一段检测锥虫特异性核酸靶序列的筛选及评价

中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (6): 588-593.

一段检测锥虫特异性核酸靶序列的筛选及评价

洪清华¹, 王韵丞¹, 李鸿雁¹, 张瑞祥¹, 李健¹, 陈木新², 刘骁³, 殷明波¹, 王敬文¹, 胡薇^1,^2,^*()

1 复旦大学生命科学学院,上海 200438
2 中国疾病预防控制中心寄生虫病预防控制所,世界卫生组织热带病合作中心,科技部国家级热带病国际联合研究中心,卫生部寄生虫病原与媒介生物学重点实验室,上海 200025
3 金弗康生物科技（有限）公司,上海 200120

收稿日期:2017-09-07 出版日期:2017-12-30 发布日期:2018-01-10
通讯作者: 胡薇
基金资助:
闵行区产学研合作计划项目（No.2014MH113）;国家寄生虫种质资源共享服务平台（平台-TDRC-22）

Screening and evaluation of a specific nucleic acid target sequence for detection of trypanosome

Qing-hua HONG¹, Yun-cheng WANG¹, Hong-yan LI¹, Rui-xiang ZHANG¹, Jian LI¹, Mu-xin CHEN², Xiao LIU³, Ming-bo YIN¹, Jing-wen WANG¹, Wei HU^1,^2,^*()

1 School of Life Science, Fudan University, Shanghai 200438, China
2 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology; Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai 200025, China
3 JinFuKang Biotechnology (Limited) Company, Shanghai 200120, China

Received:2017-09-07 Online:2017-12-30 Published:2018-01-10
Contact: Wei HU

摘要/Abstract

摘要：

目的以60S核糖体蛋白L44（60S RPL44）基因为靶基因,筛选出一段可特异性检测锥虫属（Trypanosoma）原虫的核酸靶序列,并对其敏感度和特异性进行初步评价。方法将布氏锥虫指名亚种（T.brucei brucei）的60S RPL44基因序列与公共数据库中田鼠巴贝虫（Babesia microti）、杜氏利什曼原虫（Leishmania donovani）、间日疟原虫（Plasmodium vivax）、恶性疟原虫（P.falciparum）和刚地弓形虫（Toxoplasma gondii）等24种非锥虫属常见的血源性原虫,以及8种锥虫属内其他种的同源序列进行比对。同时,利用Primer Premier 6对布氏锥虫指名亚种的60S RPL44基因序列设计多对引物,结合序列比对结果和各引物评价情况,从中筛选出一对评价情况良好且理论上仅能扩增锥虫属靶序列的引物。利用该对引物检测布氏锥虫指名亚种、布氏锥虫罗德西亚种（T.b.rhodesiense）、刚果锥虫（T.congolense）、伊氏锥虫（T.evansi）以及上述5种其他属原虫,评价其特异性。采用梯度稀释（10 ng/μl、1 ng/μl、100 pg/μl、10 pg/μl、1 pg/μl）的伊氏锥虫基因组DNA为模板评价其敏感度。结果选出一段基于60S RPL44的特异性序列,针对该序列的扩增引物为P1：5′-CCTGATGAAAGGTGCAATG-3′,P2：5′-CGTTTTCGCCTTCTTGTGGA-3′。该引物扩增布氏锥虫指名亚种、布氏锥虫罗德西亚种、刚果锥虫、伊氏锥虫的DNA均为阳性,扩增片段长156 bp;扩增田鼠巴贝虫、杜氏利什曼原虫、间日疟原虫、恶性疟原虫、刚地弓形虫等非锥虫属原虫均为阴性;该引物检测伊氏锥虫DNA的敏感度为10 pg/μl。结论筛选获得一段敏感度较高、特异性强的检测锥虫属原虫的核酸靶标序列。

关键词: 锥虫属, 序列比对, PCR检测, 靶序列

Abstract:

Objective To screen and identify a specific nucleic acid target sequence for detecting Trypanosoma based on 60S ribosomal protein L44 (60S RPL44) gene sequence of Trypanosoma brucei brucei, and evaluate its sensitivity and specificity.Methods The 60S RPL44 sequence of T.b.brucei was blasted with its homologous sequences from Babesia microti, Leishmania donovani, Plasmodium vivax, P.falciparum, Toxoplasma gondii, as well as the other genera of protozoan and trypanosome species, on a public database.Meanwhile, several pairs of primers for 60S RPL44 of T.b.brucei were designed using Primer Premier 6, of which one pair was selected for screening specific nucleic acid target sequence of Trypanosoma according to the sequences alignment and primers score result.Furthermore, the specificity of the primers was evaluated among DNA from T.b.brucei, T.b.rhodesiense, T.congolense, T.evansi and the above-mentioned other five genera of protozoan parasites.The sensitivity was assessed using gradient concentrations of T.evansi genomic DNA (10 ng/μl, 1 ng/μl, 100 pg/μl, 10 pg/μl and 1 pg/μl).Results A pair of primers for detecting Trypanosoma based on T.b.brucei 60S RPL44 sequence were selected: primer P1: 5′-CCTGATGAAAGGTGCAATG-3′; primer P2: 5′-CGTTTTCGCCTTCTTGTGGA-3′.This pair of primers could amplify T.b.brucei, T.brucei rhodesiense, T.congolense and T.evansi DNA with a product of 156 bp, rather than Babesia microti, Leishmania donovani, Plasmodium vivax, P.falciparum and T.gondii.The sensitivity in detecting T.evansi genomic DNA was 10 pg/μl.Conclusion A highly sensitive and specific nucleic acid target for detection of trypanosome is found.

Key words: Trypanosoma, Sequences alignment, PCR detection, Target sequence

中图分类号:

R382.23

洪清华, 王韵丞, 李鸿雁, 张瑞祥, 李健, 陈木新, 刘骁, 殷明波, 王敬文, 胡薇. 一段检测锥虫特异性核酸靶序列的筛选及评价[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(6): 588-593.

Qing-hua HONG, Yun-cheng WANG, Hong-yan LI, Rui-xiang ZHANG, Jian LI, Mu-xin CHEN, Xiao LIU, Ming-bo YIN, Jing-wen WANG, Wei HU. Screening and evaluation of a specific nucleic acid target sequence for detection of trypanosome[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(6): 588-593.

图/表 6

表1

表2

图1

图2

图3

图4

参考文献 19

[1]	Fèvre EM, Picozzi K, Jannin J, et al. Human African trypanosomiasis: epidemiology and control[J].Adv Parasitol, 2006, 61: 167-221.
[2]	李雍龙.人体寄生虫学[M].7版.北京:人民卫生出版社, 2008: 46-48.
[3]	Antinori S, Galimberti L, Bianco R, et al. Chagas disease in Europe: a review for the internist in the globalized world[J].Eur J Intern Med, 2017, 43: 6-15.
[4]	Ersfeld K.Genomes and genome projects of protozoan parasites[J].Curr Issues Mol Biol, 2003, 5(3): 61-74.
[5]	陈木新, 陈家旭.基因芯片技术在人体锥虫生物学特性研究方面的进展[J].中国寄生虫学与寄生虫病杂志, 2016, 34(4): 377-381.
[6]	孙懿, 黄韦华, 牛紫光, 等.1例输入性非洲锥虫病的病原学鉴定[J].中国寄生虫学与寄生虫病杂志, 2016, 34(4): 350-354.
[7]	杨金伟.我国现输入性罗得西亚锥虫病[N].健康报, 2017-09-01(2).
[8]	Radwanska M, Claes F, Magez S, et al. Novel primer sequences for polymerase chain reaction-based detection of Trypanosoma brucei gambiense[J].Am J Trop Med Hyg, 2002, 67(3): 289-295.
[9]	Radwanska M, Chamekh M, Vanhamme L, et al. The serum resistance-associated gene as a diagnostic tool for the detection of Trypanosoma brucei rhodesiense[J].Am J Trop Med Hyg, 2002, 67(6): 684-690.
[10]	Picozzi K, Carrington M, Welburn SC.A multiplex PCR that discriminates between Trypanosoma brucei brucei and zoonotic T.b.rhodesiense[J].Exp Parasitol, 2008, 118(1): 41-46.
[11]	Njiru ZK, Constantine CC, Guya S, et al. The use of ITS1 rDNA PCR in detecting pathogenic African trypanosomes[J].Parasitol Res, 2005, 95(3): 186-192.
[12]	Berriman M, Ghedin E, Hertz-Fowler C, et al. The genome of the African trypanosome Trypanosoma brucei[J].Science, 2005, 309(5733): 416-422.
[13]	El-Sayed NM, Myler PJ, Bartholomeu DC, et al. The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease[J].Science, 2005, 309(5733): 409-415.
[14]	Jackson AP, Berry A, Aslett M, et al. Antigenic diversity is generated by distinct evolutionary mechanisms in African trypanosome species[J].Proc Natl Acad Sci USA, 2012, 109(9): 3416-3421.
[15]	路媛媛, 彭恒, 朱淮民, 等.我国广西食蟹猴养殖场2种血液寄生原虫感染调查[J].中国血吸虫病防治杂志, 2016, 28(2): 141-145.
[16]	刘志华, 杨谦.球毛壳菌60S核糖体蛋白L10a基因克隆与特性分析[J].微生物学通报, 2006, 33(1): 24-28.
[17]	潘湛清, 姚琼, 唐斌, 等.甜菜夜蛾三个60S核糖体蛋白基因的获得与序列分析[J].环境昆虫学报, 2008, 30(4): 310-317.
[18]	Savage AF, Kolev NG, Franklin JB, et al. Transcriptome profiling of Trypanosoma brucei development in the tsetse fly vector Glossina morsitans[J].PLoS One, 2016, 11(12): e0168877.
[19]	Probst P, Stromberg E, Ghalib HW, et al. Identification and characterization of T cell-stimulating antigens from Leishmania by CD4 T cell expression cloning[J].J Immunol, 2001, 166(1): 498-505.

物种 Species	GenBank/EuPathDB 登录号GenBank /EuPathDB ID
巴西利什曼原虫 L.braziliensis	LBRM2903_330043800
巴拿马利什曼原虫 L.panamensis	LPAL13_330042000
豚鼠利什曼原虫 L.enriettii	LENLEM3045_330042300
蜥蜴利什曼原虫 L.tarentolae	LtaP33.3490
硕大利什曼原虫 L.major	LMJLV39_330045600
热带利什曼原虫 L.tropica	LTRL590_130021900
埃塞尔比亚利什曼原虫 L.aethiopica	LAEL147_000189300
胎儿利什曼原虫 L.infantum	LinJ.33.3380
杜氏利什曼原虫 L.donovani	XM_003864113.1
阿拉伯利什曼原虫 L.arabica	LARLEM1108_130021200
墨西哥利什曼原虫 L.mexicana	LmxM.32.3230
砂鼠利什曼原虫 L.gerbilli	LGELEM452_330043100
图兰利什曼原虫 L.turanica	LTULEM423_330043200
田鼠巴贝虫 B.microti	XM_012792916.1
间日疟原虫 P.vivax	XM_001613016.1
赖氏疟原虫 P.reichenowi	PRCDC_0303700
恶性疟原虫 P.falciparum	XM_001351084.1
加蓬疟原虫 P.gaboni	PGSY75_0304400
残遗疟原虫 P.relictum	PRELSG_0831300
鸡疟原虫 P.gallinaceum	PGAL8A_00003700
三日疟原虫 P.malariae	PmUG01_08051800
卵形疟原虫 P.ovale curtisi	PocGH01_08043600
刚地弓形虫 T.gondii	XM_002367604.2
双芽巴贝虫 B.bigemina	BBBOND_0200890

物种 Species	GenBank /EuPathDB 登录号GenBank /EuPathDB ID
活跃锥虫 T.vivax	TvY486_1103320
格氏锥虫 T.grayi	DQ04_03581020
克氏锥虫 T.cruzi	XM_806327.1
让氏锥虫 T.rangeli	TRSC58_05400
刚果锥虫 T.congolense	TcIL3000.11.3000
布氏锥虫冈比亚亚种 T.b.gambiense	XM_011773651.1
伊氏锥虫 T.evansi	TevSTIB805.2.3460