[Establishment and preliminary evaluation of recombinase-aided isothermal nucleic acid amplification combined with nanopore sequencing for identification of Plasmodium species].

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 prelimi-narily 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 transcription 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-nano-pore 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.