环介导同温扩增检测全血日本血吸虫DNA的研究

发布时间：2018-06-05 02:56

本文选题：血吸虫 + 日本　；参考：《中国病原生物学杂志》2010年10期

【摘要】：目的建立一种检测全血标本中日本血吸虫DNA的环介导同温扩增(LAMP)方法。方法选择日本血吸虫基因组重复序列DNA SjR2(839~1 138 bp、563~764 bp)、SjSH7(204~399 bp)和Sj-nonLTR1(1 678~1 877 bp)的4段DNA序列作为扩增片段,根据LAMP原理设计合成4组引物,通过对此4组引物用于DNA LAMP扩增的敏感性和特异性进行比较分析和检测条件的优化,建立特异、敏感,可用于检测全血日本血吸虫DNA的LAMP方法。应用此方法对10只日本血吸虫感染前及感染后不同时间同一家兔的配对全血DNA进行扩增,观察其应用价值。结果 4组引物中,以日本血吸虫SjR2(839~1 138 bp)作为扩增靶片段,可特异性扩增日本血吸虫中国大陆株(检出限为10-4ngDNA)、菲律宾株和曼氏血吸虫的基因组DNA,人全血基因组DNA、华支睾吸虫基因组DNA、疟原虫基因组DNA不能扩增。以Sj-nonLTR1(1 678~1 877 bp)为靶片段,LAMP扩增无特异性。以SjR2(563~764 bp)和SjSH7(204~399bp)为靶片段,可特异性扩增日本血吸虫中国大陆株基因组DNA(检出限为10-6ng和10-3ng)及菲律宾株基因组DNA,人全血基因组DNA、华支睾吸虫基因组DNA、曼氏血吸虫基因组DNA和疟原虫基因组DNA不能扩增。用SjR2(839~1 138 bp)引物对10只日本血吸虫感染兔不同时间的配对全血标本进行扩增,感染前血样均为阴性,感染后连续5周全血基因组DNA的浓缩标本均为阳性,非浓缩标本部分阳性。结论本研究建立了检测兔全血日本血吸虫兔基因组DNA的LAMP方法。该方法对浓缩DNA标本的检出效率高于非浓缩标本,具有血吸虫感染早期诊断价值。
[Abstract]:Objective to establish a method for the detection of Schistosoma japonicum DNA in whole blood samples. Methods four segments of DNA sequences of Schistosoma japonicum genomic repeat sequence (DNA SjR2(839~1 138 BP 563, 764 BP) and Sj-nonLTR1(1 678 1 877 BP) were selected as amplification fragments. Four sets of primers were designed and synthesized according to the principle of LAMP. The sensitivity and specificity of the four primers for DNA LAMP amplification were compared and the detection conditions were optimized to establish a specific and sensitive LAMP method for the detection of DNA of whole blood Schistosoma japonicum. This method was used to amplify the matched whole blood DNA of 10 rabbits before and after infection of Schistosoma japonicum. Results among the four primer groups, Schistosoma japonicum SjR2(839~1 138bp was used as the target fragment. The Chinese mainland strain of Schistosoma japonicum (the detection limit is 10-4 ng DNA), the Philippine strain and Schistosoma mansoni genomic DNA, the human whole blood genomic DNA, the Clonorchis sinensis genome DNA, the Plasmodium falciparum genome DNA can not be amplified. The amplification of lamp with Sj-nonLTR1(1 678 1 877 BP as the target fragment was not specific. Using SjR2(563~764 bp) and SjSH7 (204B) 399bp as target fragments, The genomic DNAs of Schistosoma japonicum Chinese mainland strain (detection limit: 10-6ng and 10-3 ng) and Philippine strain could be amplified specifically. The genomic DNA of human whole blood, clonorchiasis sinensis, Schistosoma mansoni and Plasmodium falciparum genome DNA could not be amplified. The whole blood samples of 10 rabbits infected with Schistosoma japonicum at different time were amplified with SjR2(839~1 138 BP primer. The blood samples were all negative before infection. The concentrated samples of whole blood genomic DNA were positive for 5 weeks after infection, and the non-concentrated samples were partially positive. Conclusion in this study, a LAMP method for the detection of genomic DNA of rabbits with whole blood Schistosoma japonicum was established. The efficiency of this method in detecting concentrated DNA samples is higher than that in non-concentrated specimens, and it is valuable for early diagnosis of schistosomiasis infection.
【作者单位】：南京医科大学病原生物学系;卫生部重要寄生虫病预防与控制高技术重点实验室江苏省血吸虫病防治研究所;
【基金】：国家“十一·五”科技重大专项项目(No.2008ZX10004-011) 江苏省卫生厅科技项目(No.H200738) 江苏省科技厅公益专项基金项目(No.BM2007704) 江苏省医学重点人才基金项目(No.RC2007095)
【分类号】：R532.21

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