血吸虫种株间18S小亚基单位核糖体核酸基因的同源性及其PCR法检测单个尾蚴的敏感性

Homology of 18S small subunit ribosomal RNA gene among species and strains of Schistosoma and sensitivity of PCR assay to detect single cercaria

目的探索日本血吸虫中国大陆株、菲律宾株及曼氏血吸虫18S小亚基单位核糖体核酸基因(18S-rRNA)序列的同源性及采用该基因建立PCR法检测水体中低密度血吸虫尾蚴的可能性。方法提取日本血吸虫中国大陆株、菲律宾株及曼氏血吸虫基因组DNA,采用PCR方法检测上述基因组中同一目的DNA片段,比较其同源性;分别以经沸水加热处理、氨水处理及NaOH、HCl、乙醇沉淀处理和未经处理的单个尾蚴标本为模板,采用PCR法扩增,比较对单个尾蚴的检出率。结果以日本血吸虫中国大陆株、菲律宾株和曼氏血吸虫基因组DNA为模板,均能扩增出长度为469bp的DNA片段。经氨水处理和NaOH、HCl、乙醇沉淀法处理的单个尾蚴标本,PCR方法均能扩增到目的基因。在未经处理或沸水加热处理的单个尾蚴标本中,仅有50%标本能扩增到目的基因。结论血吸虫不同种株间18S-rRNA基因具有广泛同源性。经氨水处理或NaOH、HCl、乙醇沉淀法处理的标本,采用PCR法对低密度尾蚴的检出率高于未经处理或经沸水加热处理的标本。

Objective To research the homology of 18S small subunit ribosomal RNA （18S-rRNA） gene about Chinese Mainland and Philippine strains of Schistosoma japonicum, and Schistosoma mansoni, and the possibility to establish the PCR assay based on the gene for detecting the cercaria in a low density level. Methods The genomie DNAs of Chinese Mainland and Philippine strains of S. japonicum, and S. mansoni were extracted. The PCR assay was used to detect the identical target DNA elements in the above genome team and the homology of their genes was compared. The single cercaria was respectively treated with the method of heating in boiling water, the method of treating with ammonia and the method of treating with NaOH, HCl and ethanol, and the single treated cercaria and the single cercaria without treating were used as the templates to amplify the target DNA by using the PCR assay, and the detection rates of the PCR assay to detect the single eercaria treated with the different methods werecalculated and compared. Results With the genomie DNAs of Chinese Mainland and Philippine strains of S. japonicum and 5; mansoni as the templates, the target DNA element of which sequence length was 469 bp was all amplified by PCR. The target DNA was all amplified by PCR to the single cerearia treated with ammonia and the method of treating with NaOH, HC1 and ethanol. However, only 50 percent of specimens of the single cercaria without treating and the single cerearia treated with the method of heating in boiling water were amplified to the target DNA by PCR. Conclusions The 18S-rRNA gene has the general homology among the species and strains of Schistosoma. The sensitivity of the PCR assay to detect the low density cercaria treated with ammonia or the method of treating with NaOH, HCl and ethanol is higher than that of the single cercaria without treating or treated with the method of heating in boiling water.