Abstract: Objective　To develop a novel assay based on recombinase⁃aided isothermal nucleic acid amplification (RAA) and nanopore sequencing for species identification of Plasmodium vivax, P. ovale, P. malariae and P. falciparum, and to preliminarily assess its detection performance. Methods　Dried blood spot samples were collected from 89 malaria patients. Genomic DNA of Plasmodium was extracted from dried blood spots using the Chelex⁃100 method, and the species of Plasmodium was identified using TaqMan real⁃time fluorescence quantitative reverse transcription PCR, real⁃time quantitative reverse transcriptoin PCR(RT⁃qPCR) and nested PCR (nPCR) assays. Then, 8 sets of specific RAA primers were designed targeting the 18S ribosomal RNA (18S rRNA) genes of P. vivax, P. ovale, P. malariae and P. falciparum. The optimal primer combination was selected for amplification of the extracted Plasmodium DNA samples, and the 49 samples with the best amplification effect were selected for nanopore sequencing. The species identification of 49 dried blood spot samples from malaria patients was compared by RT⁃qPCR assay, nPCR assay and RAA⁃nanopore sequencing, and the sensitivity, specificity and accuracy of RT⁃qPCR assay and RAA⁃nanopore sequencing were evaluated with nPCR identification as the gold standard.  Results　RAA amplification showed that among the 8 primer combinations, only the F1R2 combination produced a single fragment, and the band of the amplification product was the brightest; therefore, this primer combination was selected for RAA amplification of 89 Plasmodium genomic DNA samples. RAA⁃nanopore sequencing successfully amplified the 18S rRNA gene of 4 Plasmodium species in dried blood spot samples from malaria patients. Among the blood spot samples positive for RAA amplification, 49 samples with a single, clear and bright target band were selected for nanopore sequencing. Of these 49 samples, nPCR identified P. falciparum infection in 22 samples, P. malariae infection in 6 samples, P. vivax infection in 6 samples, P. ovale infection in 14 samples and P. falciparum⁃P. malariae mixed infection in one sample, and RT⁃qPCR detected P. falciparum infection in 25 samples, P. malariae infection in 5 samples, P. vivax infection in 6 samples and P. ovale infection in 14 samples, while RAA⁃nanopore sequencing identified P. falciparum infection in 23 samples, P. malariae infection in 6 samples, P. vivax infection in 6 samples, P. ovale infection in 13 samples and P. falciparum⁃P. malariae mixed infection in one sample. If nPCR assay served as the gold standard, the sensitivity, specificity and accuracy of RAA⁃nanopore sequencing were 92.00%, 97.33% and 96.00% for species identification of malaria parasites, which were higher than those (88.24%, 97.32%, and 95.00%) of the RT⁃qPCR assay. Conclusions　The RAA⁃nanopore sequencing established in this study is sensitive, specific and accurate for identifying Plasmodium species, which may serve as a supplementary approach to conventional techniques for detection of malaria parasites.

Key words: Plasmodium, Recombinase?aided isothermal nucleic acid amplification, Nanopore sequencing, Species identification, Detection efficiency

摘要： 目的　构建一种基于重组酶介导的等温核酸扩增（recombinase⁃aided amplification，RAA）技术和纳米孔测序的对间日疟原虫、卵形疟原虫、三日疟原虫和恶性疟原虫进行虫种鉴定的新方法，并对该方法的检测效能进行初步评价。 方法　收集89例疟疾患者干血斑样本，采用Chelex⁃100法提取干血斑中疟原虫基因组DNA，采用TaqMan实时荧光定量PCR（real⁃time quantitative reverse transcription PCR，RT⁃qPCR）法及巢式PCR（nested polymerase chain reaction，nPCR）法进行虫种鉴定。随后，针对上述4种疟原虫18S核糖体RNA（18S rRNA）基因设计8组特异性RAA引物，选择最佳组合并对上述提取的疟原虫DNA样本进行扩增后，选取49份扩增效果最佳的产物进行纳米孔测序。比较RT⁃qPCR法、nPCR法、RAA⁃纳米孔测序法对49份疟疾干血斑样本的虫种鉴定结果，以nPCR法鉴定结果为“金标准”，计算RT⁃qPCR、RAA⁃纳米孔测序法的检测灵敏度、特异度、正确率。结果　RAA扩增结果显示，8组引物组合中仅F1R2组合可产生单一片段，且产物条带最明亮，故选择该引物组合对89份疟原虫DNA样本进行RAA扩增。RAA⁃纳米孔测序法可成功扩增疟疾患者干血斑样本中的4种疟原虫18S rRNA基因。在RAA扩增所得阳性样本中，选出49份产生单一清晰明亮目的条带的样本行纳米孔测序。49份样本中，nPCR法检出恶性疟原虫感染22份、三日疟原虫感染6份、间日疟原虫感染6份、卵形疟原虫感染14份以及恶性疟原虫和三日疟原虫混合感染1份；RT⁃qPCR法检出恶性疟原虫感染25份、三日疟原虫感染5份、间日疟原虫感染6份、卵形疟原虫感染13份；RAA⁃纳米孔测序法检出恶性疟原虫感染23份、三日疟原虫感染6份、间日疟原虫感染6份、卵形疟原虫感染13份以及恶性疟原虫和三日疟原虫混合感染1份。以nPCR检测结果为“金标准”，RAA⁃纳米孔测序法的检测灵敏度、特异度、正确率分别达到92.00%、97.33%、96.00%，高于qPCR法的88.24%、97.32%、95.00%。结论　本研究建立的RAA⁃纳米孔测序法鉴定疟原虫虫种的灵敏度、特异度、正确率均较高，可作为常规疟原虫检测技术的补充方法。

关键词: 疟原虫, 重组酶介导的等温核酸扩增, 纳米孔测序, 虫种鉴定, 检测效能