Compositions and methods for detecting
Disclosed are oligonucleotides useful in methods for determining whether a sample contains Cryptococcus neoformans , a causative agent for human cryptococcosis. These oligonucleotides, which have nucleotide sequences derived from a coding segment of the gene encoding the fungal specific transcription factor gene in Cryptococcus neoformans , are useful as forward and reverse primers for a polymerase chain reaction using nucleic acids from a biological sample as templates, and as probes for detecting any resultant amplicon. Detection of an amplicon indicates the sample contains Cryptococcus neoformans . Real-time PCR and detection using florescence resonance energy transfer is disclosed.
1. An isolated hybridization probe, wherein said probe has a nucleotide sequence consisting essentially of SEQ ID NO: 5.
2. The isolated hybridization probe of claim 1 , wherein said probe comprises a fluorescent moiety.
3. The isolated hybridization probe of claim 2 , wherein said fluorescent moiety is 6-carboxy-fluorescein.
4. A kit for PCR detection of Cryptococcus neoformans , comprising the isolated hybridization probe of claim 1 , said kit further comprises:
(a) a forward primer that anneals to a Cryptococcus neoformans consensus sequence consisting essentially of nucleotides 1121-1144 of SEQ ID NO: 1;
(b) a reverse primer that anneals to a Cryptococcus neoformans consensus sequence consisting essentially of nucleotides 1512-1533 of SEQ ID NO: 2; and
(c) an instruction for using said forward primer and reverse primer in performing PCR to detect the presence of Cryptococcus neoformans in a sample.
5. The kit of claim 3 , wherein said forward primer has a nucleotide sequence consisting essentially of SEQ ID NO: 3 and said reverse primer has a nucleotide sequence consisting essentially of SEQ ID NO: 4.
6. A kit for PCR detection of Cryptococcus neoformans , comprising the isolated hybridization probe of claim 1 , said kit further comprises:
(a) a forward primer that anneals to a Cryptococcus neoformans consensus sequence consisting essentially of nucleotides 1480-1499 of SEQ ID NO: 1;
(b) a reverse primer that anneals to a Cryptococcus neoformans consensus sequence consisting essentially of nucleotides 1165-1184 of SEQ ID NO: 2; and
(c) an instruction for using said forward primer and reverse primer in performing PCR to detect the presence of Cryptococcus neoformans in a sample.
7. The kit of claim 5 , wherein said forward primer has a nucleotide sequence consisting essentially of SEQ ID NO: 6 and said reverse primer has a nucleotide sequence consisting essentially of SEQ ID NO: 7.
8. A kit for PCR detection of Cryptococcus neoformans , comprising the isolated hybridization probe of claim 1 , said kit further comprises:
(a) a forward primer that anneals to a Cryptococcus neoformans consensus sequence consisting essentially of nucleotides 2105-2124 of SEQ ID NO: 1;
(b) a reverse primer that anneals to a Cryptococcus neoformans consensus sequence consisting essentially of nucleotides 508-527 of SEQ ID NO: 2; and
(c) an instruction for using said forward primer and reverse primer in performing PCR to detect the presence of Cryptococcus neoformans in a sample.
9. The kit of claim 7 , wherein said forward primer has a nucleotide sequence consisting essentially of SEQ ID NO: 8 and said reverse primer has a nucleotide sequence consisting essentially of SEQ ID NO: 9.