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QUANTITATIVE PCR-BASED COMPOSITIONS AND METHODS FOR THE DIAGNOSIS OF INVASIVE PULMONARY ASPERGILLOSIS

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  • Publication Date:
    October 28, 2010
  • معلومة اضافية
    • Document Number:
      20100273169
    • Appl. No:
      12/768632
    • Application Filed:
      April 27, 2010
    • نبذة مختصرة :
      Provided are quantitative PCR-based compositions and methods for the diagnosis of invasive pulmonary aspergillosis (IPA) in a patient sample, such as bronchoalveolar lavage (BAL) fluid. The methods presented herein involve isolating a patient sample, optionally extracting DNA from the sample, carrying out a quantitative PCR (qPCR) reaction on the sample to generate an amplicon that includes a region of an Aspergillus spp. ribosomal RNA (rRNA) gene, and detecting the PCR amplicon. The present disclosure also provides primers and primer sets for specifically detecting an Aspergillus spp. fungal pathogen in the presence of human ribosomal DNA (rDNA).
    • Inventors:
      FREDRICKS, DAVID N. (SEATTLE, WA, US); KHOT, PRASANNA D. (COTTONWOOD HEIGHTS, UT, US); KO, DAISY L. (SEATTLE, WA, US)
    • Assignees:
      FRED HUTCHINSON CANCER RESEARCH CENTER (SEATTLE, WA, US)
    • Claim:
      1. A primer set for the diagnosis of invasive pulmonary aspergillosis (IPA), said primer set comprising a forward primer and a reverse primer wherein said forward primer and said reverse primer are capable of amplifying a region of one or more Aspergillus spp gene(s).
    • Claim:
      2. The primer set of claim 1 wherein one of said Aspergillus spp gene(s) is a ribosomal RNA (rRNA) gene.
    • Claim:
      3. The primer set of claim 2 wherein one of said Aspergillus spp gene(s) is an 18S rRNA gene.
    • Claim:
      4. The primer set of claim 3 wherein said forward primer comprises the nucleotide sequence 5′-GAT AAC GAA CGA GAC CTC GG-3′ (SEQ ID NO: 1) and said reverse primer comprises the nucleotide sequence 5′-AGA CCT GTT ATT GCC GCG C-3′ (SEQ ID NO: 2).
    • Claim:
      5. The primer set of claim 1 wherein said Aspergillus spp is selected from the group consisting of Aspergillus fumigatus, Aspergillus oryzae, Aspergillus ustus, Aspergillus candidus, Aspergillus terreus, and Aspergillus flavus.
    • Claim:
      6. A kit for the diagnosis of invasive pulmonary aspergillosis (IPA), said kit comprising (1) a primer set comprising a forward primer and a reverse primer wherein said forward primer and said reverse primer are capable of generating a PCR amplicon from a region of one or more Aspergillus spp gene(s) and (2) a probe capable of hybridizing to said PCR amplicon.
    • Claim:
      7. The kit of claim 6 further comprising an internal amplification control (IAC) primer set comprising a second forward primer and a second reverse primer wherein said second forward primer and said second reverse primer are capable of generating a PCR amplicon from a region of a second gene having a nucleotide sequence that is unrelated to said Aspergillus spp gene.
    • Claim:
      8. The kit of claim 7 wherein said second forward primer comprises the nucleotide sequence 5′-GCC TGG TGC AAA AAT TGC TTA TC-3′ (SEQ ID NO: 3) and wherein said second reverse primer comprises the nucleotide sequence 5′-CTA AGA CAA GTG TGT TTA TGG TAT TG-3′ (SEQ ID NO: 4).
    • Claim:
      9. A quantitative PCR method for the diagnosis of invasive pulmonary aspergillosis (IPA) in a patient sample, said method comprising the steps of: (a) isolating a sample from said patient, (b) collecting a cell fraction from said sample, (c) extracting DNA from said cell fraction, (d) carrying out a quantitative PCR (qPCR) reaction on the patient sample with a primer set that permits the generation of an amplicon that includes a region of an Aspergillus spp. gene, and (e) detecting said PCR amplicon; wherein the presence of said PCR amplicon indicates a positive diagnosis of IPA.
    • Claim:
      10. The quantitative PCR method of claim 9 wherein said patient sample is bronchoalveolar lavage (BAL) fluid.
    • Claim:
      11. The quantitative PCR method of claim 10 wherein said Aspergillus spp. gene is a ribosomal RNA (rRNA) gene.
    • Claim:
      12. The quantitative PCR method of claim 11 wherein said rRNA gene is an 18S rRNA gene.
    • Claim:
      13. The quantitative PCR method of claim 12 wherein said 18S rRNA gene comprises the nucleotide sequence (SEQ ID NO: 6).
    • Claim:
      14. The quantitative PCR method of claim 13 wherein said primer set comprises a forward primer comprising the nucleotide sequence 5 ′-GAT AAC GAA CGA GAC CTC GG-3′ (SEQ ID NO: 1) and a reverse primer 5′-AGA CCT GTT ATT GCC GCG C-3′ (SEQ ID NO: 2).
    • Claim:
      15. The quantitative PCR method of claim 14 wherein the step of detecting said PCR amplicon comprises the step of hybridizing a probe comprising the nucleotide sequence 5′-FAM CTT AAA TAG CCC GGT CCG C BHQ-3′ (SEQ ID NO: 5).
    • Current U.S. Class:
      435/6
    • Current International Class:
      12; 07
    • الرقم المعرف:
      edspap.20100273169