论文部分内容阅读
目的建立基于编码恶性疟原虫PHIST蛋白特有基因的环介导等温扩增技术。方法在Plasmo DB数据库中,搜索并筛选编码PHIST、在环状体或裂殖体期高表达且恶性疟原虫特有的基因。利用在线软件Primer Explorer V4设计目的基因LAMP引物。采集恶性疟原虫滤纸血并提取基因组DNA。将提取后的恶性疟原虫DNA用超纯水进行10~(-1)、10~(-2)、10~(-3)、10~(-4)倍比稀释,用LAMP法检测其敏感性;采用间日疟原虫、约氏疟原虫、牛带绦虫和日本血吸虫基因组DNA作为对照,用LAMP法评价其特异性。结果共筛选出61个编码恶性疟原虫PHIST的基因。选取环状体期高表达的特有基因PF3D7_1372300和裂殖体期高表达的特有基因PF3D7_1401600建立LAMP技术。基于PF3D7_1372300和PF3D7_1401600基因的LAMP法检测恶性疟原虫的最低限度分别为130.5个/μl和1 305.3个/μl,所获得的恶性疟原虫扩增产物其检测管染色后呈绿色,即阳性。基于PF3D7_1372300和PF3D7_1401600基因的LAMP法扩增恶性疟原虫产物检测管染色后呈绿色,即阳性;而间日疟原虫、约氏疟原虫、牛带绦虫和日本血吸虫的LAMP扩增产物检测管染色后仍呈棕色,即阴性。结论基于PF3D7_1372300基因的LAMP法检测恶性疟原虫敏感、特异、简便、实用,可用于恶性疟流行区现场调查和临床诊断。
OBJECTIVE: To develop a loop-mediated isothermal amplification technique based on a gene specific for the PHIST protein of Plasmodium falciparum. Methods In the Plasmo DB database, genes encoding PHIST, which are highly expressed in the ring or in the schizont stage and are specific to P. falciparum, are searched and screened. The target gene LAMP primer was designed by online software Primer Explorer V4. Plasmodium falciparum filter paper was collected and genomic DNA was extracted. The extracted P. falciparum DNA was diluted 10 ~ (-1), 10 ~ (-2), 10 ~ (-3), 10 ~ (-4) times with ultrapure water and detected by LAMP method Sex; Plasmodium vivax, Plasmodium yoelii, Taenia saginata and Schistosoma japonicum genomic DNA as a control, its specificity was evaluated by LAMP method. Results A total of 61 genes encoding P. falciparum PHIST were screened out. The LAMP technique was established by using the specific gene PF3D7_1372300 with high expression in the ring stage and the unique gene PF3D7_1401600 with high expression in the schizonts stage. The minimal detection limit of Plasmodium falciparum based on the LAMP method of PF3D7_1372300 and PF3D7_1401600 genes was 130.5 / μl and 1 305.3 / μl, respectively. The PCR products of P. falciparum amplification products were green and positive after staining. Plasmodium falciparum amplified by LAMP method based on the PF3D7_1372300 and PF3D7_1401600 genes amplified green, ie, positive, whereas LAMP amplification products of Plasmodium vivax, Plasmodium yoelii, Taenia saginata, and Schistosoma japonicum detect tube staining Still brown, negative. Conclusion The detection of Plasmodium falciparum based on PF3D7_1372300 gene by LAMP method is sensitive, specific, simple and practical and can be used for field investigation and clinical diagnosis of P. falciparum endemic areas.