Aradaib Imadeldin E, Mohamed Mohamed E H, Abdalla Tamadour M, Sarr Joesph, Abdalla Mohamed A, Yousof Mohamed A M, Hassan Yahia A, Karrar Abdel Rahim E
Department of Medicine, Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Khartoum, Khartoum North, Sudan.
Vet Microbiol. 2005 Dec 20;111(3-4):145-50. doi: 10.1016/j.vetmic.2005.09.014. Epub 2005 Nov 8.
The diagnostic potential of RT-PCR for detection of bluetongue virus (BTV) ribonucleic acid (RNA) sequence in cell culture and tissue samples from infected ruminants from United States, Sudan, South Africa and Senegal, was evaluated. The non structural protein 1 (NS1) gene of North American BTV serotype 11 was targeted for PCR amplification. The United States BTV serotypes 2, 10, 11, 13 and 17 and the Sudanese BTV serotypes 1, 2, 4 and 16 and BTV serotype 4 from South Africa and BTV serotype 2 from Senegal were studied. RNAs from all BTV field isolates used in this study, propagated in cell cultures, were detected by the described RT-PCR-based assay. The first specific 790bp BTV PCR products were amplified using a pair of outer primers (BTV1 and BTV2). Specificity of the PCR products was confirmed by a nested amplification of a 520bp PCR product using a pair of internal (nested) primers (BTV3 and BTV4). The BTV PCR products were visualized on ethidium bromide-stained agarose gels. Amplification products were not detected when the RT-PCR-based assay was applied to RNAs from closely related orbiviruses including, epizootic hemorrhagic disease virus (EHDV) prototypes serotypes 1, 2, 4; RNA from Sudanese isolate of palyam orbiviruses serogroup and total nucleic acid extracts from uninfected Vero cells. Application of the nested BTV RT-PCR to clinical samples resulted in amplification of BTV RNA from blood and serum samples from goats experimentally infected with BTV4 and from naturally infected sheep, goats, cattle and deer. The results of this study indicated that this RT-PCR assay could be applied for rapid detection of BTV, in cell culture and clinical samples from susceptible ruminants during an outbreak of the disease, in the United States and African.
对逆转录聚合酶链反应（RT-PCR）检测来自美国、苏丹、南非和塞内加尔受感染反刍动物的细胞培养物和组织样本中蓝舌病病毒（BTV）核糖核酸（RNA）序列的诊断潜力进行了评估。以北美BTV血清型11的非结构蛋白1（NS1）基因作为PCR扩增的靶标。研究了美国的BTV血清型2、10、11、13和17，苏丹的BTV血清型1、2、4和16，南非的BTV血清型4以及塞内加尔的BTV血清型2。本研究中使用的所有在细胞培养物中增殖的BTV野外分离株的RNA，均通过所述基于RT-PCR的检测方法检测到。使用一对外部引物（BTV1和BTV2）扩增出了第一条特异性的790bp BTV PCR产物。通过使用一对内部（巢式）引物（BTV3和BTV4）对520bp PCR产物进行巢式扩增，确认了PCR产物的特异性。BTV PCR产物在溴化乙锭染色的琼脂糖凝胶上进行可视化。当将基于RT-PCR的检测方法应用于来自密切相关的环状病毒的RNA时，未检测到扩增产物，这些环状病毒包括：流行性出血病病毒（EHDV）原型血清型1、2、4；来自苏丹帕利亚姆环状病毒血清群分离株的RNA以及未感染的非洲绿猴肾细胞（Vero细胞）的总核酸提取物。将巢式BTV RT-PCR应用于临床样本，从实验感染BTV4的山羊的血液和血清样本以及自然感染的绵羊、山羊、牛和鹿中扩增出了BTV RNA。本研究结果表明，这种RT-PCR检测方法可用于在美国和非洲疾病暴发期间，快速检测易感反刍动物的细胞培养物和临床样本中的BTV。
