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

Table 3 Symmetric, asymmetric and specificity assessment of different mixes

Parameter	Species ^a	Mix
Parameter	Species ^a	A	B	C	D	E	F	G	H	I	J	K
DNA amount (ng) ^b,c	A. creber	5	LOD^c	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

^agDNA extracted from pure strains from the BCCM/IHEM collection
^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
^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
^dDetection parameters i.e., clusters defined with the Biorad Precision Melt Analysis software 1.2 (Temse, Belgium). ND: no amplicon obtained, no cluster detected
^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
^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)

ISSN: 1471-2180