关键词: 疟原虫, 硫代磷酸化dNTP, 重组酶聚合酶扩增, CRISPR/Cas12a

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