TABLE 3.

Screening data for the top 11 fungal broad-range amplicons

Species or parameter	Screening dataa
	ITS1 (End18SF-5.8SR)	ITS2 (5.8SF-1R)	ITS2 (5.8SF-2R)	ITS2 (5.8SF-3R)	28S (9F-12R)	28S (10F-12R)	28S (12F-13R)	28S (15F-22R)	28S (18F-22R)	28S (18F-23R)	28S (23F-25R)
Absidiacorymbifera	−	+	−	−	+++	+++	+++	−	++	+++	+
Aspergillus candidus	+++	+++	+	+	+++	+++	+++	+++	+++	+++	+
Aspergillus flavus	+++	+++	+++	+++	+++	+++	++	+++	+++	+++	++
Aspergillus fumigatus	+++	+++	+++	+++	+++	+++	++	+++	+++	+++	+
Aspergillus oryzae	+++	+++	+++	+++	+++	+++	++	+++	+++	+++	++
Aspergillus terreus	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	++
Aspergillus ustus	+++	+++	+++	+++	+++	+++	++	+++	+++	+++	++
Candida albicans	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++
Candida dubliniensis	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++
Candida glabrata	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++
Candida guilliermondii	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++
Candida kefyr	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++
Candida krusei	+++	+++	+++	+++	+++	+++	++	+++	+++	+++	+++
Candida lusitaniae	+++	+++	+++	+++	+++	+++	+	+++	+++	++	−
Candida tropicalis	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++
Cryptococcus neoformans	+++	+++	+	++	+++	+++	++	+++	+++	++	−
Cunninghamellaberthollotiae	−	++	++	+	+++	+++	+++	−	++	+++	−
Fusarium solani	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	++
Mucorracemosus	+++	++	++	+++	+++	+++	+++	−	+++	+++	+
Paecilomycesvariotii	−	+++	+++	+++	+++	+++	++	+++	+++	+++	+
Penicilliumnotatum	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++
Rhizomucormiehei	−	++	+	−	+	+++	+	−	++	+++	+
Rhizopusoryzae	+++	+++	+++	+++	+++	+++	+++	−	−	+++	++
Rhodotorulaglutinis	+++	+++	+++	+++	−	−	+++	+++	+++	−	+++
Saccharomyces cerevisiae	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++
Scedosporiumapiospermum	++	+++	+++	+++	+++	+++	++	+++	+++	+++	+
No. of +++ bands	21	22	20	21	24	25	16	21	22	23	10
No. of ++ bands	1	3	2	1	0	0	8	0	3	2	6
No. of + bands	0	1	3	2	1	0	2	0	0	0	7
No. of with absent band	4	0	1	2	1	1	0	5	1	1	3
Effect of 1 μg human DNAb	None	None	None	None	None	None	None	None	None	None	None
Sum for distance matrixc	642.5d	1,055.8	NDe	ND	ND	310.8	470.6	67.5	74.5	117.3	358.3d
Sum for distance matrixf	97,914d	113,388	ND	ND	ND	82,452	61,321	17,522	9,629	28,716	66,445d
Amplicon lengthg	295 ± 70d	254 ± 43	ND	ND	ND	339 ± 7	198 ± 25	289 ± 1	147 ± 1	308 ± 46	263 ± 10d

^a+++, ++, +, and − indicate the PCR yield reflected by the intensity level of product bands on a 1.5% agarose gel. +++, ++, and + indicate decreasing intensity levels, and − indicates no amplification. An example of a gel image is shown in Fig. 2.

^bCross-reactivity was evaluated based on the ability to amplify 10 fg of A. fumigatus DNA in the presence of 1 μg of human genomic DNA.

^cDistances based on the Tajima-Nei algorithm and the neighbor-joining tree-building method. The sum for distance matrix was estimated from amplicons of 51 fungal species representing 30 genera. A higher value indicates greater sequence diversity among fungi for the selected amplicon.

^dDue to unavailability of some complete sequences spanning the ITS1(End18SF-5.8SR) and 28S(23F-25R) amplicons, 49 and 45 sequences, respectively, were used to estimate the sum of the distance matrix and mean amplicon length.

^eND, distance not estimated because the amplicon overlaps significantly with another amplicon in the table which has a greater breadth of fungi detected.

^fDistances based on absolute nucleotide differences and the neighbor-joining tree-building method.

^gMeans ± standard deviations for 51 fungal species representing 30 genera. The S. apiospermum sequence for the 28S(15F-22R), 28S(18F-22R), and 28S(18F-23R) amplicons was excluded from estimating the mean ± standard deviation due to the presence of a very large insert (nearly 400 bp) in the amplicon which disproportionately skewed the standard deviation estimate.