基于RPA-CRISPR/Cas12a技术的十二指肠钩虫核酸检测方法的建立和评价

doi:10.12140/j.issn.1000-7423.2024.06.009

中国寄生虫学与寄生虫病杂志 ›› 2024, Vol. 42 ›› Issue (6): 748-755.doi: 10.12140/j.issn.1000-7423.2024.06.009

基于RPA-CRISPR/Cas12a技术的十二指肠钩虫核酸检测方法的建立和评价

闫书宁¹(), 杨汉银¹, 蔡玉春¹, 徐斌¹, 俞铖航¹, 莫子冉³, 卢艳¹, 杨硕¹, 辛怡¹, 郑彬¹^,²^,^*()

1 中国疾病预防控制中心寄生虫病预防控制所（国家热带病研究中心），传染病溯源预警与智能决策全国重点实验室，国家卫生健康委员会寄生虫病原与媒介生物学重点实验室，世界卫生组织热带病合作中心，科技部国家级热带病国际研究中心，上海 200025
2 上海交通大学医学院-国家热带病研究中心全球健康学院，上海 200025
3 内蒙古大学生命科学学院，内蒙古呼和浩特 010070

收稿日期:2024-07-08 修回日期:2024-08-16 出版日期:2024-12-30 发布日期:2025-01-14
通讯作者: *郑彬（1972—），女，博士，研究员，从事寄生虫病防治研究。E-mail：zhengbin@nipd.chinacdc.cn
作者简介:闫书宁（2000—），女，硕士研究生，从事寄生虫病监测与预警研究。E-mail：ysn3123@163.com
基金资助:
中国疾病预防控制中心寄生虫病预防控制所科技创新支撑计划(TF2024008)

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

YAN Shuning¹(), YANG Hanyin¹, CAI Yuchun¹, XU Bin¹, YU Chenghang¹, MO Ziran³, LU Yan¹, YANG Shuo¹, XIN Yi¹, ZHENG Bin¹^,²^,^*()

1 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; Chinese Center for Tropical Diseases Research; National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases; Key Laboratory on Parasite and Vector Biology, Ministry of Health; WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology, Shanghai 200025, China
2 School of Global Health, Chinese Center for Tropical Diseases Research and Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
3 College of Life Sciences, Inner Mongolia University, Hohhot 010070, Inner Mongolia, China

Received:2024-07-08 Revised:2024-08-16 Online:2024-12-30 Published:2025-01-14
Contact: *E-mail: zhengbin@nipd.chinacdc.cn
Supported by:
Science and Technology Innovation Support Program of the Institute of Parasitic Disease Prevention and Control, Chinese Centre for Disease Control and Prevention(TF2024008)

摘要/Abstract

摘要：

目的基于重组酶聚合酶扩增（RPA）技术和簇状规则间隔短回文重复序列及其相关蛋白12a（CRISPR/Cas12a）技术，建立快速、便捷、高效检测人粪样中十二指肠钩虫的方法，以及时干预、降低疾病传播风险，提高公共卫生水平。方法参考十二指肠钩虫线粒体细胞色素c氧化酶亚基Ⅰ（cox1）序列，设计并筛选RPA引物和CRISPR RNA（crRNA），优化crRNA浓度、ssDNA浓度和其他反应条件，建立RPA-CRISPR/Cas12a荧光检测体系。以10⁵、10⁴、10³、10²、10¹、10⁰、10^-1、10^-2拷贝/µl的十二指肠钩虫阳性质粒为模板，分别采用RPA法和RPA-CRISPR/Cas12a法检测最低检出限，评价该方法的敏感度。分别以十二指肠钩虫（10⁵拷贝/µl）、美洲钩虫（20 ng/µl）、异尖线虫（30 ng/µl）、旋毛形线虫（60 ng/µl）基因组DNA为模板，采用RPA-CRISPR/Cas12a法进行检测，评价该方法的特异性。以Kato-Katz法结合半巢式PCR法结果为标准，检测42份粪样（经Kato-Katz法检测31份为钩虫阳性，11份为钩虫阴性），计算RPA-CRISPR/Cas12a法的灵敏度和特异度，Kappa值比较检测结果的一致性。结果建立了基于十二指肠钩虫cox1序列的RPA-CRISPR/Cas12a荧光检测体系，在优化的CRISPR/Cas12a体系中，LbCasl2a核酸酶终浓度为50 nmol/L、crRNA探针终浓度为50 nmol/L、ssDNA报告分子终浓度为100 nmol/L。该检测体系的反应条件为37 ℃ 40 min。RPA的最低检出限为10²拷贝/μl，RPA-CRISPR/Cas12a法的最低检出限为10拷贝/μl，后者敏感度更高，且与美洲钩虫、异尖线虫、旋毛形线虫无交叉反应。与Kato-Katz法结合半巢式PCR法比较，RPA-CRISPR/Cas12a检测的灵敏度为19/19，特异度为91.30%（21/23），一致性较好（Kappa = 0.904 8）。结论该研究建立的RPA-CRISPR/Cas12a荧光检测体系具备快速、灵敏、特异的检测能力，为公共卫生监测和钩虫病防控提供了可靠技术支持。

关键词: 十二指肠钩虫, 重组酶聚合酶扩增, CRISPR/Cas12a, 核酸检测

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

中图分类号:

R532.12

闫书宁, 杨汉银, 蔡玉春, 徐斌, 俞铖航, 莫子冉, 卢艳, 杨硕, 辛怡, 郑彬. 基于RPA-CRISPR/Cas12a技术的十二指肠钩虫核酸检测方法的建立和评价[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(6): 748-755.

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

基于RPA-CRISPR/Cas12a技术的十二指肠钩虫核酸检测方法的建立和评价

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

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RPA-CRISPR/Cas12a	Kato-Katz阳性时半巢式PCR结果 Semi-nested PCR results for Kato-Katz positive samples		Kato-Katz阴性时半巢式PCR结果 Semi-nested PCR results for Kato-Katz negative samples		合计Total
RPA-CRISPR/Cas12a	+	-	+	-	合计Total
+	19	2	0	0	21
-	0	10	0	11	21
合计 Total	19	12	0	11	42

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