Li Mei, Wang Zhen-yu, Zhang Tao, Xia Zhi-gui
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2016 Dec;34(6):500-5.
To explore the application and specificity of one-step reverse transcription PCR (RT-PCR) in detecting four species of human Plasmodium parasites.
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.
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.
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.
探讨一步法逆转录聚合酶链反应（RT-PCR）在检测四种人体疟原虫中的应用及特异性。
采集恶性疟病例、间日疟病例、卵形疟病例各1例以及三日疟病例5例的血样。提取RNA和DNA。对用DNase消化后的RNA进行一步法RT-PCR和一步法实时RT-PCR，以扩增疟原虫18S rRNA。用传统PCR和一步法RT-PCR对不同稀释度的RNA（有无DNase消化）和DNA中的18S rRNAs和1S rDNAs进行扩增。比较两种扩增系统的最低可检测稀释浓度。
一步法RT-PCR产生了310、394和323 bp的特异性条带，测序结果表明分别为恶性疟原虫、卵形疟原虫和间日疟原虫的18S rRNA。未发现三日疟原虫的特异性条带。一步法实时RT-PCR结果显示四种疟原虫均有荧光信号，除三日疟原虫外，其余均有单峰熔解曲线。一步法RT-PCR的最低可检测稀释浓度根据DNA或RNA模板量在1至10⁻⁴之间变化。具体而言，DNA的最低可检测稀释浓度与原始RNA相似或低一个数量级，且两者均低于DNase消化后的RNA。此外，以最低可检测稀释浓度评估的一步法RT-PCR的灵敏度比传统PCR高10至1000倍。
一步法RT-PCR技术可用于新鲜血样中恶性疟原虫、卵形疟原虫和间日疟原虫的检测。但其在检测三日疟原虫方面的应用需要进一步评估。
