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Table 3 Symmetric, asymmetric and specificity assessment of different mixes

From: Discrimination of three genetically close Aspergillus species by using high resolution melting analysis applied to indoor air as case study

Parameter Species a Mix           
A B C D E F G H I J K
DNA amount (ng) b,c A. creber 5 LODc 5 5 25 5 5 0 5 5 0
  A. sydowii 5 5 LOD 5 5 25 5 5 0 5 0
  A. versicolor 5 5 5 LOD 5 5 25 5 5 0 0
  P. chrysogenum 0 0 0 0 0 0 0 25 25 25 5
Cluster d   4 4 4 4 4 4 4 5 6 7 ND
Peaks in Melt Peak Chart observed e   72.40 ± 0.00 72.40 ± 0.00 72.40 ± 0.00 72.40 ± 0.00 72.40 ± 0.00 72.50 ± 0.14 72.50 ± 0.14 78.40 ± 0.00 77.20 ± 0.28 78.10 ± 0.14 ND
   80.60 ± 0.00 80.60 ± 0.00 80.60 ± 0.00 80.60 ± 0.00 80.60 ± 0.00 80.60 ± 0.00 80.60 ± 0.00 81.60 ± 0.00 85.00 ± 0.00 82.40 ± 1.41  
   85.40 ± 0.00 85.40 ± 0.00 85.60 ± 0.00 85.60 ± 0.00 85.50 ± 0.14 85.40 ± 0.00 85.40 ± 0.00     
Confidence (%) f   99.85 ± 0.07 99.75 ± 0.07 99.55 ± 0.35 96.65 ± 2.33 99.85 ± 0.07 99.85 ± 0.07 99.25 ± 1.06 99.30 ± 0.00 99.60 ± 0.00 99.10 ± 0.56 ND
  1. agDNA extracted from pure strains from the BCCM/IHEM collection
  2. bEach assay contained 4 pure strains i.e., BCCM/IHEM 2646 for A. creber, BCCM/IHEM 895 for A. sydowii, BCCM/IHEM 10351 for A. versicolor, and BCCM/IHEM 4151 for P. chrysogenum. The former 3 were used as positive control and resulted in cluster 1 (A. versicolor), cluster 2 (A. sydowii) and cluster 3 (A. creber). For P. chrysogenum, used as negative control, no amplification and hence no cluster was obtained. Each HRM reaction was done with 5 μl of mix
  3. cLOD defined as the limit of detection defined for each species i.e., 0.1 pg for A. versicolor and 0.5 pg for A. creber and A. sydowii
  4. dDetection parameters i.e., clusters defined with the Biorad Precision Melt Analysis software 1.2 (Temse, Belgium). ND: no amplicon obtained, no cluster detected
  5. ePeak observed in the Melt Peak chart given by the Biorad Precision Melt Analysis software 1.2 (Temse, Belgium) – this does not correspond to the Tm of each separate amplicon of the different species
  6. fConfidence (%) is the average of each percentage of confidence defined with the Biorad Precision Melt Analysis software 1.2 (Temse, Belgium) obtained for each repetition and standard deviation (±SD)