基于重组酶聚合酶扩增的曼氏血吸虫核酸可视化检测技术的建立及初步评价

doi:10.12140/j.issn.1000-7423.2022.03.009

中国寄生虫学与寄生虫病杂志 ›› 2022, Vol. 40 ›› Issue (3): 337-343.doi: 10.12140/j.issn.1000-7423.2022.03.009

基于重组酶聚合酶扩增的曼氏血吸虫核酸可视化检测技术的建立及初步评价

王丽萍(), 吕超, 秦志强, 许静, 邓王平()

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）,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,国家级热带病国际研究中心,上海 200025

收稿日期:2021-07-26 修回日期:2021-12-13 出版日期:2022-06-30 发布日期:2022-07-06
通讯作者: 邓王平
作者简介:王丽萍（1995-）,女,硕士研究生,从事血吸虫病防治研究。E-mail： wlpnlnl@163.com
基金资助:
上海市卫生健康委员会卫生行业临床研究专项(202040053);国家科技重大专项(2018ZX10101002-002);国家自然科学基金(82073619);上海市公共卫生体系建设三年行动计划第五轮重点学科(GWV-10.1-XK13)

Establishment and preliminary evaluation of a visualized detection technique for Schistosoma mansoni nucleic acid based on recombinase polymerase amplification

WANG Li-ping(), LV Chao, QIN Zhi-qiang, XU Jing, DENG Wang-ping()

National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research); NHC Key Laboratory of Parasite and Vector Biology; WHO Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Shanghai 200025, China

Received:2021-07-26 Revised:2021-12-13 Online:2022-06-30 Published:2022-07-06
Contact: DENG Wang-ping
Supported by:
Special Clinical Research Project of Health Industry of Shanghai Municipal Health Commission(202040053);the National Science and Technology Major Project(2018ZX10101002-002);National Natural Science Foundation of China(82073619);the Fifth Round Key Disciplines of Three-year Action Plan of Public Health System Construction in Shanghai(GWV-10.1-XK13)

摘要/Abstract

摘要：

目的结合重组酶聚合酶扩增技术（RPA）和侧流层析试纸条（LFD）,建立一种快速、便捷的曼氏血吸虫核酸可视化检测方法,并初步评价其检测效能。方法以曼氏血吸虫细胞色素c氧化酶亚基1（SmCOX1）基因为靶序列,利用Primer Primer 5软件结合手工辅助,设计特异性引物和探针并进行筛选,建立曼氏血吸虫核酸LFD-RPA检测方法。制备不同浓度（1 ng/μl、100 pg/μl、10 pg/μl、1 pg/μl、100 fg/μl、10 fg/μl、1 fg/μl、0.1 fg/μl）的曼氏血吸虫成虫基因组DNA和含有不同拷贝数浓度（10⁵、10⁴、10³、10²、10¹、10⁰、10^-1拷贝/μl）的SmCox1重组质粒DNA,评价所建立方法的敏感度。以曼氏血吸虫成虫、日本血吸虫成虫、埃及血吸虫虫卵、感染曼氏血吸虫双脐螺（阳性双脐螺）和阴性双脐螺、感染日本血吸虫钉螺（阳性钉螺）和阴性钉螺、华支睾吸虫成虫、大片形吸虫成虫、卫氏并殖吸虫成虫基因组DNA为模板,评价所建立方法的特异性。将30只雌性BALB/c小鼠随机分为40尾感染组、80尾感染组和健康对照组,每组10只,建立感染小鼠模型,提取感染后1~8周的小鼠粪样和血样DNA,评价LFD-RPA方法检测曼氏血吸虫早期感染的效能。结果以SmCox1基因片段为靶标建立的曼氏血吸虫LFD-RPA检测方法以39 ℃、20 min为最佳反应温度和时间。敏感度检测结果显示,SmCox1-LFD-RPA方法对曼氏血吸虫成虫基因组、SmCox1重组质粒的检出限分别为10 fg/μl和10拷贝/μl。特异性检测结果显示,SmCox1-LFD-RPA方法仅对曼氏血吸虫和阳性双脐螺DNA特异,与其他吸虫DNA无交叉反应。SmCox1-LFD-RPA方法对感染小鼠1~8周血样及粪样DNA的检测结果显示,40尾组小鼠自感染后3周开始出现阳性条带,80尾组小鼠自感染后1周开始出现阳性条带,且条带颜色随感染时间延长逐渐加深。结论建立了曼氏血吸虫LFD-RPA检测方法,其检出限低、特异性强,操作简单快捷。

关键词: 曼氏血吸虫, 重组酶聚合酶,等温扩增, 可视化, 侧流试纸条

Abstract:

Objective To establish and preliminarily evaluate a fast, convenient and visualized method for detection of Schistosoma mansoni nucleic acid by combining the recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD). Methods Selecting the cytochrome c oxidase subunit 1 (COX1) gene of S. mansoni as the target sequence, specific primers and probes were designed using Primer Primer5 with manual assistance and screened to establish a LFD-RPA method for detection of S. mansoni. The sensitivity of the established method was evaluated by detecting the genomic DNA of adult S. mansoni at different concentrations (1 ng/μl, 100 pg/μl, 10 pg/μl, 1 pg/μl, 100 fg/μl, 10 fg/μl, 1 fg/μl, 0.1 fg/μl) and the recombinant SmCox1 plasmids with different copies (10⁵, 10⁴, 10³, 10², 10¹, 10⁰, 10^-1 copies/μl). The specificity of LFD-RPA was evaluated by detecting the genomic DNA of S. japonicum adult worms, S. haematobium eggs, S. mansoni infected and non-infected Biomphalaria spp., S. japonicum infected and non-infected Oncomelania hupensis and other trematodes. Thirty female BALB/c mice were randomly divided into low infection group (40 cercariae per mouse), high infection group (80 cercariae per mouse) and control group (no infection) to establish the mouse infection model. The DNA was extracted from the feces and serum samples from the mice at different time points post infection (1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks) and detected by LFD-RPA to evaluate its effectiveness in early detection of S. mansoni infection. Results The specific band of S. mansoni could be detected by the established SmCox1-LFD-RPA method after 20 min of reaction at 39 ℃. The detectable limits of LFD-RPA method for S. mansoni were 10 fg/μl for the genomic DNA of adult worms and 10 copies/μl for SmCox1 recombinant plasmid, respectively. The established SmCox1-LFD-RPA method was specific to the DNA of S. mansoni and S. mansoni infected Biomphalaria spp., no cross-reaction with other flukes were observed. When detecting DNA samples extracted from mouse blood and feces from the 40 cercariae group 1 week to 8 weeks post infection, SmCox1-LFD-RPA presented positive reaction since 3 weeks post infection. While for the mice of the 80 cercariae group, positive band began to appear from 1-week post infection. The color of the band was gradually deepened with the infection time. Conclusion A LFD-RPA based visualized rapid detection method for S. mansoni nucleic acid was developed, with higher sensitivity, specificity, rapidity and easy-to-use.

Key words: Schistosoma mansoni, Recombinase polymerase, Isothermal amplification, Visualization, Lateral flow dipstick

中图分类号:

R383.24

王丽萍, 吕超, 秦志强, 许静, 邓王平. 基于重组酶聚合酶扩增的曼氏血吸虫核酸可视化检测技术的建立及初步评价[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(3): 337-343.

WANG Li-ping, LV Chao, QIN Zhi-qiang, XU Jing, DENG Wang-ping. Establishment and preliminary evaluation of a visualized detection technique for Schistosoma mansoni nucleic acid based on recombinase polymerase amplification[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(3): 337-343.

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参考文献 31

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基于重组酶聚合酶扩增的曼氏血吸虫核酸可视化检测技术的建立及初步评价

Establishment and preliminary evaluation of a visualized detection technique for Schistosoma mansoni nucleic acid based on recombinase polymerase amplification

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