Methods and compositions for detecting fungi and mycotoxins

Abstract

The invention relates to a method of identifying a specific fungal species in patient tissue or body fluid. The method comprises the steps of extracting and recovering DNA of the fungal species from the patient tissue or body fluid, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the fungal species, and specifically identifying the fungal species. The invention also relates to a method of identifying a mycotoxin in patient tissue or body fluid. The method comprises the steps of extracting and recovering the mycotoxin from the patient tissue or body fluid, contacting the mycotoxin with an antibody directed against the mycotoxin, and identifying the myocotoxin. Both of these methods can be used to determine if a patient is at risk for or has developed a disease state related to a fungal infection, and to develop an effective treatment regimen for the patient.

What is claimed is: 
     
         1 . A method of identifying a specific fungal species in patient tissue or body fluid, the method comprising the steps of:
 extracting and recovering DNA of the fungal species from the patient tissue or body fluid;   amplifying the DNA;   hybridizing a probe to the DNA to specifically identify the fungal species; and   specifically identifying the fungal species.   
     
     
         2 . The method of  claim 1  wherein the amplifying step is performed with primers that hybridize to the DNA. 
     
     
         3 . The method of  claim 1  wherein the body fluids are selected from the group consisting of urine, nasal secretions, nasal washes, bronchial lavages, bronchial washes, spinal fluid, sputum, gastric secretions, seminal fluid, other reproductive tract secretions, lymph fluid, whole blood, serum, and plasma. 
     
     
         4 . The method of  claim 1  wherein the DNA is amplified using PCR. 
     
     
         5 . The method of  claim 4  wherein the PCR is real-time PCR. 
     
     
         6 . The method of  claim 1  wherein the probe is fluorescently labeled. 
     
     
         7 . The method of  claim 5  wherein the probe is fluorescently labeled. 
     
     
         8 . The method of  claim 1  wherein the fungal species are selected from the group consisting of  S.   chartarum ,  A.   versicolor ,  A.   niger ,  P.   chrysogenum ,  P.   verrucosum ,  G.   candidum ,  A.   flavus ,  A.   fumigatus ,  A.   nidulans ,  A.   ochraceus ,  A.   paraciticus ,  A.   sydowii ,  A.   ustus ,  F.   solani ,  P.   aurantiogriseum ,  P.   citrinum ,  P.   corylophilum ,  P.   crustosum ,  P.   expansum ,  P.   fellutanum ,  P.   roquefortii ,  P.   simplicissimum ,  S.   echinata , and  E.   amstelodami . 
     
     
         9 . The method of  claim 2  wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 1, the forward primer comprises the sequence of SEQ ID NO: 2, and the reverse primer comprises the sequence of SEQ ID NO: 3. 
     
     
         10 . The method of  claim 2  wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 4, the forward primer comprises the sequence of SEQ ID NO: 5, and the reverse primer comprises the sequence of SEQ ID NO: 6. 
     
     
         11 . The method of  claim 2  wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 7, the forward primer comprises the sequence of SEQ ID NO: 8, and the reverse primer comprises the sequence of SEQ ID NO: 9. 
     
     
         12 . The method of  claim 2  wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 10, the forward primer comprises the sequence of SEQ ID NO: 11, and the reverse primer comprises the sequence of SEQ ID NO: 12. 
     
     
         13 . The method of  claim 2  wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 13, the forward primer comprises the sequence of SEQ ID NO: 14, and the reverse primer comprises the sequence of SEQ ID NO: 15. 
     
     
         14 . The method of  claim 2  wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 18, the forward primer comprises the sequence of SEQ ID NO: 16, and the reverse primer comprises the sequence of SEQ ID NO: 17. 
     
     
         15 . The method of  claim 2  wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 19, the forward primer comprises the sequence of SEQ ID NO: 20, and the reverse primer comprises the sequence of SEQ ID NO: 21. 
     
     
         16 . The method of  claim 2  wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 22, the forward primer comprises the sequence of SEQ ID NO: 23, and the reverse primer comprises the sequence of SEQ ID NO: 24. 
     
     
         17 . The method of  claim 2  wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 25, the forward primer comprises the sequence of SEQ ID NO: 26, and the reverse primer comprises the sequence of SEQ ID NO: 27. 
     
     
         18 . The method of  claim 2  wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 28, the forward primer comprises the sequence of SEQ ID NO: 29, and the reverse primer comprises the sequence of SEQ ID NO: 30. 
     
     
         19 . The method of  claim 2  wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 31, the forward primer comprises the sequence of SEQ ID NO: 32, and the reverse primer comprises the sequence of SEQ ID NO: 33. 
     
     
         20 . The method of  claim 2  wherein the probe, a forward primer, and a reverse primer are used during the amplification step and the probe comprises the sequence of SEQ ID NO: 34, the forward primer comprises the sequence of SEQ ID NO: 35, and the reverse primer comprises the sequence of SEQ ID NO: 36.