Broad range PCR-based compositions and methods for the detection and identification of fungal pathogens

A method of broad range detection of at least one type of fungal nucleic acid in a patient sample, said method comprising:

• (a) carrying out a combination of PCR reactions on an isolated patient sample, wherein the combination comprises amplification of at least a region of a fungal ITS2 ribosomal RNA (rRNA) gene and a region of a fungal 28S rRNA gene;
• i. wherein the fungal ITS2 rRNA gene is amplified with a first PCR reaction comprising a first primer set comprising a first forward primer and a first reverse primer, wherein said first reverse primer is complementary to a fungal ITS2 rRNA gene in a region of said fungal ITS2 rRNA gene;
• ii. wherein the fungal 28S rRNA gene is amplified with a second PCR reaction comprising a second primer set comprising a second forward primer and a second reverse primer, wherein said reverse primer is complementary to a fungal 28S rRNA gene in a region of said fungal 28S rRNA gene that is 3′ to a D1-D2 highly variable region, wherein said second forward primer comprises a sequence selected from the group consisting of:
• 5′-GTAAAAGTCGTAACAAGGTTTC-3′,
• (SEQ ID NO:
• 1)
• 5′-GTGAATCATCGARTCTTTGAAC-3′,
• (SEQ ID NO:
• 2)
• 5′-GAAGTGGGGAAAGGTTCC-3′,
• (SEQ ID NO:
• 11)
• 5′-GACATGGGTTAGTCGATCCTA-3′,
• (SEQ ID NO:
• 12)
• 5′-GTTGATAGAAYAATGTAGATAAGG-3′,
• (SEQ ID NO:
• 14)
• 5′-CCGGGTTGAWGACATTGTCA-3′,
• (SEQ ID NO:
• 17)
• 5′-TTTGATTTTCAGTGTGAATACAAACCA-3′,
• (SEQ ID NO:
• 20)
• and
• 5′-GATAATTGGTWTTTGCGGCTG-3′,
• (SEQ ID NO:
• 25)
• and
• wherein said second reverse primer comprises a sequence selected from the group consisting of:
• 5′-GTTCAAAGAYTCGATGATTCAC-3′,
• (SEQ ID NO:
• 29)
• 5′-TATGCTTAAGTTCAGCGGGTA-3′,
• (SEQ ID NO:
• 30)
• 5′-TTTCCTCCGCTTATTGATATGC-3′,
• (SEQ ID NO:
• 31)
• 5′-CCGCTTCACTCGCCGYTACT-3′,
• (SEQ ID NO:
• 32)
• 5′-CCTTATCTACATTRTTCTATCAAC-3′,
• (SEQ ID NO:
• 41)
• 5′-GACAGTCAGATTCCCCTTG-3′,
• (SEQ ID NO:
• 42)
• 5′-CTTACCGAATTCTGCTTCGGT-3′,
• (SEQ ID NO:
• 51)
• 5′-CAGCCGCAAAWACCAATTATC-3′,
• (SEQ ID NO:
• 52)
• 5′-AGTAGCCTTGTTGYTACGA-3′,
• (SEQ ID NO:
• 54)
• and
• 5′-CCTTATCTACATTATTCTATGGAC-3′;
• (SEQ ID NO:
• 108)
• wherein said second primer set comprises a combination of forward and reverse primers selected from the group consisting of (SEQ ID NO:
• 2 and SEQ ID NO:
• 31), (SEQ ID NO:
• 2 and SEQ ID NO:
• 32), (SEQ ID NO:
• 11 and SEQ ID NO:
• 41), (SEQ ID NO:
• 1 and SEQ ID NO:
• 29), (SEQ ID NO:
• 2 and SEQ ID NO:
• 30), (SEQ ID NO:
• 12 and SEQ ID NO:
• 41), (SEQ ID NO:
• 12, SEQ ID NO:
• 41, and SEQ ID NO:
• 108), (SEQ ID NO:
• 14 and SEQ ID NO:
• 42), (SEQ ID NO:
• 17 and SEQ ID NO:
• 51), (SEQ ID NO:
• 20 and SEQ ID NO:
• 51), (SEQ ID NO:
• 20 and SEQ ID NO:
• 52), and (SEQ ID NO:
• 25 and SEQ ID NO:
• 54);
• iii. wherein if fungal DNA is present in said patient sample, said second PCR reaction generates a fungal PCR amplicon of between 50 and 1000 base pairs that comprises a region of said fungal ITS2 rRNA gene or a region of said fungal 28S rRNA gene that is 3′ to a D1-D2 highly variable region, and
• iv. wherein if human DNA is present in said patient sample, said second PCR reaction does not generate a detectable PCR amplicon that comprises a region of said human DNA;
• and
• (b) detecting said fungal PCR amplicon;
• wherein detection of the fungal PCR amplicon is indicative of the presence of at least one type of fungal nucleic acid;
• and
• wherein the at least one type of fungal nucleic acid is from one or more fungal pathogens selected from the group consisting of Absidia corymbifera;
• Cunninghamella bertholletiae;
• Fusarium solani;
• Mucor racemosus;
• Paecilomyces variotii;
• Penicillium chrysogenum;
• Rhizomucor miehei;
• Rhodotorula glutinis;
• Scedosporium apiospermum;
• Antrodia vaillantii;
• Aspergillus fumigatus;
• Aspergillus niger;
• Aspergillus oryzae;
• Aspergillus terreus;
• Batrachochytrium dendrobatidis;
• Botrytis cinerea;
• Candida albicans;
• Candida dublineinsis;
• Candida glabrata;
• Candida gulliermundei;
• Candida kefyr;
• Candida krusei;
• Candida hpolytica;
• Candida lusitaniae;
• Candida parapsilosis;
• Candida tropicalis;
• Chaetomium globosum;
• Coccidioides immitis;
• Coccidioides posadasii;
• Cryptococcus neoformans;
• Fusarium graminearum;
• Fusarium oxysporum;
• Histoplasma capsulatum;
• Hypocrea jecorina;
• Lodderomyces elongisporus;
• Magnaporthe grisea;
• Metarhizium anisopliae;
• Microsporum gypseum;
• Neurospora crassa;
• Paracoccidioides brasthens;
• Pneumocystis carinii;
• Penicillium verrucosum;
• Pichia aponica;
• Rhizopus oryzae;
• Saccharomyces cerevisiae;
• Schizosaccharomyces aponicas;
• Schizosaccharomyces pombe;
• Sclerotinia sclerotiorum;
• Stagonospora nodorum;
• Umbilicaria esculenta;and Uncinocarpus reesii.