Multiplex PCR for Detection of Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger.
RATIONAL: Aspergillus species mainly A.fumigatus, A.flavus and A.niger are clinically important as they induce hypersensitivity reactions in immunocompetent and invasive diseases in immunocompromised hosts. A.fumigatus is known to be the main causative agent in both allergic and invasive aspergillosis. Current report is based on multiplex PCR using unique sequences of A.fumigatus, Polyketide Synthase A (pks) gene of A.flavus and homologous region of pks gene in A.fumigatus, A.flavus and A.niger for thier detection in a single step. This can be extended to clinical samples. METHODS: Immunoscreening of cDNA clones from a cDNA library of A.fumigatus (Af293) with pooled sera of allergic bronchopulmonary aspergillosis (ABPA) patients was carried out. Sequences for cDNA clones were obtained and analyzed (submitted to Gene bank). Multiplex PCR assay has been designed based on two unique sequences of (BM032616, CB331858), pksA gene of A. flavus and homologous region of pks gene (from NCBI database) in A.fumigatus, A.flavus and A.niger. RESULTS: PCR products of 600 bp and 300 bp specific to A. fumigatus for unique sequences of A.fumigatus, product of 362 bp specific to A.flavus for pksA and product of 500 bp common to A. fumigatus, A.flavus and A.niger based on homologus PKS gene were amplified by multiplex PCR. CONCLUSIONS: The multiplex PCR reported can detect specifically A. fumigatus, and A. flavus and also A. fumigatus, A.flavus and A. niger together. This detection method can be standardized for screening clinical samples suspected for invasive aspergillosis.
Full conference title:
American Academy of Allergy Asthma & Immunology