Development of RT-PCR-ELISA assay for detecting Cryptosporidium hominis
10.3969/j.issn.1002-2694.2010.02.014
- VernacularTitle:人隐孢子虫RT-PCR-ELISA检测方法的建立
- Author:
Guoqing LI
;
Yijian YE
;
Xiangjie LIANG
;
Zhenyong GAO
;
Cailing YUE
;
Jialin CHEN
;
Haibo ZHU
;
Qianming XU
;
Qianming XU
- Publication Type:Journal Article
- Keywords:
Cryptosporidium hominis;
ELISA;
RT-PCR;
detection
- From:
Chinese Journal of Zoonoses
2010;(2):150-153
- CountryChina
- Language:Chinese
-
Abstract:
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.
