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

Abstract

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

CLC Number:

R382.23

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.

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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