Aradaib Imadeldin E, Mohamed Mohamed E H, Abdalla Mohamed A
Molecular Biology Laboratory, Department of Medicine, Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Khartoum, Khartoum North, Sudan.
J Virol Methods. 2009 Oct;161(1):70-4. doi: 10.1016/j.jviromet.2009.05.019. Epub 2009 Jun 3.
A single-tube nested reverse transcriptase (nRT) polymerase chain reaction (nRT-PCR) was developed and evaluated for detection of palyam serogroup orbiviruses ribonucleic acid (RNA) in cell cultures and clinical samples. A pair of outer primers (pal1 and pal2), designed from genome segment three of Chuzan virus of the palyam viruses serogroup, resulted in amplification of a primary 660-base pair (bp) PCR product. Using a pair of internal (nested) primers (pal3 and pal4), the nRT-PCR produced a 350-bp PCR product. The primary and the nested PCR products were amplified from RNA extracted from Sudanese and South African isolates of palyam viruses, propagated in cell cultures. Application of this nRT-PCR to clinical samples resulted in direct detection of palyam virus RNA in blood and serum samples from infected cattle and goats. The nested amplification increased the sensitivity of the assay by 1000-fold, and specific PCR products were detected from as little as 0.1fg of viral RNA. Amplification products were not detected when the nRT-PCR was applied to RNA from closely related orbiviruses including, bluetongue virus (BTV) serotypes 1, 2, 4, 6; epizootic hemorrhagic disease of deer virus prototype serotype 1 (EHDV-1); Sudanese isolates of EHDV-318; total nucleic acid extracts from non-infected Vero cells; and blood and sera from goats and calves from which virus was not isolated. This nRT-PCR provides a reliable, sensitive and specific assay for rapid detection and differentiation of palyam viruses from other related orbiviruses. In addition, the assay is recommended for inclusion in epidemiological surveys and during investigation of an epizootic of the disease among susceptible ruminants.
开发并评估了一种单管巢式逆转录酶（nRT）聚合酶链反应（nRT-PCR），用于检测细胞培养物和临床样本中的帕利亚姆血清群环状病毒核糖核酸（RNA）。根据帕利亚姆病毒血清群中楚赞病毒基因组片段3设计的一对外部引物（pal1和pal2），扩增出一条660碱基对（bp）的初级PCR产物。使用一对内部（巢式）引物（pal3和pal4），nRT-PCR产生了一条350 bp的PCR产物。初级和巢式PCR产物是从在细胞培养物中繁殖的苏丹和南非帕利亚姆病毒分离株提取的RNA中扩增得到的。将这种nRT-PCR应用于临床样本，可直接检测感染牛和山羊血液及血清样本中的帕利亚姆病毒RNA。巢式扩增使检测灵敏度提高了1000倍，从低至0.1 fg的病毒RNA中即可检测到特异性PCR产物。当将nRT-PCR应用于密切相关的环状病毒的RNA时，未检测到扩增产物，这些病毒包括蓝舌病毒（BTV）血清型1、2、4、6；鹿流行性出血病病毒原型血清型1（EHDV-1）；EHDV-318的苏丹分离株；未感染的非洲绿猴肾细胞（Vero细胞）的总核酸提取物；以及未分离到病毒的山羊和小牛的血液及血清。这种nRT-PCR为快速检测帕利亚姆病毒并将其与其他相关环状病毒区分开来提供了一种可靠、灵敏且特异的检测方法。此外，建议将该检测方法纳入流行病学调查以及易感反刍动物疾病流行调查中。
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