Compositions and methods for detection of candida species

Abstract

The invention features compositions and methods for the detection of Candida species in biological samples, such as human tissue samples. The invention features species specific nucleic acid probes that can hybridize to target nucleic acid molecules in Candida species and can be used in a variety of detection assays, such as fluorescence based assays or NMR based assays. The compositions and methods of the invention can be used to rapidly and accurately detect one or more Candida species in patients and can be used to assist in point-of care-decisions for treating Candida infections.

1 .- 60 . (canceled) 
     
     
         61 . A nucleic acid molecule comprising a sequence with at least 90% sequence identity the sequence of any one of SEQ ID NOs: 2, 4, 5, 14-15, 17-19, 22, 23, and 39. 
     
     
         62 . The nucleic acid molecule of  claim 61 , wherein the sequence has at least 95% sequence identity to the sequence of any one of SEQ ID NOs: 2, 4, 5, 14-15, 17-19, 22, 23, and 39. 
     
     
         63 . The nucleic acid molecule of  claim 61 , wherein the nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 2, 4, 5, 14-15, 17-19, 22, 23, and 39. 
     
     
         64 . The nucleic acid molecule of  claim 61 , wherein the nucleic acid molecule consists of the sequence of any one of SEQ ID NOs: 2, 4, 5, 14-15, 17-19, 22, 23, and 39. 
     
     
         65 . The nucleic acid molecule of  claim 61 , wherein the nucleic acid molecule is conjugated to a detection moiety. 
     
     
         66 . The nucleic acid molecule of  claim 64 , wherein the detection moiety comprises a fluorescent label. 
     
     
         67 . A composition comprising at least one said nucleic acid molecule of  claim 61 . 
     
     
         68 . The composition of  claim 67 , wherein the sequence of the nucleic acid molecule has at least 95% sequence identity to the sequence of any one of SEQ ID NOs: 2, 4, 5, 14-15, 17-19, 22, 23, and 39. 
     
     
         69 . The composition of  claim 67 , wherein the nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 2, 4, 5, 14-15, 17-19, 22, 23, and 39. 
     
     
         70 . The composition of  claim 67 , wherein the nucleic acid molecule consists of the sequence of any one of SEQ ID NOs: 2, 4, 5, 14-15, 17-19, 22, 23, and 39. 
     
     
         71 . The composition of  claim 67 , wherein the composition comprises two or more of the nucleic acid molecules. 
     
     
         72 . The composition of  claim 67 , wherein the nucleic acid molecule is conjugated to a detection moiety. 
     
     
         73 . The nucleic acid molecule of  claim 72 , wherein the detection moiety comprises a fluorescent label. 
     
     
         74 . A kit comprising the nucleic acid molecule of  claim 61  or a composition comprising the nucleic acid molecule. 
     
     
         75 . A method for detecting at least one  Candida  species in a sample comprising:
 a) contacting said sample with at least one nucleic acid molecule of  claim 61  under conditions which allow the at least one nucleic acid molecule to hybridize to a nucleic acid molecule of said  Candida  species; and   b) detecting hybridization between said at least one nucleic acid molecule and said nucleic acid molecule of said  Candida  species, thereby detecting said at least one  Candida  species in said sample.   
     
     
         76 . The method of  claim 75 , wherein each said nucleic acid molecule comprises a detection moiety that is conjugated to the nucleic acid molecule. 
     
     
         77 . The method of  claim 76 , wherein said detection moiety comprises a fluorescent label. 
     
     
         78 . The method of  claim 77 , wherein each said nucleic acid molecule comprises the same label or is differentially labelled. 
     
     
         79 . The method of  claim 75 , wherein the sample is a biological sample. 
     
     
         80 . The method of  claim 79 , wherein the biological sample is selected from the group consisting of whole blood, sweat, tears, urine, saliva, semen, serum, plasma, cerebrospinal fluid (CSF), feces, vaginal fluid or tissue, sputum, nasopharyngeal aspirate or swab, lacrimal fluid, mucous, or epithelial swab (buccal swab), organ tissue, bone tissue, teeth, and a tumor. 
     
     
         81 . The method of  claim 75 , wherein:
 (a) the sample is a human sample;   (b) at least one cell in said sample is lysed using a hypotonic solution or a detergent, wherein optionally, said lysis step is followed by centrifuging the lysed sample and removing the resulting supernatant;   (c) at least one cell of said  Candida  species is lysed by chemical or mechanical lysis;   (d) at least one cell of said  Candida  species is lysed by heat denaturation;   (e) said method further comprises amplifying said nucleic acid molecule of said  Candida  species prior to said detecting step;   (f) said method further comprises placing a container comprising a composition resulting from said contacting step into a device capable of detecting said hybridization between said nucleic acid molecule and said nucleic acid molecule of said  Candida  species;   (h) each said nucleic acid molecules comprises a fluorescent label and said hybridization is detected by heating said sample through a melting temperature (Tm) of at least one said nucleic acid molecule to obtain a melting curve, wherein a decrease in fluorescence of said sample at the Tm of at least one said nucleic acid molecule indicates the presence of said at least one  Candida  species;   (i) said hybridization is detected by fluorescence of said nucleic acid molecule using a thermal cycler;   (j) detection of said at least one  Candida  species in said sample can be completed within 3 hours; or   (k) the method is capable of detecting said at least one  Candida  species in said sample when present at a concentration of at least 3 cells/ml.   
     
     
         82 . The method of  claim 81 , wherein:
 (a) said chemical lysis comprises treatment with one or more enzymes;   (b) said mechanical lysis comprises using any one or more of a finned tube, small particles, ultrasound, freeze-boil, freeze-thaw, and a bead; or   (c) said heat denaturation comprises heating said at least one cell to about 95° C.   
     
     
         83 . The method of  claim 82 , wherein:
 (a) said bead comprises a glass bead; or   (b) said amplifying comprises enzymatic amplification or asymmetric amplification.