POLYMERASE-CHAIN-REACTION FOR DIAGNOSIS AND SPECIES DIFFERENTIATION OF MICROSPORIDIA

Polymerase chain reaction (PCR) techniques have been developed for the detection of microsporidian DNA in different biological samples. We u sed sequence data of the rRNA gene for the identification of Enterocyt ozoon bieneusi. Encephalitozoon intestinalis, E. cuniculi, and E. hell em in different biological samples of HIV-infected patients by PCR, So uthern blot hybridization, restriction endonuclease digestion analysis , cloning, and comparative genetic sequencing. One primer pair was use d for amplification of the entire small subunit (SSU)-rRNA gene of E. bieneusi, E. intestinalis, and E. hellem from samples with electron mi croscopy confirmed infection. The amplified 1.2 kb SSU-rRNA ene fragme nts were ligated into a pMOSBlue T-vector, transfected into pMOSBlue c ompetent, cells, and were used as positive controls. Several primer pa irs and hybridization probes were used to amplify and identify microsp oridian DNA from different samples. Light microscopical examination of samples was performed in all patients and transmission electron micro scopy was done on a subset of patient samples. DNA products were obtai ned from all samples with confirmed microsporidial infections. The ide ntity of the DNA fragments was determined by Southern blot hybridizati on or by restriction endonuclease digestion analysis or by DNA sequenc ing. The results show that PCR is a reliable and sensitive indicator f or the presence of microsporidian DNA in different biological samples of HIV-infected patients. PCR can be used further for species differen tiation of, microsporidia, even between species which cannot be differ entiated by light and/or electron microscopy.