Establishment and assessment of nucleic acid detection method for Ancylostoma duodenale based on RPA-CRISPR/Cas12a technology

doi:10.12140/j.issn.1000-7423.2024.06.009

Abstract

Abstract:

Objective To develop a rapid, convenient, and efficient nucleic acid method based on RPA-CRISPR/Cas12a technology for detecting Ancylostoma duodenale in human stool samples to facilitate timely intervention, reduce the risk of disease transmission, and enhance the level of public health. Methods Based on the conserved sequence of mitochondrial cytochrome c oxidase subunit Ⅰ (cox1) of A. duodenale, RPA primers and CRISPR RNA (crRNAs) were designed and screened, while the concentration of crRNA and ssDNA, along with other reaction conditions, were optimized to establish the RPA-CRISPR/Cas12a fluorescence detection system. The minimum detection limits were determined using A. duodenale plasmid samples as templates, at the concentrations of 10⁵, 10⁴, 10³, 10², 10¹, 10⁰, 10⁻¹ and 10⁻² copies/μl, employing both the RPA method and the RPA-CRISPR/Cas12a method. The specificity of the RPA-CRISPR/Cas12a method was determined using genomic DNA templates from A. duodenale (10⁵ copies/μl), Necator americanus (20 ng/µl), Anisakis (30 ng/µl), and Trichinella spiralis (60 ng/µl). To assess the sensitivity and specificity of the RPA-CRISPR/Cas12a method, Kato-Katz combined with semi-nested PCR were used as reference standards, and 42 fecal samples (31 positive and 11 negative for A. duodenale based on the Kato-Katz method) were tested. Sensitivity and specificity values for the RPA-CRISPR/Cas12a method were calculated, and the agreement between detection results was analyzed using Kappa statistics. Results A RPA-CRISPR/Cas12a fluorescence detection system targeting the A. duodenale cox1 gene sequence was developed. In the optimized CRISPR/Cas12a system, the final concentrations of LbCas12a nuclease and crRNA probe were both 50 nmol/L, while the ssDNA reporter concentration was 100 nmol/L. The reaction was performed at 37 ℃ for 40 minutes. The minimum detection limit for the RPA assay was 10⁰ copies/μl, while the RPA-CRISPR/Cas12a system achieved a lower detection limit of 10 copies/μl, demonstrating enhanced sensitivity. The system showed no cross-reactivity with N. americanus, Anisakis, and T. spiralis. When compared with the Kato-Katz combined with semi-nested PCR, the RPA-CRISPR/Cas12a assay exhibited a sensitivity of 100% (19/19), a specificity of 91.30% (21/23), and good consistency (Kappa = 0.9048). Conclusion The RPA-CRISPR/Cas12a fluorescent detection system established in this study possesses rapid, sensitive, and specific detectability, providing reliable technical support for public health surveillance and the prevention and control of hookworm disease.

Key words: Ancylostoma duodenale, Recombinase polymerase amplification, CRISPR/Cas12a, Nucleic acid testing

CLC Number:

R532.12

YAN Shuning, YANG Hanyin, CAI Yuchun, XU Bin, YU Chenghang, MO Ziran, LU Yan, YANG Shuo, XIN Yi, ZHENG Bin. Establishment and assessment of nucleic acid detection method for Ancylostoma duodenale based on RPA-CRISPR/Cas12a technology[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2024, 42(6): 748-755.

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名称 Name	序列 Sequence
F1	CTTTGGGTATGGTTTATGCAATTTTAAGTA
F2	CTCACCATATGTATACTGTAGGTATGGATTTG
R1	CATGTAAAATAATATCCAACCTTGAATTTGAC
R2	CTTGAATTTGACAATACAACACCAGTTAAACC
crRNA1	UAAUUUCUACUAAGUGUAGAUCGGCUGCAACUAUGGUGAUU
crRNA2	UAAUUUCUACUAAGUGUAGAUCUAUUGGUGGUUUAACUGGU
crRNA3	UAAUUUCUACUAAGUGUAGAUGGUGGUUAGCCACAUUAUUU
ssDNA	FAM-TTATT-BHQ1

引物名称 Primer name	序列（5′→3′） Sequence (5′→3′)
NC1	ACGTCTGGTTCAGGGTTGTT
NC2	TTAGTTTCTTTTCCTCCGCT
AD	CGACTTTAGAACGTTTCGGC
NA	ATGTGCACGTTATTCACT