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目的对卵形疟原虫(Plasmodium ovale)curtisi亚种和wallikeri亚种编码的细胞色素b(cytochrome b,Cytb),细胞色素c氧化酶Ⅰ(cytochrome c oxidaseⅠ,CoxⅠ)和乳酸脱氢酶(lactate dehydrogenase,LDH)基因的单核苷酸多态性进行分析。方法选择浙江省疟疾诊断参比实验室2011-2014年确诊的卵形疟原虫感染患者血样,提取卵形疟原虫DNA,巢式PCR扩增卵形疟原虫Cytb、CoxⅠ和LDH基因的部分片段,对扩增产物进行双向测序。测序序列经校对、拼接后,应用MEGA软件对测序序列和相应的氨基酸序列进行比对,分析单核苷酸多态性位点以及氨基酸突变位点。结果 19例卵形疟原虫感染者(其中curtisi亚种11例,wallikeri亚种8例)均为赴非洲返国人员。PCR扩增Cytb、CoxⅠ和LDH基因的产物长度分别为735、1 323和355 bp。curtisi和wallikeri两个亚种在Cytb、CoxⅠ和LDH基因分别发现10、13、13个单核苷酸二态性位点,且大多数的二态性位点属于同义突变。此外,两个亚种内部也存在核苷酸多态性,同时发现1例血样的CoxⅠ基因存在693个碱基的缺失。结论对卵形疟患者血样的Cytb、CoxⅠ和LDH基因进行了单核苷酸多态性分析,丰富了卵形疟原虫基因多态性信息。
Objective To investigate the cytochrome b (Cytb), cytochrome c oxidase I (Cox I) and lactate dehydrogenase (Cox I) encoded by Plasmodium ovale subsp. Curtisi and wallikeri subsp. , LDH) gene single nucleotide polymorphisms were analyzed. Methods Blood samples were collected from malaria-infected malaria patients in Zhejiang Reference Laboratory for Diagnosis of Malaria from 2011 to 2014. Plasmodium falciparum DNA was extracted, and some fragments of Cytb, CoxⅠ and LDH genes of Plasmodium ovale were amplified by nested PCR. The amplified product was bi-directionally sequenced. Sequencing and alignment of the sequencing sequence, MEGA software was used to align the sequence and the corresponding amino acid sequence to analyze single nucleotide polymorphism and amino acid mutation sites. Results 19 cases of Plasmodium ovale infection (including 11 curtisi subspecies and 8 wallikeri subspecies) were returned to Africa. The PCR products of Cytb, Cox Ⅰ and LDH were 735, 1332 and 355 bp, respectively. Two subspecies curtisi and wallikeri, respectively, found 10,13,13 single nucleotide polymorphisms in Cytb, Cox I and LDH genes, and most of the two polymorphic sites belong to synonymous mutations. In addition, there were nucleotide polymorphisms in both subspecies. One sample of blood was found to have a 693-bp deletion in the CoxⅠ gene. Conclusion Single nucleotide polymorphisms (SNPs) of Cytb, Cox Ⅰ and LDH genes in blood samples from oval-shaped malaria patients were analyzed, which enriched the genetic information of Plasmodium ovale.