Development of a MIQE-compliant Aspergillus terreus specific qPCR assay

Ref ID: 19386

Author:

A. Ferrini,1 G. L. Johnson,1 S. G. Agrawal1 and S. A. Bustin2

Author address:

1Blizard Institute, Queen Mary University Of London, United
Kingdom and 2Faculty Of Health, Social Care And Education,
Anglia Ruskin University, Chelmsford, United Kingdom

Full conference title:

6th Trends in Medical Mycology 2013

Date: 11 October 2014

Abstract:

Objectives A reliable method for the early and accurate identifica-
tion of Aspergillus terreus is becoming more important as it increas-
ingly emerges as a causative agent of Invasive Aspergillosis.
Moreover, most members of this species have decreased susceptibility
to the antifungal drug Amphotericin B in vitro and in vivo (1). As a
consequence, the ability to distinguish A. terreus from other species
of Aspergillus is important to the clinician for therapeutic decision-
making and prognosis. Although in theory real-time PCR (qPCR) is
an effective method for the sensitive and accurate identification of
this pathogen, the practical usefulness of qPCR in fungal diagnostics
remains uncertain. Lack of specificity, poor amplification efficiencies
and absence of standardised protocols have resulted in assays that
are unreliable, not sufficiently optimised or validated and not fit for
purpose as routine diagnostic tests. The MIQE guidelines aim to pro-
vide a blueprint for best practice in assay design and reporting and
are widely viewed as an important contribution to translating qPCR-
based assays from a research into a practical diagnostic setting (2).
We followed these guidelines to generate a robust, sensitive and rapid
assay that is specific for Aspergillus terreus.
Methods The first step was the in silico analysis of primers and
amplicon, with stringent design criteria which resulted in two assays
taken forward for several empirical optimisation tests where the reac-
tion conditions and the assay performance were evaluated. The qPCR
assays were then validated using DNA extracted from fungal cultures
to assess their specificity. The two assays were comparable in their
performance, but the probe assessment was carried out for only one
of them, given its higher specificity. No amplification was seen in the
negative controls – human DNA and non-template control wells – in
any plates.
Results We obtained an A.terreus-specific assay that has an effi-
ciency of 98% which translates into a limit of detection of 0.6 copies
of A.terreus genome and that can be completed in less than 90 min-
utes. Furthermore, the results of the specificity analysis run show
that there was no amplification of any non-Aspergillus species or any
other Aspergillus species such as A. fumigatus, A.niger, A.nidulans or
A.flavus. Amplification only occurs with A. terreus, at a Cq of 27 for
the 2 pg/ll dilution, which corresponds to 60 copies of A. terreus
genome/ll.
Conclusion This highly sensitive and specific A.terreus-specific assay
is suitable for clinical application and the rapid identification of this
particular Amphotericin B-resistant Aspergillus species.
1) Steinbach et al., Infections due to Aspergillus terreus: A multi-
center retrospective analysis of 83 cases. Clinical Infectious Dis-
ease,2004; 39(2): 192-198
2) Bustin et al., The MIQE Guidelines: Minimum Information for
Publication of Quantitative Real-Time PCR Experiments, 2009, Clini-
cal Chemistry, 55:4, 611-622

Abstract Number: p059

Conference Year: 2013

Link to conference website: NULL

New link: NULL

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