荧光实时定量PCR定量水体中日本血吸虫尾蚴方法的建立

摘要

目的 建立快速、高效、特异的定量水体中尾蚴的数量和检测水体中日本血吸虫尾蚴残余基因组DNA的方法,来评估水体受日本血吸虫尾蚴污染的程度.方法 根据日本血吸虫基因组DNA中的3个多拷贝序列Sjrh1.0(序列号:U92488.1)、18S小亚基单位核楷体核酸基因(18SrRNA)序列(序列号:AY157226.1)和逆转录转座子SjR2的G55A序列(G55A)(序列号:AF412221.1),设计常规PCR引物和实时定量PCR引物,选取较好的靶序列建立SYBR GreenI实时定量PCR方法,绘制尾蚴数的对数与Ct值的标准曲线,并对疫水中日本血吸虫尾蚴残余的基因组DNA进行检测.结果 尾蚴数的对数与Ct值的标准曲线有良好的线性关系,相关系数r2为0.918 6,重复性良好.结论 本方法特异性高,灵敏,可定量水体中尾蚴数,对疫水检测有一定的预警作用.%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.