基于线粒体细胞色素氧化酶亚单位3基因鉴定贵州省首例输入性卵形疟病例

提取贵州省首例镜检诊断为输入性卵形疟病例的血样DNA,以疟原虫的核糖体RNA小亚基(SSU r RNA)和线粒体细胞色素氧化酶亚单位3基因(cox3)为靶标,分别设计属、种特异性引物及种特异性引物, PCR扩增、测序,比较2种引物对疟原虫种类PCR鉴定的特异性。以卵形疟原虫wallikeri亚种参考序列(GenBank登录号:HQ712053.1)和curtisi亚种参考序列(GenBank登录号:HQ712052.1)为模板,应用DNAMAN V6软件对扩增序列进行Blast比对。利用Mega 7.0.26软件,采用邻接法,基于疟原虫线粒体cox3基因DNA序列构建系统进化树。PCR结果显示,基于疟原虫SSU r RNA属、种特异性引物扩增,仅获得1 200 bp的属特异性条带,未出现种特异性条带。基于疟原虫cox3种特异性引物扩增,可获得880 bp的目的条带。Blast比对结果显示,PCR扩增获得的cox3基因序列与卵形疟原虫wallikeri亚种和curtisi亚种的序列一致度为99.4%和97.4%。

Mitochondrial cox3 based PCR detection of the first imported case of Plasmodium ovale in Guizhou province

To develop a PCR-based detection of Plasmodium ovale infection, DNA was extracted from blood of a patient diagnosed as the first imported case of P. ovale infection in Guizhou province, two sets of PCR based on the Plasmodium ribosomal RNA small subunit (SSU rRNA) and mitochondrial cytochrome oxidase subunit 3 gene (cox3) were established. The genus, species-specific primers were designed accordingly, the specificity of Plasmodium species detection was performed using the designed primers and the amplified cox3 PCR products were sequenced. The obtained sequences were aligned with reference sequence of the P. ovale wallikeri subspecies (GenBank accession number: HQ712053.1) and the curtisi subspecies (GenBank accession number: HQ712052.1) using DNAMAN V6. A phylogenetic tree was constructed based on the Plasmodium, cox3 gene using the neighbor-joining method and Mega 7.0.26 software. The PCR results showed that only a genus-specific band with 1 200 bp was obtained based on Plasmodium SSU rRNA and no species-specific bands were amplified. However, a PCR product with 880 bp was amplified from the patient sample based on Plasmodium cox3 gene. The Blast alignment showed that the obtained cox3 sequence shared 99.4% identity with P. ovale wallikeri subspecies and 97.4% with curtisi subspecies.