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

doi:10.12140/j.issn.1000-7423.2024.03.019

Abstract

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

CLC Number:

R382.221

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.

Figures/Tables 4

References 23

[1]	World Health Organization. Control of the leishmaniasis[R]. Geneva: WHO, 2010: 5-12.
[2]	Bates PA. Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies[J]. Int J Parasitol, 2007, 37(10): 1097-1106. doi: 10.1016/j.ijpara.2007.04.003 pmid: 17517415
[3]	Wang ZJ, Xiong GH, Guan LR. Epidemiology and control achievements of kala-azar in New China[J]. Chin J Epidemiol, 2000(1): 51-54. (in Chinese) pmid: 15459981
	(王兆俊, 熊光华, 管立人. 新中国黑热病流行病学与防治成就[J]. 中华流行病学杂志, 2000(1): 51-54.)
[4]	Zhou ZB, Li YY, Zhang Y, et al. Prevalence of visceral leishmaniasis in China during 2015-2018[J]. Chin J Parasitol Parasit Dis, 2020, 38(3): 339-345. (in Chinese)
	(周正斌, 李元元, 张仪, 等. 2015—2018年我国内脏利什曼病疫情分析[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(3): 339-345.) doi: 10.12140/j.issn.1000-7423.2020.03.013
[5]	Zhou ZB, Pan GQ, Li YY, et al. Prevalence of visceral leishmaniasis in China in 2021[J]. Chin J Parasitol Parasit Dis, 2023, 41(2): 149-155. (in Chinese)
	(周正斌, 潘改芹, 李元元, 等. 2021年我国内脏利什曼病疫情分析[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(2): 149-155.) doi: 10.12140/j.issn.1000-7423.2023.02.004
[6]	Li YY, Zhou ZB, Yang LM, et al. Epidemiological characteristics of visceral leishmaniasis in China in 2022[J]. Chin J Parasitol Parasit Dis, 2023, 41(6): 669-676. (in Chinese)
	(李元元, 周正斌, 杨丽敏, 等. 2022年全国内脏利什曼病疫情特征分析[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(6): 669-676.) doi: 10.12140/j.issn.1000-7423.2023.06.002
[7]	Zhou ZB, Li YY, Zhang Y, et al. Prevalence of visceral leishmaniasis in China in 2019[J]. Chin J Parasitol Parasit Dis, 2020, 38(5): 602-607. (in Chinese)
	(周正斌, 李元元, 张仪, 等. 2019年我国内脏利什曼病疫情分析[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(5): 602-607.) doi: 10.12140/j.issn.1000-7423.2020.05.012
[8]	Zhou ZB, Li YY, Zhang Y, et al. Prevalence of visceral leishmaniasis in China in 2018[J]. Chin J Parasitol Parasit Dis, 2020, 38(2): 175-180, 187. (in Chinese)
	(周正斌, 李元元, 张仪, 等. 2018年全国内脏利什曼病疫情分析[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(2): 175-180, 187.) doi: 10.12140/j.issn.1000-7423.2020.02.007
[9]	Zhou ZB, Lyu S, Zhang Y, et al. Visceral leishmaniasis-China, 2015-2019[J]. China CDC Wkly, 2020, 2(33): 625-628.
[10]	Li LJ, Ren H. Infectious diseases[M]. 8th ed. Beijing: People’s Medical Publishing House, 2013: 287. (in Chinese)
	(李兰娟, 任红. 传染病学[M]. 8版. 北京: 人民卫生出版社, 2013: 287.)
[11]	Zuo XP, Yi MM, Kai SE, et al. Application of rK39 dipstick on diagnosis and epidemiological survey of kala-azar in Xinjiang, China[J]. J Trop Dis Parasitol, 2007, 5(1): 19-22. (in Chinese)
	(左新平, 伊马木, 开赛尔, 等. rK39免疫层析试条在新疆黑热病诊断和流行病学调查中的应用[J]. 热带病与寄生虫学, 2007, 5(1): 19-22.)
[12]	Feng XP, Gao CH, Shi F, et al. Evaluation of colloid gold labeled immunochromatographic strip test for rapid detection of canine leishmanial infection in China[J]. Chin J Zoonoses, 2016, 32(1): 24-27. (in Chinese)
	(冯晓平, 高春花, 石锋, 等. 检测特异抗体胶体金免疫层析试条诊断内脏利什曼病患者现场评价[J]. 中国人兽共患病学报, 2016, 32(1): 24-27.)
[13]	Shi F, Ding D, Gao CH, et al. Establishment and evaluation of a colloidal gold labeled immunochromatographic strip test based on the Leishmania K26 recombinant antigen for rapid detection of visceral leishmaniasis[J]. Chin J Zoonoses, 2023, 39(10): 981-985. (in Chinese)
	(石锋, 丁丹, 高春花, 等. 基于利什曼原虫K26重组抗原检测黑热病特异抗体的免疫层析试条方法的建立与效果评价[J]. 中国人兽共患病学报, 2023, 39(10): 981-985.)
[14]	Shi YD, Zhao ZF, Zhu D, et al. Principle of LAMP (loop mediated isothermal amplification) and its application in parasite detection[J]. Chin J Zoonoses, 2011, 27(10): 935-939. (in Chinese)
	(史亚东, 赵子方, 朱丹, 等. LAMP(环介导等温扩增)的原理及在寄生虫检测上的应用[J]. 中国人兽共患病学报, 2011, 27(10): 935-939.)
[15]	Ma L, Zhang Z, Wang AL, et al. Establishment and application of real-time fluorescence quantitative PCR for detection of Leishmania[J]. Chin J Parasitol Parasit Dis, 2021, 39(4): 548-552. (in Chinese)
	(马琳, 张铮, 王安礼, 等. 检测利什曼原虫的实时荧光定量PCR的建立及应用[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(4): 548-552.) doi: 10.12140/j.issn.1000-7423.2021.04.021
[16]	Wang JY, Gao CH. Interpretation of diagnostic criteria for kala-azar[J]. Chin J Schisto Control, 2017, 29(5): 541-543. (in Chinese)
	(汪俊云, 高春花. 《黑热病诊断标准》解读[J]. 中国血吸虫病防治杂志, 2017, 29(5): 541-543.)
[17]	Liu JX, Wang JY. Applications of molecular techniques in classification and identification of Leishmania[J]. Chin J Parasitol Parasit Dis, 2020, 38(4): 508-512, 517. (in Chinese)
	(刘建秀, 汪俊云. 分子技术在利什曼原虫分类与鉴定上的应用[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(4): 508-512, 517.) doi: 10.12140/j.issn.1000-7423.2020.04.019
[18]	Zhang CY, Huang YX, Yuan Y, et al. Heat-shock protein 70 gene sequences for the phylogeny of Chinese Leishmania isolates[J]. Chin J Zoonoses, 2014, 30(2): 163-168, 190. (in Chinese)
	(张春莹, 黄玉霞, 袁余, 等. 热休克蛋白70(hsp70)基因对利什曼原虫中国分离株的系统发育分析基因对利什曼原虫中国分离株的系统发育分析[J]. 中国人兽共患病学报, 2014, 30(2): 163-168, 190.)
[19]	Akhoundi M, Downing T, Votýpka J, et al. Leishmania infections: molecular targets and diagnosis[J]. Mol Aspects Med, 2017, 57: 1-29.
[20]	Tian Y, Chen JP, Hu XS. Cloning and sequence analysis of the ribosomal DNA ITS gene of Leishmania donovani isolates from hill foci of China[J]. Chin J Parasitol Parasit Dis, 2004, 22(5): 294-296. (in Chinese)
	(田玉, 陈建平, 胡孝素. 山丘疫区杜氏利什曼原虫核糖体基因内转录间隔区的克隆及序列分析[J]. 中国寄生虫学与寄生虫病杂志, 2004, 22(5): 294-296.)
[21]	Abbasi I, Kirstein OD, Hailu A, et al. Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples[J]. Acta Trop, 2016, 162: 20-26.
[22]	Xu S. Study and evaluation of a novel visualized loop-mediated isothermal amplification (LAMP) for Plasmodium detection[D]. Wuxi: Jiangsu Institue of Parasitic Disease, 2012: 25-28. (in Chinese)
	(徐岁. 一种新的可视化环介导等温扩增技术检测疟原虫的研究及其应用评价[D]. 无锡: 江苏省血吸虫病防治研究所, 2012: 25-28.)
[23]	Luo ZW, Zhou ZB, Gong YF, et al. Current status and challenges of visceral leishmaniasis in China[J]. Chin J Parasitol Parasit Dis, 2022, 40(2): 146-152. (in Chinese)
	(罗卓韦, 周正斌, 公衍峰, 等. 我国内脏利什曼病的流行现状和防控挑战[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(2): 146-152.) doi: 10.12140/j.issn.1000-7423.2022.02.003

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