1. DNA-probe based detection for differential
diagnosis of dermatomycoses Lysann Mehlig (1), Anne Wiedenfeld (1), Elisabeth Bröse (1), Angela Zeiler (2),
Cornelia Weber (2), Eric Aichinger (2), Werner Brabetz (2) and Andrea Bauer (1)
(1) Department of Dermatology, University Hospital "Carl Gustav Carus", Technical University Dresden, Fetschwerstraße 74 , Dresden, Germany
(2) Biotype Diagnostic GmbH, Moritzburger Weg 67, Dresden, Germany
Background
The sample pad contains the gold conjugated antibodies which bind the labeled probes hybridized to the species specific amplicons.
The thus formed hapten-amplicon-antibody complex binds to the Anti-hapten-antibodies found in the detection zone of the LFT leading to the visualization of the complex on the nitrocellulose membrane. The detection zone of each LFT contains five rows of Anti-hapten-antibodies enabling the detection of up to four specific pathogens at a time. The fifth row of the Anti-hapten-antibodies functions as a control position.
Controls
Prior to DNA extraction, patient samples are spiked with a control DNA sample which serves as extraction control. From the very same DNA, a hapten-labelled amplicon is generated during the Multiplex PCR serving as an internal amplification control for the PCR itself. Given the possibility of negative clinical samples, this hapten-amplicon-antibody complex will also serve as control for the functionality of the LFT. Its visualization at the fifth row of the Anti-hapten-antibodies in the detection zone proves the functionality of the lyophilized and liquid reagents (running buffer and the colloidal gold on the membrane) and a sufficient gold labeling of all PCR amplicons.
Sensitivity
Pathogens are detected from only 5 pg up to 1 ng DNA after DNA extraction.
* * able to detect also A. vanbreuseghemii,
T. mentagrophytes
PCR 1: Screen
dermatophytes/ yeasts/ moulds
PCR 1
QS-Ex *
T. rubrum
Dermatophyton spp. (TME)
Candida spp.
Scopulariopsis brevicaulis
PCR 2:
Trichophyten
PCR 2
T. Interdigitale* *
T. tonsurans
T. violaceum
A. benhamiae/
T. erinacei + T. verrucosum
PCR 3:
Microsporum/ Epidermophyton
PCR 3
M. audouinii
M. gypseum
M. canis
Epidermophyton floccosum
* QS-Ex = quality control for extraction of DNA and PCR (conform to RiLiBÄK and MiQ)
Worldwide, dermatomycosis and fungal in-fections of skin and mucosa are the most common diseases with increasing prevalence up to 26% and a high number of unreported cases1-3. The causative agent of superficial mycoses are mainly dermatophytes, yeasts and to a lesser extend non-dermatophyte mould. Depending on clinical pattern and geographic area different pathogens are dominating. In central europe most frequent for Tinea capitis is Microsporum canis 4, for onychomycoses Trichophyton rubrum, T. interdigitale 5 and Epidermophyton flocossum 6 but also Scopulariopsis brevicaulis, Aspergillus spp. or Candida spp. 7-9 (Fig.1).
Figure 2: Dermatophytes, yeasts and non-dermatophyte moulds detectable with the multiplex PCR Mentype® MycoDermQS NALFT.
Method
Pathogen specific amplification
The pathogen amplification is based on a modular multiplex PCR approach which consists of three multiplex PCRs (Fig.2). Having the same amplification parameters, they can either be run simultaneously or consecutively. PCR 1 is de-signed as a screen, determining whether the infection is caused by dermatophytes, fungi, yeast or a combination. Simultaneously, T. rubrum, the most common pathogen, is identified as well. With PCR 2 and 3 a possible dermatophytic infection is analyzed. T. inter-digitale, T. tonsurans, T. violaceum and T. verru-cosum are identified on species level by PCR 2, which also detects T. verrucosum, T. erinacei and A. benhamiae. PCR 3 identifies the Microsporum species M. audouinii, M. gypseum and M. canis as well as Epidermophyton floccosum. All generated amplicons are individually tagged with specific haptens.
Pathogen detection
The amplicons are detected on a lateral flow platform combining the nucleic acid lateral flow immunoassay (NALFIA) and nucleic acid lateral flow test strip (NALFT) technologies (Fig. 3 (1)). In our two-step model, at first the hapten-tagged amplicons from the multiplex PCR hybridized with labelled amplicon-specific probes (Fig. 3 (2)). In the second step the hapten-antibody-complex will be detected by a colour reaction on the lateral flow test strip (LFT) (Fig.3 (3)). LFTs are composed of sample pad, reagent pad, detection zone and wick (Fig.4 (f,g)).
Figure 1: Most frequent dermatophytes: Scopulariopsis brevicaulis (A), Microsporum gypseum (B), Trichophyton rubrum (C); depicted as culture macroscopic (left), culture microscopic (middle) and clinical picture (right).
An accurate and rapid detection of fungi is most important for the choice of antimycotics and the success of treatment. For a long time, identification of fungi was almost based on morphological features examined by microscopy or microbial culture. The success of these conventional laboratory procedures requires long-term expertise due to technical challenges as well as the interspecific morphological similarities and growth variabilities. Meanwhile,
molecular methods using different genetic targets and techniques are available. To fulfill quality standards for pathogen detection based on DNA techniques the affirmation of target amplification by means of a DNA probe (MiQ) is required.
Figure 4: Workflow in pictures: multiplex-PCR (a), centrifugation (b), hybridization with DIG-labelled probes (c) in a thermo cycler (d) and detection via lateral flow test strips (LFT) (f,g)
(a) (b) (c) (d) (e)
(f) (g)
Objectives
Sample
Pad
Reagent
(Gold conj. Ab)
Detection
Zone
Wick
(1) multiplex PCR
(3) Lateral Flow Detektion of
hapten-antibody-complex
(2) hybridization
LF diagnostic medical dipstick + LF buffer
haptene:
probes labeled with DIG:
Anti-
Hapten-Ab
Figure 3: NALFT with hybridization haptene + antibody (Ab) (compliant with MiQ 2011 )
The aim of this study was to improve a recently evaluated multiplex-based PCR diagnostic test kit 10 to fulfill the above mentioned quality standards while maintaining already achieved performance. The PCR diagnostic kit has been developed as a rapid screening assay allowing the detection and differentiation of the most relevant human pathogenic dermatophytes, yeasts and moulds present in Europe (Fig.2).
At the same time we pursued improvement of further test parameter such as user-optimized handling, time saving and species specific recognition.
References
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