中国人兽共患病学报2011，Vol.27Issue(12)：1075-1081,7.
荧光实时定量PCR定量水体中日本血吸虫尾蚴方法的建立
Fluorescent quantitative real-time PCR for quantitative detection of Schistosoma japonicum cercariae in water
摘要
Abstract
The objective was to establish a rapid, sensitive and specific method to detect the number of cercariea in water and evaluate level of water stained by Schistosoma japonicum cercariea. Convenience PCR primer sequences were designed tar-geting pSjrH1.0(U92488. 1), Sjl8SrRNA( AY157226. 1) and the clone G55A of the highly repetitive retrotransposon SjR2 (AF412221. 1) in S. japonicum genome, and sequence the PCR product. Based on conserved sequence of pSjrHl. 0, Sjl8SrRNA and clone G55A of highly repetitive retrotransposon SjR2 (G55A), design primers and the quantitative real-time PCR essays were established, by which, the amplifying products were 150 to 170 bp. The sequence in S. japonicum genome and the best annealing temperature were selected by comparing their threshold cycle (Ct value). Then the quantitative real-time PCR essays were established under the better annealing temperature, generate standard curve between the logarithms of gradi-ent diluted DNA templates and Ct value. Five DNA samples extracted from 1, 5, 10, 20 and 80 cercariae were used as quanti-tative template to generate standard curve between the logarithm for the number of S. japonicum cercariea and Ct value. Using the modified ROSE method, extract genome DNA from 200uL water samples containing cercariea and establish flourescent quantitative real-time PCR essays to detect the residue cercariae genome DNA in water containing S. japonicum cercariae after 24 hours. The results of the essays showed that pSjrHl. 0 had higher sensitivity and lower Ct, in the same template concentra-tion condition, comparing with Sjl8SrRNA and G55A, and could quantitatively detect as low as 2 fg S. japonicum genome DNA in the study. The standard curve showed a fine linear relationship between the logarithms of gradient diluted DNA tem-plate and Ct value, and the correlation coefficient r2was 0. 997 7. The standard curve showed a fine linear relationship between the logarithm of the number of S. japonicum cercariae and Ct value, and the correlation coefficient r2 was 0. 918 6. Repeated experiments prove that the method of quantitative detection of S. japonicum cercariea has high repetitiveness, which is a rapid, highly sensitive and highly repetitive method for evaluation of the water containing S. japonicum cercariae.关键词
日本血吸虫/尾蚴/荧光实时定量PCR/预警Key words
Schistosoma japonicum /cercariae/fluorescence real-time quantitative PCR/precaution分类
医药卫生引用本文复制引用
王本敬,王文波,周霞,陈艳勤,张静,刘晨晨,梁幼生,诸葛洪祥..荧光实时定量PCR定量水体中日本血吸虫尾蚴方法的建立[J].中国人兽共患病学报,2011,27(12):1075-1081,7.基金项目
江苏省自然科学基金(BK2009076)资助 （BK2009076）
