Abstract:

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.

Key words: Reverse-transcription PCR, Plasmodium parasite, 18S rRNA