Detection of African Horsesickness Virus by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using Primers for Segment 5 (NS1 Gene)

Journal of Veterinary Medical Science

Akira IWATA¹  Masanobu KAMADA¹  Susumu UEDA¹  Kenichi SAKAMOTO¹  Akio FUKUSHO¹  Noriko MIZUKOSHI¹ 

关键词: African horsesickness;    NS1;    RT-PCR;    segment 5;   

DOI  :  10.1292/jvms.56.347

学科分类：兽医学

来源: Japanese Society of Veterinary Science

【 摘 要 】

References(18)Cited-By(8)The reverse transcription followed by the polymerase chain reaction (RT-PCR) technique was applied to the detection of African horsesickness virus (AHSV) using primers specific for attenuated AHSV serotype 4 segment 5 (NS1 gene). Total RNA which contains both messenger RNA and genomic dsRNA was extracted by the acid guanidinium-phenol-chloroform method from the AHSV infected Vero cells and was used as templates to optimize the RT-PCR. A pair of primer (NP2-NP32) amplified the product of the expected size from all serotypes of attenuated AHSV when four pairs of primers were tested. Using this primer pair, no RT-PCR product was detected from the RNA samples extracted from ten other orbiviruses infected cells and their virions. In addition, RT-PCR using a serial dilution of RNA samples suggested that AHSV was efficiently detected from 1 to 2 cells of the cell monolayer infected with 106TCID50 of AHSV. The RT-PCR concerning with total RNAs of AHSV NS1 gene was found to be a specific and sensitive method for the detection of AHSV.

【 授权许可】

Unknown