基于恶性疟原虫PHIST特有基因的环介导等温扩增技术的建立与评价

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。

Establishment and evaluation of loop - mediated isothermal amplification based on Plasmodium falciparum unique genes coding PHIST proteins

Objective Objective To establish a novel convenient loop ?mediated isothermal amplification（LAMP）method with the unique genes coding Plasmodium helical interspersed sub?telomeric superfamily（PHIST）for the rapid molecular diagnosis of P. falciparum. Methods Methods The unique genes coding PHIST with high expression mRNA profile during the ring form or schizont period of P. falciparum were screened and selected from the PlasmoDB database. The LAMP primers of targeted genes were de? signed by the online software（PrimerExplorer V4） . The LAMP assay was executed by the color?displaying method with SYBR Green. The dried blood spots of P. falciparum from clinical isolates were collected and the genomic DNA（gDNA）was extracted. For evaluation of sensitivity，the gDNA was diluted to four gradients（10?1 ，10?2 ，10?3 ，and 10?4 ） . For assessment of specificity， the gDNA （s）of P. vivax，P. yoelii，Taenia saginata，and Schistosoma japonicum were also extracted. Results Results Totally，61 P. falciparum unique genes coding PHIST were found. The PF3D7_1372300 with high expression value during the ring form and PF3D7_1401600 with high expression value during the schizont period were selected for LAMP assay. The lowest detectable lim? its of PF3D7_1372300 and PF3D7_1401600 were 130.5 parasite/μl and 1 305.3 parasite/μl，respectively. Specific tests showed the amplified products of P. falciparum was positive and all the others including P. vivax，P. yoelii，T. saginata，and S. japoni? cum were negative. Conclusions Conclusions The established LAMP method with PF3D7_1372300 gene is sensitive，specific，simple and useful. It can be applied to the field investigation and clinical diagnosis for falciparum malaria.