Polymerase chain reaction (PCR) techniques have been developed for the detection of microsporidian DNA in different biological samples. We used sequence data of the rRNA gene for the identification of Enterocytozoon bieneusi, Encephalitozoon intestinalis, E. cuniculi, and E. hellem in different biological samples of HIV-infected patients by PCR, Southern blot hybridization, restriction endonuclease digestion analysis, cloning, and comparative genetic sequencing. One primer pair was used for amplification of the entire small subunit (SSU)-rRNA gene of E. bieneusi, E, intestinalis, and E. hellem from samples with electron microscopy confirmed infection. The amplified 1.2 kb SSU-rRNA gene fragments were ligated into a pMOSBlue T-vector, transfected into pMOSS/ме competent cells, and were used as positive controls. Several primer pairs and hybridization probes were used to amplify and identify microsporidian DNA from different samples. Light microscopical examination of samples was performed in all patients and transmission electron microscopy was done on a subset of patient samples. DNA products were obtained from all samples with confirmed microsporidial infections. The identity of the DNA fragments was determined by Southern blot hybridization or by restriction endonuclease digestion analysis or by DNA sequencing. The results show that PCR is a reliable and sensitive indicator for the presence of microsporidian DNA in different biological samples of HIV-infected patients. PCR can be used further for species differentiation of microsporidia, even between species which cannot be differentiated by light and/or electron microscopy.
The objective of this study was to determine if primers, probes, and pHCl, a plasmid containing a 2.3 kilobasc insert of genomic DNA from Cryptosporidium parvum, would be useful for the detection of Cryptosporidium wrairi DNA using the polymerase chain reaction. C. wrairi DNA was prepared from oocysts recovered from guinea pigs and C. parvum DNA was prepared from the Iowa strain of C. parvum. Two 26-(bp) primers were used to amplify a 452-base pair bp target sequence within the cloned DNA. Similarly-sized PCR products were obtained with the pHCl plasmid DNA, C. parvum DNA, and C. wrairi DNA. However, a 20-base pair probe did not detect C. wrairi DNA. Sequencing of C. wrairi DNA homologous with the 452-bp segment of C. parvum DNA showed 18 bp changes including bp changes in the segment homologous with the probe. A new probe based on homologous sequences was useful for detection of both species of Cryptosporidia. The sequences of the homologous 452-bp segment from the Iowa strain of C. parvum and that segment of C. parvum DNA from the pHC I plasmid were very similar. Nine base pairs identical in the homologous bp segment of the Iowa strain of C. parvum, C. wrairi and the pHCl plasmid differed from those previously reported. Previously reported primers and a newly designed probe proved to be useful for detecting C. wrairi DNA.