罕见输入性卵形疟原虫wallikeri亚种的PCR鉴定和测序分析

PCR identification and sequencing of Plasmodium ovale wallikeri in a rare case of imported malaria

目的应用PCR技术对与间日疟原虫形态相识的卵形疟原虫亚种wallikeri进行鉴定。方法采集1例镜检可疑间日疟输入性疟疾患者滤纸血,采用PCR扩增18核糖体小亚单位RNA(18Small Subunit ribosomal RNA,18S SurRNA)片段,并进行测序分析。结果采用5对引物(rFAL1、rFAL2,rVIV1、rVIV2,rMAL1、rMAL2,rOVA1、rOVA2,rOVA1v、rOVA2v)进行PCR扩增,其中rOVA1v、rOVA2v引物扩增阳性,PCR产物大小为760bp;测序分析该基因片段(GenBank accession number KJ786425)与GenBank数据库中Plasomdium ovale wallikeri克隆RSH10 18S rRNA基因部分序列(KF219561.1)及卵形疟ssrRNA基因(AJ001527.1)的序列一致性都为100%。结论卵形疟与间日疟原虫形态相似,可采用PCR技术确认。本例患者感染的疟原虫经PCR鉴定和测序分析确定为卵形疟原虫变形P.ovale wallikeri变形亚种。

Objective To use PCR to identify imported Plasmodium ovale wallikeri,which is morphologically similar to P.vivax. Methods Microscopy suggested that a patient had imported P.vivaxmalaria.Blood was collected from the patient and filtered.PCR was used to amplify fragments of the small subunit（SSU）18Sribosomal RNA gene（18S SSUrRNA）,which were then sequenced. Results Five 5pairs of primers（rFAL1and rFAL2,rVIV1 and rVIV2,rMAL1 and rMAL2,rOVA1 and rOVA2,and rOVA1 vand rOVA2v）were used to perform PCR amplification.Only the primers rOVA1 vand rOVA2vresulted in amplification,and the PCR product was 760 bp in size.Sequencing revealed that the gene fragment（GenBank accession number KJ786425）had 100% similarity to part of the sequence of an RSH1018 SrRNA clone of P.ovale wallikeri（GenBank accession number KF219561.1）and part of the sequence of an SSU RNA gene of P.ovale（GenBank accession number AJ001527.1）. Conclusion P.ovale is morphologically similar to P.vivaxbut its identity can be verified with PCR.PCR identification and sequencing identified the malaria pathogen in the current case as a subtype of P.ovale wallikeri.