一步反转录PCR技术在检测4种人疟原虫中的初步应用

Exploration of Using One-step Reverse Transcription PCR in Detection of Four Species of Human Malaria Parasites

目的 探讨应用一步反转录PCR（RT-PCR）技术检测4种人疟原虫的可行性和特异性。 方法 取恶性疟（1例）、间日疟（1例）、卵形疟（1例）和三日疟（5例）患者全血，提取疟原虫总RNA和基因组DNA。应用一步RT-PCR和一步实时荧光RT-PCR分别扩增经脱氧核糖核酸酶（DNase）消化的疟原虫RNA样品中的18S rRNA序列，应用普通PCR和一步RT-PCR技术分别扩增不同稀释浓度的疟原虫RNA（未经DNase消化和经DNase消化）和DNA样品中的疟原虫18S rRNA序列和18S rDNA序列，比较2种检测技术可检测到目标序列的样品最低稀释浓度。 结果 患者全血基因组DNA经一步RT-PCR分别扩增出310、394和323 bp条带，测序结果显示分别为恶性疟原虫（Plasmodium falciparum）、卵形疟原虫（P. ovale）和间日疟原虫（P. vivax）的18S rRNA序列特异性条带，但未扩增出三日疟原虫（P. malariare）特异条带。一步实时荧光RT-PCR结果显示，4种人疟原虫RNA样品均出现相应的荧光信号，除三日疟原虫样品外，各溶解曲线均仅有单一的扩增峰。因4种人疟原虫DNA和RNA原液样品中模板量的差异，一步RT-PCR可检测到的最低稀释浓度从1至10-4不等，但可检测到的DNA样品的最低稀释浓度与未经DNase消化的RNA样品相同或较后者低一个数量级，且两者最低稀释浓度均低于经DNase消化的RNA样品。与普通PCR的检测结果比较发现，同一样品均以一步RT-PCR可检测到的稀释浓度最低，较普通PCR的敏感性提高10～1 000倍。 结论 一步RT-PCR技术可检测出人血液样品中的恶性疟原虫、卵形疟原虫和间日疟原虫DNA，但在检测三日疟原虫样品中的适用性有待进一步分析。

Objective To explore the application and specificity of one-step reverse transcription PCR（RT-PCR）in detecting four species of human Plasmodium parasites. Methods Blood samples were collected from a falciparum malaria case, a vivax malaria case, an ovale malaria case, and five quartan malaria cases. RNA and DNA were isolated. One-step RT-PCR and one-step real-time RT-PCR were performed on the RNA digested with DNase to amplify the Plasmodium 18S rRNA. Traditional PCR and one-step RT-PCR were used to amplify 18S rRNAs and 18S rDNAs in differentially diluted RNAs（with or without DNase digestion） and DNAs. The lowest detectable dilution concentrations for the two amplification systems were compared. Results One-step RT-PCR produced specific bands of 310, 394 and 323 bp, which were sequenced to be 18S rRNA of P. falciparum, P. ovale, and P. vivax. No specific band for P. malariare was found. The one-step real-time RT-PCR results showed fluorescence for all the four species, and all had a melting curve with a single peak except for P. malariare. The lowest detectable dilution concentration by one-step RT-PCR varied from 1 to 10-4 based on the DNA or RNA template amount. Specifically, the lowest detectable dilution concentration of DNA was similar to or lower than that of original RNA by an order of magnititude, and both were lower than that of DNase-digested RNA. Further, the sensitivity of one-step RT-PCR evaluated in terms of lowest detectable dilution concentration was 10-1 000 times higher than that of the traditional PCR. Conclusions The one-step RT-PCR technique can be applied in the detection of P. falciparum, P. ovale, and P. vivax in fresh blood samples. But its use in detecting P. malariae parasites needs further evaluation.