一段检测锥虫特异性核酸靶序列的筛选及评价  

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

作　　者：洪清华 王韵丞 李鸿雁 张瑞祥[1] 李健[1] 陈木新[2] 刘骁 殷明波[1] 王敬文 胡薇[1,2] HONG Qing-hua;WANG Yun-cheng;LI I-Iong-yan;ZHANG Rui-xiang;LI Jian;CHEN Mu-xin;LIU Xiao;YIN Ming-bo;WANG Jing-wenl;HU Wei(School of Life Science, Fudan University, Shanghai 200438, China;National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention;WHO Collaborating Centre for Tropical Disease;National Center for International Research on Tropical Diseases, Ministry of Science and Technolog;Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai 200025, China;3 JinFuKang Biotechnology （Limited） Company, Shanghai 200120, Chin)

机构地区：[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

出　　处：《中国寄生虫学与寄生虫病杂志》2017年第6期588-593,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：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)

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。Objective To screen and identify a specific nucleic acid target sequence for detecting Trypanosoma based on 60S ribosomal protein L44 （60S RPIA4） gene sequence of Trypcmosoma 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 RP1A4 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/Ixl, 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 PI： 5＇-CCTGATGAAAGGTGCAATG-3＇; primer P2： 5＇-CGTTTTCGCCTCGCCTTGTGGA-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 Bobesia 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.

关 键 词：锥虫属 序列比对 PCR检测 靶序列 

分 类 号：R382.23[医药卫生—医学寄生虫学]