关键词: 约氏疟原虫, 18S核糖体DNA, 检测, 逆转录-聚合酶链反应

Abstract: 　Objective To determine sequence of sporogony stage-specific (S type) 18S ribosomal RNA gene of Plasmodium yoelii (P.y) By265 strain, and by using it to detect the malaria parasites within vector mosquito. Methods A pair of conserved DNA primers, universe primer (Pu) and reverse transcription one (Pr), was designed and synthesized according to sequence of the 18S rRNA gene of Plasmodium berghei (P.b). The segment of the S type 18S rDNA of P.y was amplified by reverse transcript-polymerase chain reaction (RT-PCR) from dissected midguts of Anopheles stephensi infected with P.y on the 7th day after infective blood-meal, and its sequence was then determined. One P.y sporogony stage-specific primer (Pys) was selected according to the sequence. Using this primer and Pr, the parasites within mosquitoes were semi-quantitatively detected through RT-PCR between 1-7 d post-infection. Results The length of the amplified segment was 920 bp. Alignment in match region of the 18S rDNA among S type of P.y (PyS), S type of P.b (PbS) and asexual blood stage-specific one of P.y (PyA) revealed that the similarity between the former and the latter two reached 95.3% and 94.0% respectively. The density of amplified band was significantly concordance with the intensity of oocyst in the midgut. Sensitivity of RT-PCR method was higher than that of the traditional dissection and oocyst observation also. The assay could detect the 18S rRNA molecule of the parasites on the third day post-infection while their oocysts were difficult to be recognized under an optical microscope at that time. Conclusion This S type 18S rDNA sequence in P.y species was first reported (AF266261). As a molecular marker, it could be applied to monitoring the parasite development in its vector at an earlier stage semi-quantitatively with an adequate sensitivity and specificity.

Key words: Plasmodium yoelii, 18S ribosomal DNA, detection, reverse transcript-polymerase chain reaction