

Harkanwal Halait ◽

Kelli DeMartin ◽

Sweta Shah ◽

Stephen Soviero ◽

Rachel Langland ◽

...

Keyword(s):

Metastatic Melanoma ◽

Real Time ◽

Diagnostic Test ◽

Real Time Pcr ◽

Braf Inhibitor ◽

Analytical Performance ◽

The Novel ◽

Braf Gene ◽

Companion Diagnostic

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Evaluation of the Novel Helicobacter pylori ClariRes Real-Time PCR Assay for Detection and Clarithromycin Susceptibility Testing of H. pylori in Stool Specimens from Symptomatic Children

Journal of Clinical Microbiology ◽

10.1128/jcm.00103-07 ◽

2007 ◽

Vol 45 (6) ◽

pp. 1718-1722 ◽

Cited By ~ 42

Author(s):

C. Lottspeich ◽

A. Schwarzer ◽

K. Panthel ◽

S. Koletzko ◽

H. Russmann

Keyword(s):

Helicobacter Pylori ◽

Real Time ◽

Real Time Pcr ◽

Susceptibility Testing ◽

The Novel ◽

Pcr Assay ◽

Stool Specimens ◽

H Pylori

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Rapid Screening Method for Compounds That Affect the Growth and Germination of Candida albicans, Using a Real-Time PCR Thermocycler

Applied and Environmental Microbiology ◽

10.1128/aem.06227-11 ◽

2011 ◽

Vol 77 (22) ◽

pp. 8193-8196 ◽

Cited By ~ 6

Author(s):

Lucja M. Jarosz ◽

Bastiaan P. Krom

Keyword(s):

Candida Albicans ◽

Real Time ◽

Real Time Pcr ◽

Fluorescent Protein ◽

Screening Method ◽

Rapid Screening ◽

Content Type ◽

Wide Range ◽

Hyphal Formation ◽

Green Fluorescent

ABSTRACTWe propose a screening method for compounds affecting growth and germination inCandida albicansusing a real-time PCR thermocycler to quantify green fluorescent protein (GFP) fluorescence. Using PACT1-GFPand PHWP1-GFPreporter strains, the effects of a wide range of compounds on growth and hyphal formation were quantitatively assessed within 3 h after inoculation.

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