Morphological and molecular identification of Cyclospora-like organism from dogs / 中国人兽共患病学报

The morphological and molecular identifications of Cyclospora-like oocysts of dogs were underwent in the present study, in which the morphological characteristics of the Cyclospora-like oocysts firstly found in the stool samples of dogs, such as shape, size, acid-fast staining,sporulation and auto-fluorescene, were observed. According to the published sequence of the rDNA gene of Cyclospora in GenBank, 3 primers were designed and were used to amplify part of the 18S rDNA gene of dog-associated Cyclospora-like organism by nested PCR.The amplicons were purified and cloned into vector pMD19T. Then, the positive clones screened were sequenced and subjected to sequence homology and phylogenetic analysis. It was found that the morphological charactertistics of the Cyclospora-like oocysts in dogs were similar to that of the human Cyclospoa oocysts and the size of-the amplified fragment of 18S rDNA was proved to be 715 bp, that was identical to that of the target fragment. Based on the results of sequence homology and phylogenetic analysis, the dog-associated Cyclospora-like organism was identified as the Cyclospora species.

Development of RT-PCR-ELISA assay for detecting Cryptosporidium hominis / 中国人兽共患病学报

To establish a highly sensitive and specific method to detect the presence of Cryptosporidium homini, the RT-PCR-ELISA assay was tried, in which the primer with a biotin-labeled probe was designed to amplify fragment containing the highly variable region by multiple alignment between p23 gene of C.hominis and other Cryptosporidium spp. The RT-PCR was used to amplify the target fragment, and the amplified product was used to hybridize with the probe primer. The hybridized product was then captured on micro-plate wells coated with streptavidin and reacted with anti-digoxin antibody labeled with horse-radish peroxidase. This method of testing was then used for the detection of C.hominis in 22 clinical specimens and compared with the conventional methods of testing. It was demonstrated that the RT-PCR--ELISA for the detection of C.hominis was proved to be quite sensitive and specific. Its sensitivity was 100 times higher than that of the general PCR. From the result of clinic detection, the detection rate of RT-PCR-ELISA assay attained to 86%(19/22), while those of RT-PCR, sucrose floating method and anti-acid staining were 27%, 27% and 50% respectively. This result indicates that the RT-PCR-ELISA assay is more sensitive to detect C.hominis than the other three methods of testing.