Assessment of the Critical Components of a Pan-fungal Pcr Assay

P. D. Khot, E. S. Slechta, E. Kish-Trier, K. E. Hanson

Author address: 

ARUP Lab., Salt Lake City, UT

Abstract: 

Background: Broad-range detection of fungal nucleic acid directly in human tissue has potential utility for the diagnosis of invasive fungal infections. The purpose of this study was to assess impact of primer design, master mix composition and DNA extraction method on interference from environmental contamination.Methods: Real Time PCR was performed on the Quant Studio (Life Technologies), with amplicons analyzed by post-PCR melt-curve analysis and Sanger sequencing. Two master mixes were evaluated including a standard mix and a fungal DNA-free custom mix, both from Promega. Primers targeted the 18S, 28S and ITS2 regions. Automated (Promega and Chemagen) and manual (Zymo and MoBio) extraction methods using fresh tissue with were also compared.Results: Initial studies were performed with 62 different fungal organisms and the standard master mix. All organism identifications were correct, but the number of specimens detected at

Tables: 

2016

abstract No: 

MONDAY-242

Full conference title: 

ASM Microbe 2016
    • ASM microbe 1st (2016)