Aradaib Imadeldin E
Molecular Biology Laboratory, Department of Medicine, Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Khartoum, P.O. Box 32, Khartoum North, Sudan.
J Virol Methods. 2009 Jul;159(1):1-5. doi: 10.1016/j.jviromet.2009.02.012. Epub 2009 Feb 21.
A nested reverse transcriptase (RT) polymerase chain reaction (RT-PCR), for rapid detection of African horse sickness virus (AHSV) double-stranded ribonucleic acid (dsRNA) in cell culture and tissue samples, was developed and evaluated. Using an outer pair of primers (P1 and P2), selected from genome segment three of AHSV serotype 6 (AHSV-6), the RT-PCR-based assay resulted in amplification of a 890 base pair (bp) primary PCR product. RNAs from the nine vaccine strains of AHSV, and a number of AHSV field isolates including the Central African isolates of AHSV-9 and AHSV-6, propagated in cell cultures, were detected by this assay. A second pair of nested primers (P3 and P4) was used to produce a 240-bp PCR product. The RT-PCR described below detected as little as 0.1 fg of AHSV RNA, which is equivalent to six viral particles. The nested amplification confirmed the integrity of the primary PCR product and increased the sensitivity of the PCR assay by at least 1000-fold. Application of this RT-PCR assay to clinical samples resulted in direct detection of AHSV dsRNA from blood and a variety of tissue samples collected from equines infected experimentally and naturally. The specificity studies indicated that the primary or the nested PCR products were not amplified from, closely related orbiviruses including, bluetongue virus (BTV) prototypes serotypes 1, 2, 4, 10, 16 and 17; epizootic hemorrhagic disease of deer virus (EHDV) prototypes serotypes 1 and 2; EHDV-318, Sudanese isolates of palyam serogroup of orbiviruses; total nucleic acid extracts from uninfected Vero cells; or unfractionated blood from horses and donkeys that were AHSV-seronegative and virus isolation negative. The RT-PCR provides a valuable tool for study of the epidemiology of AHSV and can be recommended for rapid diagnosis during an outbreak of the disease among susceptible equines.
为了快速检测细胞培养物和组织样本中的非洲马瘟病毒（AHSV）双链核糖核酸（dsRNA），我们开发并评估了一种巢式逆转录酶（RT）聚合酶链反应（RT-PCR）。使用从AHSV血清型6（AHSV-6）基因组片段3中选择的一对外部引物（P1和P2），基于RT-PCR的检测方法扩增出了一个890碱基对（bp）的初级PCR产物。通过该检测方法可检测到在细胞培养物中增殖的AHSV的9种疫苗株以及包括AHSV-9和AHSV-6的中非分离株在内的许多AHSV野外分离株的RNA。使用第二对巢式引物（P3和P4）产生了一个240 bp的PCR产物。以下所述的RT-PCR可检测低至0.1 fg的AHSV RNA，这相当于6个病毒颗粒。巢式扩增证实了初级PCR产物的完整性，并将PCR检测的灵敏度提高了至少1000倍。将这种RT-PCR检测方法应用于临床样本，可直接从感染实验性和自然感染马匹的血液和各种组织样本中检测到AHSV dsRNA。特异性研究表明，初级或巢式PCR产物不会从密切相关的环状病毒中扩增出来，这些环状病毒包括蓝舌病病毒（BTV）原型血清型1、2、4、10、16和17；鹿流行性出血病病毒（EHDV）原型血清型1和2；EHDV-318、环状病毒帕利姆血清群的苏丹分离株；未感染的Vero细胞的总核酸提取物；或AHSV血清阴性且病毒分离阴性的马和驴的未分级血液。该RT-PCR为研究AHSV的流行病学提供了一个有价值的工具，可推荐用于易感马匹疾病爆发期间的快速诊断。
