Interlaboratory reproducibility of a microsatellite-based typing assay for Aspergillus fumigatus through the use of allelic ladders: proof of concept.
An interlaboratory study was performed with the aim of investigating the reproducibility of a multiplex microbial microsatellite-based typing assay for Aspergillus fumigatus in different settings using a variety of experimental and analytical conditions and with teams having variable prior microsatellite typing experience. In order to circumvent problems with exchange of sizing data, allelic ladders are introduced as a straightforward and universally applicable concept for standardization of such typing assays. Allelic ladders consist of mixtures of well-characterized reference fragments to act as reference points for the position in an electrophoretic trace of fragments with established repeat numbers. Five laboratories independently analysed six microsatellite markers in 18 samples that were provided either as DNA or as A. fumigatus conidia. Allelic data were reported as repeat numbers and as sizes in nucleotides. Without the use of allelic ladders, size differences of up to 6.7 nucleotides were observed, resulting in interpretation errors of up to two repeat units. Difficulties in interpretation were related to non-specific amplification products (which were resolved with explanation) and bleed-through of the different fluorescent labels. In contrast, after resolution of technical or interpretive problems, standardization of sizing data by using allelic ladders enabled all participants to produce identical typing data. The use of allelic ladders as a routine part of molecular typing using microsatellite markers provides robust results suitable for interlaboratory comparisons and for deposition in a global typing database.
de Valk
H A
HA
Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands.
Meis
J F G M
JF
Bretagne
S
S
Costa
J-M
JM
Lasker
B A
BA
Balajee
S A
SA
Pasqualotto
A C
AC
Anderson
M J
MJ
Alcázar-Fuoli
L
L
Mellado
E
E
Klaassen
C H W
CH
D001232:Aspergillus fumigatus / Q000145:classification* / Q000235:genetics; D016172:DNA Fingerprinting / Q000379:methods* / Q000592:standards* / Q000706:statistics & numerical data; D004271:DNA, Fungal / Q000235:genetics*; D005838:Genotype; D018895:Microsatellite Repeats; D016533:Mycological Typing Techniques / Q000379:methods* / Q000592:standards* / Q000706:statistics & numerical data; D015588:Observer Variation; D015203:Reproducibility of Results
D004271:DNA, Fungal