Methods and reagents for detecting fungal pathogens in a biological sample

Abstract

The present invention provides novel methods and materials for detecting the presence of a fungus in a biological sample. The inventive methods and materials exploit the fact that the amino acid sequence of the α-aminoapidate reductase molecule is highly conserved in fungi. Inventive hybridization probes, nucleic acids, PCR primers, antibodies, epitopes, reagents and methods are provided.

We claim:  
     
         1 . A nucleic acid hybridization probe comprising an isolated and purified nucleic acid having a nucleotide sequence selected from the group consisting of nucleotide sequences coding for a polypeptide fragment wherein the isolated and purified nucleic acid is not homologous to and does not cross react with mammalian genetic material and wherein the polypeptide fragment is (a) derived from the α-aminoapidate reductase molecule expressed by  C. albicans  and (b) conserved in fungi.  
     
     
         2 . A nucleic acid of  claim 1  wherein the hybridization probe has a nucleotide sequence that either is identical to a sequence contained in the LYS 2 gene of  C. albicans  or cross hybridizes with a portion of the LYS 2 gene of  C. albicans  under conditions of high stringency.  
     
     
         3 . A hybridization probe of  claim 1  wherein the probe has the nucleotide sequence illustrated in SEQ ID NO.: 7.  
     
     
         4 . A hybridization probe of  claim 1  wherein the probe is a labeled probe.  
     
     
         5 . A hybridization probe of  claim 1  wherein the probe is labeled with a radioactive label, a fluorescent label or an antigenic label.  
     
     
         6 . A method of screening a biological sample for the presence of a fungal pathogen, the method comprising the step of hybridizing a nucleic acid isolated from the biological sample with a hybridization probe of  claim 1 .  
     
     
         7 . A method of screening a biological sample for the presence of a fungal pathogen, the method comprising the step of hybridizing a nucleic acid isolated from the biological sample with a hybridization probe of  claim 2 .  
     
     
         8 . A method of screening a biological sample for the presence of a fungal pathogen, the method comprising the step of hybridizing a nucleic acid isolated from the biological sample with a hybridization probe of  claim 3 .  
     
     
         9 . A method of screening a biological sample for the presence of a fungal pathogen comprising detecting hybridization of a nucleic acid hybridization probe wherein the hybridization probe 
 (a) comprises an isolated and purified nucleic acid having a nucleotide sequence selected from the group consisting of nucleotide sequences coding for polypeptide fragments that are (i) derived from the α-aminoapidate reductase molecule expressed by  C. albicans  and (ii) conserved in fungi; and    (b) is not homologous to and does not cross react with mammalian genetic material.    
     
     
         10 . The method of  claim 9  wherein the method is performed by machine.  
     
     
         11 . The method of  claim 9  wherein the hybridization is carried out on a filter.  
     
     
         12 . The method of  claim 9  wherein the hybridization is carried out in solution.  
     
     
         13 . The method of  claim 9  wherein hybridization is detected by autoradiography.  
     
     
         14 . The method of  claim 9  wherein the nucleic acid is isolated from the biological sample prior to hybridization with the hybridization probe.  
     
     
         15 . The method of  claim 9  wherein the hybridization probe is applied directly to a biological sample.  
     
     
         16 . The method of  claim 9  wherein the hybridization probe has a nucleotide sequence that either is identical to a sequence contained in the LYS 2 gene of  C. albicans  or cross hybridizes with a portion of the LYS 2 gene of  C. albicans  under conditions of high stringency.  
     
     
         17 . The method of  claim 9  wherein the nucleotide sequence of the nucleic acid hybridization probe comprises the sequence illustrated in SEQ ID NO.:7.  
     
     
         18 . A kit for detecting a fungal pathogen in a biological sample comprising a hybridization probe of  claim 1 .  
     
     
         19 . A kit for detecting a fungal pathogen in a biological sample comprising a hybridization probe of  claim 2 .  
     
     
         20 . A kit for detecting a fungal pathogen in a biological sample comprising a hybridization probe of  claim 3 .  
     
     
         21 . A reagent for detecting the presence of a fungal pathogen in a biological sample comprising a hybridization probe of  claim 1 .  
     
     
         22 . A reagent for detecting the presence of a fungal pathogen in a biological sample comprising a hybridization probe of  claim 2 .  
     
     
         23 . A reagent for detecting the presence of a fungal pathogen in a biological sample comprising a hybridization probe of  claim 3 .  
     
     
         24 . A pair of nucleic acid primers, each member of the pair having a nucleotide sequence selected from the group consisting of nucleotide sequences coding for a polypeptide fragments wherein the primer is not homologous to and does not cross react with mammalian genetic material and wherein the polypeptide fragment is (a) derived from the α-aminoapidate reductase molecule expressed by  C. albicans  and (b) conserved in fungi.  
     
     
         25 . A pair of nucleic acid primers, each member of the pair having a nucleotide sequence selected from the group consisting of nucleotide sequences that either are identical to a sequences contained in the LYS 2 gene of  C. albicans  or cross hybridize with a portion of the LYS 2 gene of  C. albicans  under conditions of high stringency.  
     
     
         26 . A nucleic acid primer of  claim 24  wherein the nucleotide sequence either (a) is identical to, or (b) cross-hybridizes with under conditions of high stringency, nucleotide sequence selected from the group consisting of VB21 (SEQ ID NO.:2) and VB22 (SEQ ID NO.: 3).  
     
     
         27 . A method of screening a biological sample for the presence of a fungal pathogen comprising detecting hybridization of a hybridization probe to an amplified sample of genetic material, wherein the genetic material is amplified using nucleic acid primer pairs each member of the pair having a nucleotide sequence selected from the group consisting of nucleotide sequences coding for a polypeptide (a) derived from the α-aminoapidate reductase molecule expressed by  C. albicans  and (b) conserved in fungi, wherein neither member of the primer pair is homologous to or cross reacts with mammalian nucleic acids.  
     
     
         29 . The method of  claim 27  wherein each member of the nucleic acid primer pairs has a nucleotide sequence that either is identical to a sequence contained in the LYS 2 gene of  C. albicans  or cross hybridizes with a portion of the LYS 2 gene of  C. albicans  under conditions of high stringency.  
     
     
         30 . The method of  claim 27  wherein each member of the nucleic acid primer pair has a nucleic acid sequence selected from the group consisting of VB21 (SEQ ID NO.:2) and VB22 (SEQ ID NO.: 3).  
     
     
         31 . The method of  claim 27  wherein the hybridization is conducted on a filter.  
     
     
         32 . The method of  claim 27  wherein the hybridization is conducted in solution.  
     
     
         33 . The method of  claim 27  wherein hybridization is detected by autoradiography.  
     
     
         34 . The method of  claim 27  wherein the method is performed by machine.  
     
     
         35 . A reagent for detecting the presence of a fungal pathogen in a biological sample comprising a nucleic acid primer of  claim 24 .  
     
     
         36 . A reagent for detecting the presence of a fungal pathogen in a biological sample comprising a nucleic acid primer of  claim 25 .  
     
     
         37 . A reagent for detecting the presence of a fungal pathogen in a biological sample comprising a nucleic acid primer of  claim 26 .  
     
     
         38 . A kit for detecting the presence of a fungal pathogen in a biological sample comprising a nucleic acid primer of  claim 24 .  
     
     
         39 . A kit for detecting the presence of a fungal pathogen in a biological sample comprising a nucleic acid primer of  claim 25 .  
     
     
         40 . A kit for detecting the presence of a fungal pathogen in a biological sample comprising a nucleic acid primer of  claim 26 .  
     
     
         41 . An antibody capable of binding to an epitope of α-aminoapidate reductase expressed by wild type  C. albicans.    
     
     
         42 . An antibody of  claim 41  wherein said epitope comprises the amino acid sequence LTKRDCLKIRGFT or a fragment thereof.  
     
     
         43 . An antibody of  claim 41  wherein the antibody is labeled.  
     
     
         44 . An antibody of  claim 43  wherein the antibody label is selected from the group consisting of a radioactive isotope, a fluorescent compound or an enzyme.  
     
     
         45 . A method of screening a biological sample for the presence of a fungal pathogen, the method comprising detecting antibody binding to a biological sample, wherein the antibody binds selectively to α-aminoapidate reductase expressed by wild type  C. albicans.    
     
     
         46 . The method of  claim 45  wherein the method is an enzyme linked immunosorbent assay.  
     
     
         47 . The method of  claim 45  wherein the method is carried out by machine.  
     
     
         48 . A kit for detecting a fungal pathogen in a biological sample wherein the kit comprises an antibody of  claim 41 .  
     
     
         49 . A reagent comprising an antibody of  claim 41 .  
     
     
         50 . An epitope comprising a polypeptide having an amino acid sequence homologous to the amino acid sequence LTKRDCLKIRGFT or fragment thereof.  
     
     
         51 . A method of using the antibody of  claim 41  comprising using the antibody to detect the presence of a fungal epitope in a biological sample wherein the antibody is used to immunoprecipitate the fungal epitope.  
     
     
         52 . The method of  claim 45  wherein the method is a radioimmunoassay.  
     
     
         53 . The method of  claim 45  wherein the method is an sandwich binding assay.  
     
     
         54 . A reagent comprising the epitope of  claim 50 .  
     
     
         55 . The epitope of  claim 50  wherein the epitope is labeled.  
     
     
         56 . A method of using the antibody of  claim 41  wherein the antibody is used im a radioimmunoassay.  
     
     
         57 . A method of using the epitope of  claim 50  wherein the method comprises a radioimmunoassay.  
     
     
         58 . A method of using the hybridization probe of  claim 1 , wherein the probe is used to identify restriction fragment length polymorphisms in nucleic acid isolated from a biological sample and digested.