Immuno-polymerase chain reaction for detection ofAspergillus fumigatus

摘要

A number of Aspergillus infections are caused by the opportunistic fungal pathogenAspergillus fumigatus in humans especially under immunosuppressed conditions. Major forms of the disease include invasive aspergillosis, allergic bronchopulmonary aspergillosis and aspergilloma. A procedure that uses chitinase and microwave treatment is described for the extraction of genomic DNA of Aspergillus species from the sputum and bronchial aspirate of patients with established aspergillosis. Detection ofA.fumigatus was compared by culture, microscopy, serology by ELISA, immunodiffusion, agarose gel electrophoresis of PCR products and colorimetric immuno-PCR. A colorimetric method for the detection of PCR product was developed based on immunoaffinity reactions. Out of the clinical samples tested from nineteen patients, fourteen were positive and five were negative by all the methods tested. It was established that at least 1 pg of DNA was extractable from the clinical samples sufficient to produce enough quantities of PCR product for detection on agarose gel or by immunoaffinity based color reaction. An absorbance value of 0.9 to 1.5 against 0.2 for negative control was obtained at 405 nm for colorimetric immuno-PCR. This method can be exploited for screening large number of clinical samples from immunocompromized as well as from suspected cases of aspergillosis.