Immuno-polymerase chain reaction for detection ofAspergillus fumigatus
- 24 Downloads
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.
Key WordsA.fumigatus PCR diagnosis Colorimetric PCR
Unable to display preview. Download preview PDF.
- 4.Arruda, L.K., Mann, B.J. and Chapman, M.D. (1992). Selective expression of major allergen, and cytotoxin, Asp 1, inAspergillus fumigatus. J. immunology. 149, 3354–3359.Google Scholar
- 6.Sarma, P.U. and Reddy, P. Unpublished data.Google Scholar
- 8.Sambrook, J., Fritsch, E.F. and Manatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Vol. 1, p. 1.21Google Scholar
- 9.Sarma, P.U., Bir, N., Paliwal, A. and Reddy, P. (1998) Detection ofA. fumigatns by immuno-PCR from clinical samples. (Communicated).Google Scholar
- 10.Ouchterlony, D. (1953) Antigen-antibody reaction in gels: Types of reaction in coordinated system of diffusion. Acta Pathol. Microbiol. Scand. 31, 231–237.Google Scholar
- 11.Banerjee, B., Madan, T., Sharma, G.L., Prasad, K., Nath, I. and Sarma, P.U. (1995). Characterization of 45 kDa glycoprotein antigen ofA.fumigatus. Serodiagnosis and immunotherapy. Infectious Diseases. 7, 147–152.Google Scholar
- 12.Helmuth, R. (1990). Nonisotopic detection of PCR products In: PCR protocols. Eds. Innis, M.A., Gelfanel, D.H., Sninsky, J.J. and White, T.J., Academic Press, Toronto p 119–128.Google Scholar
- 13.Dell Portillo, P., Murillo, L.A. and Patarroyo, M.E. (1991). Amplification of a species specific DNA fragment ofMycobacterium tuberculosis and its possible use in diagnosis. J. Clin. Microbiol. 29, 2163–2168.Google Scholar
- 20.Manos, M.M., Ting, Y., Wright, D.K., Lewis, A., Broker, T.R. and Wolinsky, S.M. (1989). The use of polymerase chain reaction amplification for the detection of genital human papillomaviruses. Cancer Cells 7, 209–214.Google Scholar