METHODS AND COMPOSITIONS FOR IDENTIFYING AND QUANTIFYING MICROBIAL DNA
This invention relates to methods and compositions for identifying microbial DNA in the tissues or body fluid samples of patients. More particularly, the invention relates to two-step polymerase chain reaction based methods for identifying microbial DNA in the tissues or body fluid samples of patients, and compositions therefor. Microbial DNA can also be quantified using the methods described herein.
1 - 30 . (canceled)
31 . A method of identifying a specific fungal species in a sample, the method comprising:
extracting and recovering fungal DNA of the fungal species from the sample,
amplifying the fungal DNA to produce an amplicon,
hybridizing an isolated probe to the amplicon to identify the specific fungal species, wherein the probe consists of SEQ ID NO: 57, or a compliment of SEQ ID NO: 57, and
identifying the specific fungal species, wherein the fungal species is Candida auris.
32 . The method of claim 31 wherein the amplifying step is performed with primers that hybridize to the fungal DNA.
33 . The method of claim 31 wherein the fungal DNA is amplified using PCR.
34 . The method of claim 33 wherein the PCR is real-time PCR.
35 . The method of claim 32 wherein the primers consist of SEQ ID NO: 58 and SEQ ID NO: 59.
36 . The method of claim 31 wherein the sample is a patient tissue or body fluid.
37 . The method of claim 36 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.
38 . The method of claim 31 wherein the amplified sequence is from internal transcribed spacer regions of nuclear ribosomal DNA.
39 . The method of claim 31 wherein the probe is labeled with at least one fluorescent dye.
40 . The method of claim 39 wherein the at least one fluorescent dye is selected from the group consisting of a fluorescence resonance energy transfer pair, FAM, dCAL FLUOR Orange 560, and BHQ.
41 . A method of determining if a patient is at risk for or has developed a disease state related to a fungal infection, the method comprising:
extracting and recovering fungal DNA of the fungal species from a sample,
amplifying the fungal DNA to produce an amplicon,
hybridizing an isolated probe to the amplicon to identify the specific fungal species, wherein the probe consists of SEQ ID NO: 57, or a compliment of SEQ ID NO: 57, and
identifying the specific fungal species to determine if the patient is at risk for or has developed the disease state related to a fungal infection, wherein the fungal species is Candida auris.
42 . The method of claim 41 wherein the amplifying step is performed with primers that hybridize to the fungal DNA.
43 . The method of claim 41 wherein the fungal DNA is amplified using PCR.
44 . The method of claim 43 wherein the PCR is real-time PCR.
45 . The method of claim 42 wherein the primers consist of SEQ ID NO: 58 and SEQ ID NO: 59.
46 . The method of claim 41 wherein the sample is a patient tissue or body fluid.
47 . The method of claim 46 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.
48 . The method of claim 41 wherein the amplified sequence is from internal transcribed spacer regions of nuclear ribosomal DNA.
49 . The method of claim 41 wherein the probe is labeled with at least one fluorescent dye.
50 . The method of claim 49 wherein the at least one fluorescent dye is selected from the group consisting of a fluorescence resonance energy transfer pair, FAM, dCAL FLUOR Orange 560, and BHQ.