基于SSUrRNA基因序列的间日疟原虫LAMP检测技术的建立

Establishment of a loop-mediated isothermal amplification assay for detecting blood-stage Plasmodium vivax based on the specific sequence of an SSUrRNA-encoding gene

目的克隆分析间日疟原虫小亚基亚单位核糖体核糖核酸(SSUrRNA)特异性基因序列,建立间日疟原虫环介导等温扩增(LAMP)检测技术。方法针对间日疟原虫SSUrRNA种特异性基因序列设计1对引物,采用聚合酶链反应(PCR)从血样核酸提取物中扩增SSUrRNA基因片段,纯化后与pGEM-Teasy载体连接,构建重组质粒并转化大肠埃希菌JM109,PCR与双酶切鉴定筛选阳性克隆并测序,设计6条寡核苷酸片段,LAMP检测感染血样中间日疟原虫DNA,扩增产物作琼脂糖电泳分析或直接荧光染色肉眼观察。结果间日疟原虫SSUrRNA基因扩增片段大小约为235 bp;阳性克隆重组质粒插入的SSUrRNA基因扩增片段含有235个核苷酸,与GenBank中的Sal-1株、Belem株间日疟原虫相同序列进行比对,同源性为100%,与PV2008/TR/DEL株、PVK1294株、E1 Salvador株的同源性均为99%,与三日疟原虫、卵形疟原虫及恶性疟原虫的序列同源性均低于95%。将含有SSUrRNA靶基因的重组质粒以蒸馏水倍比稀释后做LAMP,试验的灵敏度为10 copy/μl;特异性检验显示间疟原虫患者血样DNA呈阳性反应,恶性疟原虫、弓形虫、日本血吸虫、华支睾吸虫感染者及正常人血DNA为阴性。结论扩增、克隆的间日原虫SSUrRNA基因序列不同地理株间同源性高,以其为靶基因建立的间日疟原虫LAMP检测技术灵敏度高、特异性好,且无需昂贵的仪器设备,具有推广应用价值。

Objective To establish a loop-mediated isothermal amplification（LAMP） method for detecting Plasmodium vivax based on the cloning and analysis of a specific SSUrRNA-encoding gene（SSUrDNA） fragment.Methods A pair of primers was designed according to a specific P.vivax SSUrDNA sequence,and the SSUrDNA fragment was amplified by PCR from the DNA extracts of a P.vivax-infected blood sample.After purification,the gene fragment was ligated with plasmid pGEM-Teasy to construct a recombinant plasmid,which was transformed into E.coli JM109.Positive bacteria clones were identified by PCR and double enzyme digestion,and the sequence of the inserted SSUrDNA fragment was determined.Based on the determined sequence,6 primers for LAMP were designed to detect P.vivax DNA in blood samples.The amplified products was analyzed with gel electrophoresis or directly observed with the naked eye following addition of fluorescent dye SYBR Green I.Results The amplified SSUrDNA fragment was about 235 bp in length and matched the corresponding sequence of P.vivax deposited in the GenBank database.Nucleotides were 100% homologous to those of the Sal-1 and Belem strains,99.0% homologous to those of the PV2008/TR/DEL,PVK1294,and E1 Salvador strains,and less than 95% homologous to those of other species of human malaria parasites,such as P.falciparum,P.malariae,and P.ovale.Using 10-fold serial dilution of the SSUrDNA recombinant plasmid with sterile water as a template,the LAMP method was sensitive enough to detect 10 copies/μl of the target gene,and testing of specificity revealed no cross-reactivity in blood samples infected with P.falciparum,Toxoplasma gondii,Schistosoma japonicum,or Clonorchis sinensis.Conclusion The cloned SSUrRNA gene sequence of blood-stage P.vivax was conserved among different strains,and the established LAMP method was sensitive and specific enough to detect P.vivax in blood samples without the need for expensive amplification instruments.