Phosphorothioate-dNTP assisted RPA coupled with CRISPR/Cas12a for rapid genotyping of Plasmodium

doi:10.12140/j.issn.1000-7423.2026.01.010

Abstract

Abstract:

Objective To establish a rapid nucleic acid assay for genotyping for Plasmodium (PRCP) based on phosphorothioate dNTPs (dNTPαS) enhancement of the specificity and sensitivity of recombinase polymerase amplification (RPA), combined with the target nucleic acid recognition and signal amplification capabilities of CRISPR/Cas12a. Methods Universal RPA primers were designed with the software Primer Premier 6, and specific gRNAs targeting different species of Plasmodium were designed within the universal primer region using the software SnapGene 6.0. Then, dNTPαS was added to the reaction system to generate an S-RPA amplification reaction, and CRISPR/Cas12a was employed for typing recognition and signal amplification output of the amplification product. The dNTPαS concentration, RPA primer concentration, gRNA concentration, Cas12a concentration, reaction temperature, reaction time, and final Cas12a cleavage time in the PRCP reaction system were optimized in sequence. PRCP was performed with P. falciparum plasmids at concentrations of 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10¹ copies/µL as templates to evaluate its sensitivity, and hepatitis B virus, Babesia, Trypanosoma brucei, influenza A virus, influenza B virus, Mycoplasma pneumoniae, and Chlamydia pneumoniae served as controls to evaluate the specificity, and was conducted with addition of 2 g/L hemoglobin, 0.1 mmol/L triglyceride, and 1 μmol/L bilirubin to evaluate its anti-interference ability. In addition, mixed plasmid samples were used to detect the ability of the PRCP system to distinguish mixed infections, and the consistency was compared between detection of mixed plasmid samples and clinical samples (10 samples of Plasmodium infections and 10 negative samples) with thick and thin blood smears. Results A dNTPαS-assisted RPA assay was established based on 3F3R screened as the universal Plasmodium nucleic acid RPA primer to construct a PRCP system. The optimized parameters for the PRCP system included the optimal proportion of dNTPαS as 70%, the optimal final concentration of primers as 0.50 μmol/L (Rate10 as 676.36), the final concentration of Cas12a as 0.10 μmol/L (Rate10 as 338.28), and the final concentration of gRNA as 0.10 μmol/L (Rate10 as 718.90), and the RPA reaction conditions included 39 ℃ (grayscale value of 32 570 ± 5 045) and 20 minutes (grayscale value of 22 513 ± 156), with Cas12a cleavage for 15 minutes as the detection endpoint (grayscale value 8 624 ± 359). The detection sensitivity of the PRCP system was 100 copies/μL or below, and no cross-reactivity was found with hepatitis B virus, Babesia, T. brucei, influenza A virus, influenza B virus, M. pneumoniae, or C. pneumoniae. The PRCP system was found to resist interference from hemoglobin, triglyceride, and bilirubin, and was able to detect mixed infections. Compared with thick and thin blood smears, the PRCP system showed a 20/20 consistency for detection. Conclusion A rapid, sensitive and specific PRCP assay has been successfully established for rapid genotyping of Plasmodium genes, which provides a novel protocol for early screening and precise diagnosis and treatment of Plasmodium.

Key words: Plasmodium, Phosphorothioate-dNTP, Recombinase polymerase amplification, CRISPR/Cas12a

CLC Number:

R382.31

HUANG Xiao, CHEN Ying, WANG Maoquan, CHEN Yating, LUO Guangcheng. Phosphorothioate-dNTP assisted RPA coupled with CRISPR/Cas12a for rapid genotyping of Plasmodium[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2026, 44(1): 64-71.

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引物/gRNA Primers/ gRNAs	序列（5′→3′） Sequences (5′→3′)
RPA-1F	AACGAAAGTTAAGGGAGTGAAGACGATCAGATACCG
RPA-1R	ACTCGCCCCAGAACCCAAAGACTTTGATTTCTCATAAGG
RPA-2F	AAGTTAAGGGAGTGAAGACGATCAGATACCGTCG
RPA-2R	TCGCCCCAGAACCCAAAGACTTTGATTTCTCATAAG
RPA-3F	AGAACGAAAGTTAAGGGAGTGAAGACGATCAGATACC
RPA-3R	AATACTCGCCCCAGAACCCAAAGACTTTGATTTCTC
恶性疟原虫gRNA P. falciparum gRNA	UAAUUUCUACUAAGUGUAGAUAAAAAUAAAAGUCAUCUUUC
卵形疟原虫gRNA P. ovale gRNA	UAAUUUCUACUAAGUGUAGAUCCGAAGAGACUAUCCUUAUU
间日疟原虫gRNA P. vivax gRNA	UAAUUUCUACUAAGUGUAGAUAAUAAGAAAAUUCCUUUCGG
三日疟原虫gRNA P. malariae gRNA	UAAUUUCUACUAAGUGUAGAUAUAUAUAUGAGUGUUUCUU