Simultaneous detection and differentiation of Theileria and Babesia parasites infecting small ruminants by reverse line blotting
Simultaneous detection and differentiation of Theileria and Babesia parasites infecting small ruminants by reverse line blotting
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DOI：
10.1007/s00436-003-0980-9
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发表时间：
2004-02-01
影响因子：
2
通讯作者：
Ahmed, JS
中科院分区：
文献类型：
--
作者：
Schnittger, L;Yin, H;Ahmed, JS
在绵羊/山羊旋毛虫物种的小核糖体RNA基因的高变区4(V4区)内发现了特征序列特征，这些物种包括泰勒氏锥虫、绵羊锥虫、分离锥虫、绵羊巴贝西亚、莫塔西、粗毛芽孢杆菌[包括B.crassa(伊朗)和B.crassa(土耳其)]和几个新物种：Theileria sp.1(中国)，泰勒虫(Theileria sp.)2(中国)和巴贝斯虫(Babesia sp.)(中国)，由巴贝斯虫[Babesia sp.(Lintan)和巴贝斯虫Babesia sp.(宁县)]如前所述。基于已查明的基因变异，建立了一种反向线杂交(RLB)分析方法，可以直接、同时、高度特异和灵敏地鉴定几乎所有目前已知的绵羊/山羊旋体物种。所有的探针只与它们各自的靶序列结合，因此没有观察到交叉反应，从而对单个菌株、物种或组进行了明确的识别。以绵羊和山羊的基因组DNA为对照时，未观察到信号，表明这些信号是由于被调查样品中存在寄生虫DNA所致。此外，通过重新扩增聚合酶链式反应产物(套式聚合酶链式反应)，可以显著提高RLB的灵敏度，以检测至少10(-12)%的寄生虫血症水平，从而显著增加识别携带者动物的可能性。
Characteristic sequence signatures were identified within the hypervariable region 4 (V4 region) of the small ribosomal RNA gene of ovine/caprine piroplasm species including Theileria lestoquardi, T. ovis , T. separata , Babesia ovis , B. motasi , B. crassa [comprising strains B. crassa (Iran) and B. crassa (Turkey)] and several novel species: Theileria sp. 1 (China), Theileria sp. 2 (China) and Babesia sp. (China), [comprising strain Babesia sp. (Lintan), and Babesia sp. (Ningxian)] as defined previously. Based on the ascertained gene variations a reverse line blotting (RLB) assay was developed enabling direct, concurrent, highly specific and sensitive identification of virtually all presently known ovine/caprine piroplasm species. All probes bound to their respective target sequence only, therefore, no cross-reaction was observed resulting in clear recognition of either individual strains, species or groups. No signal was observed when ovine and caprine genomic DNA was used as the control, demonstrating that the signals are due to the presence of parasite DNA in investigated samples. Furthermore, the sensitivity of RLB could be considerably enhanced to detect a parasitemia level of at least 10(-12)% by reamplification of PCR products (nested PCR) thereby substantially increasing the possibility of identifying carrier animals.