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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 clone and analyze the specific gene sequence of small subunit subunit ribulose ribonucleic acid (SSUrRNA) of Plasmodium vivax, and to establish the ring-mediated isothermal amplification of Plasmodium vivax (LAMP) detection technique. Methods A pair of primers was designed according to the specific gene sequence of SSUrRNA in Plasmodium vivax. The SSUrRNA gene fragment was amplified from the nucleic acid extract of blood by polymerase chain reaction (PCR) and ligated with pGEM-Teasy vector to construct the recombinant plasmid The recombinant plasmid was transformed into Escherichia coli JM109. The positive clones were screened by PCR and restriction enzyme digestion. Six oligonucleotide fragments were designed and LAMP was used to detect the DNA of Plasmodium vivax in blood samples. The amplified product was analyzed by agarose gel electrophoresis or direct fluorescent staining Eye observation. Results The SSU rRNA gene amplified fragment of Plasmodium vivax was about 235 bp in length. The amplified fragment of SSU rRNA gene contained 235 nucleotides inserted into the positive cloned recombinant plasmid, which was the same as Sal-1 and Belem P. vivax in GenBank The homology was 100% with 99% identity with PV2008 / TR / DEL strain, PVK1294 strain and El Salvador strain. The homology with the P. vivax, P. ovale and P. falciparum Sequence homology is less than 95%. The recombinant plasmid containing SSUrRNA target gene was diluted with distilled water to make LAMP, and the sensitivity of the test was 10 copy / μl. The specific test showed that the DNA of the Plasmodium falciparum was positive for blood DNA, and that Plasmodium falciparum, Toxoplasma gondii, Schistosoma japonicum, Clonorchis sinensis infection and normal blood DNA negative. Conclusion The amplication and cloning of the protozoan SSUrRNA gene sequences of different geographical isolates with high homology, with its target gene established P. vivax LAMP detection technology is highly sensitive and specific, and without expensive equipment, with Promote the value of application.