利什曼原虫环介导等温扩增检测方法的建立及应用

doi:10.12140/j.issn.1000-7423.2024.03.019

中国寄生虫学与寄生虫病杂志 ›› 2024, Vol. 42 ›› Issue (3): 413-417.doi: 10.12140/j.issn.1000-7423.2024.03.019

利什曼原虫环介导等温扩增检测方法的建立及应用

贾西帅(), 周水茂^*(), 罗华堂, 刘聪, 王帅, 徐文秀

武汉市疾病预防控制中心，湖北武汉 430015

收稿日期:2024-01-08 修回日期:2024-03-01 出版日期:2024-06-30 发布日期:2024-07-16
通讯作者: *周水茂（1967—），男，本科，主任技师，从事寄生虫病防治和诊断工作。E-mail：867494784@qq.com
作者简介:贾西帅（1988—），男，硕士，主管技师，从事寄生虫病防治工作。E-mail：578261805@qq.com
基金资助:
武汉市卫生健康委医学科研项目(WG17B04);武汉市卫生健康委医学科研项目(WG18Q05);武汉市卫生健康委第七批中青年医学骨干人才培养工程

Establishment and application of loop-mediated isothermal amplification for detection of Leishmania

JIA Xishuai(), ZHOU Shuimao^*(), LUO Huatang, LIU Cong, WANG Shuai, XU Wenxiu

Wuhan Center for Disease Prevention and Control, Wuhan 430015, Hubei, China

Received:2024-01-08 Revised:2024-03-01 Online:2024-06-30 Published:2024-07-16
Supported by:
Wuhan Municipal Health and Family Planning Commission Medical Research Project(WG17B04);Wuhan Municipal Health and Family Planning Commission Medical Research Project(WG18Q05);Seventh Batch of Young and Middle-aged Medical Backbone Talent Training Projects of Wuhan Municipal Health and Health Commission

摘要/Abstract

摘要：

建立一种基于环介导等温扩增（LAMP）检测利什曼原虫的方法，为内脏利什曼病防治提供技术支持。根据利什曼原虫动基体5.8S核糖体RNA（GenBank：OP829811）序列，设计合成LAMP扩增特异性引物，建立LAMP法。用LAMP法检测恶性疟原虫、间日疟原虫、三日疟原虫、卵形疟原虫感染者和健康人血样DNA，以及日本血吸虫、刚地弓形虫和杜氏利什曼原虫前鞭毛体DNA，评价LAMP法的特异性评价特异性；杜氏利什曼原虫前鞭毛体DNA稀释为1 ng/μl、100 pg/μl、10 pg/μl、1 pg/μl、100 fg/μl、10 fg/μl、1 fg/μl，确定LAMP法的最低检测限及有、无钙黄绿素对检测限的影响。用建立的LAMP法和实时荧光定量PCR（qPCR）检测不明原因发热患者和健康人血样；取阳性患者骨髓涂片吉氏染色后镜检，查找利什曼原虫无鞭毛体。建立的LAMP法可检出杜氏利什曼原虫DNA，反应结果呈绿色；检测4种疟原虫感染者和健康人血样以及日本血吸虫、刚地弓形虫DNA的结果均为阴性，呈橙色。LAMP法检测健康人血样46份，均呈阴性，无交叉反应，特异性高。65 ℃反应60 min，未加和加钙黄绿素的检出限分别为1 pg/μl和1 ng/μl，浊度速率峰值均值分别为0.194、0.120，加钙黄绿素后出现浊度时间较未加钙黄绿素平均延迟23.6 min。LAMP法和qPCR法检测发热患者血样67份的结果一致，均检出相同的2份阳性，2份阳性血样对应的骨髓涂片镜检均查见利什曼原虫无鞭毛体。本研究建立的了检测利什曼原虫的LAMP法操作简便、灵敏度和特异性均较高，检测结果可视，具有较好的推广应用价值。

关键词: 利什曼原虫, 环介导等温扩增, 可视化

Abstract:

A method for detecting Leishmania based on loop-mediated isothermal amplification (LAMP) was established to support for the prevention and treatment of visceral leishmaniasis. According to the sequence of the kinetoplast 5.8S ribosomal RNA of Leishmania (GenBank: OP829811), specific primers for LAMP were designed and synthesized, and the LAMP method was established. The DNA of blood samples from patients infected with Plasmodium falciparum, P. vivax, P. malariae, P. ovale and healthy individuals, as well as the promastigote DNA of Schistosoma japonicum, Toxoplasma gondii, and L. donovani, were detected by the LAMP method to evaluate the specificity. The promastigote DNA of L. donovani was diluted to 1 ng/μl, 100 pg/μl, 10 pg/μl, 1 pg/μl, 100 fg/μl, 10 fg/μl, and 1 fg/μl to determine the minimum detection limit of the LAMP method and the effect of the presence or absence of calcein on the detection limit. LAMP method and real-time fluorescence quantitative PCR (qPCR) were used to detect blood samples from patients with unexplained fever and healthy people. Bone marrow smears of positive patients were stained by Giemsa to find Leishmania amastigotes. The established LAMP method could detect L. donovani DNA, and the reaction results were green; the results of detecting blood samples of patients infected with 4 species of Plasmodium and healthy people, as well as S. japonicum and T. gondii DNA were all negative, and orange. A total of 46 healthy blood samples were tested by the LAMP method, all of which were negative, without cross-reaction, and with high specificity. After reaction at 65 ℃ for 60 min, the detection limits without and with calcein were 1 pg/μl and 1 ng/μl, respectively. The average peak values of turbidity rates without and with calcein were 0.194 and 0.120, and the time of turbidity after adding calcein was delayed by an average of 23.6 min compared with that without calcein. The LAMP method and qPCR method were used to detect 67 blood samples from patients with fever, of which the same 2 samples were positive. The bone marrow smears corresponding to the same two positive blood samples were examined under a microscope and Leishmania amastigotes were found. The LAMP method for detecting Leishmania is easy to operate, has high sensitivity and specificity, and the test results are visible, which has good promotion and application value.

Key words: Leishmania, Loop-mediated isothermal amplification, Visualization

中图分类号:

R382.221

贾西帅, 周水茂, 罗华堂, 刘聪, 王帅, 徐文秀. 利什曼原虫环介导等温扩增检测方法的建立及应用[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(3): 413-417.

JIA Xishuai, ZHOU Shuimao, LUO Huatang, LIU Cong, WANG Shuai, XU Wenxiu. Establishment and application of loop-mediated isothermal amplification for detection of Leishmania[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2024, 42(3): 413-417.

图/表 4

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引物	序列（5'→3'）
F3	GTCGCGATGGATGACTTG
B3	ATGCAGAAGAGAGGAGGC
FIP	TTGCGTTCAAAGATTCGGTAATTGATATTTCGTTGAAGAACGC-AGT
BIP	GCGCATGGGAGAAGCTCTATTTTTTTGTTCGACACTGAGAA
LF	GCAATTGATACCACTTATCGCACT
LB	ATCCCCGTGCATGCCATA

利什曼原虫环介导等温扩增检测方法的建立及应用

Establishment and application of loop-mediated isothermal amplification for detection of Leishmania

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