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Immuno-polymerase chain reaction for detection ofAspergillus fumigatus
Abstract:
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.
Insights
A new method efficiently extracts Aspergillus fumigatus DNA from patient samples for accurate detection. This advance aids in diagnosing aspergillosis, especially in immunocompromised individuals.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Clinical Microbiology
Background:
- Aspergillus fumigatus is an opportunistic pathogen causing severe infections in immunocompromised individuals.
- Aspergillosis presents in various forms, including invasive disease, allergic bronchopulmonary aspergillosis, and aspergilloma.
- Accurate and rapid detection of A. fumigatus is crucial for timely patient management.
Purpose of the Study:
- To develop and evaluate a novel DNA extraction method for Aspergillus species from clinical samples.
- To compare the efficacy of this new method with existing diagnostic techniques for A. fumigatus detection.
- To establish a sensitive and specific diagnostic assay for aspergillosis.
Main Methods:
- Genomic DNA extraction using chitinase and microwave treatment from sputum and bronchial aspirate.
- Detection of A. fumigatus using culture, microscopy, ELISA, immunodiffusion, PCR with agarose gel electrophoresis, and colorimetric immuno-PCR.
- Development of a colorimetric detection method based on immunoaffinity reactions for PCR products.
Main Results:
- The described DNA extraction method yielded sufficient DNA (at least 1 pg) for PCR amplification from clinical samples.
- Fourteen out of nineteen patient samples were positive across all tested methods.
- The colorimetric immuno-PCR achieved high absorbance values (0.9–1.5) compared to negative controls (0.2).
Conclusions:
- The chitinase and microwave-assisted DNA extraction is effective for Aspergillus species.
- Colorimetric immuno-PCR offers a sensitive and specific method for detecting A. fumigatus.
- This approach is suitable for screening large numbers of clinical samples, aiding in aspergillosis diagnosis.
