Detection of African horsesickness virus by reverse transcriptase polymerase chain reaction (RT-PCR) using primers for segment 5 (NS1 gene).
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 10(6) TCID50 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.
Mizukoshi
N
N
Exotic Diseases Research Division, National Institute of Animal Health, Tokyo, Japan.
Sakamoto
K
K
Iwata
A
A
Ueda
S
S
Kamada
M
M
Fukusho
A
A
D000355:African Horse Sickness / Q000175:diagnosis*; D000356:African Horse Sickness Virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D006736:Horses; D008969:Molecular Sequence Data; D018163:Orbivirus / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D012680:Sensitivity and Specificity; D012703:Serotyping; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000235:genetics*
D017931:DNA Primers; C089209:NS1 protein, African horse sickness virus; D012333:RNA, Messenger; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins