Detection of African horsesickness virus by reverse transcriptase polymerase chain reaction (RT-PCR) using primers for segment 5 (NS1 gene).

The study discusses the use of the reverse transcriptase polymerase chain reaction (RT-PCR) technique to detect African horsesickness virus (AHSV) by applying primers specific to a certain gene. A particular pair of primers was found effective at amplifying the expected results. The method demonstrated high specificity and sensitivity for AHSV detection.

Methodology

• The research applied a technology known as reverse transcription followed by the polymerase chain reaction (RT-PCR), aiming to detect the African horsesickness virus (AHSV).
• The RT-PCR technique utilized primers specific for attenuated AHSV serotype 4 segment 5. This segment is also known as the NS1 gene.
• Ribonucleic acid (RNA), encompassing both messenger RNA and genomic double-stranded RNA, was extracted from cells infected by AHSV. This extraction was performed using the acid guanidinium-phenol-chloroform method.
• With the extracted RNA serving as templates, the RT-PCR technique was optimized.

Findings

• The RT-PCR technique was tested with four different pairs of primers. Among them, only one pair named NP2-NP32 could amplify the expected product from all serotypes of attenuated AHSV.
• When using this NP2-NP32 primer pair, no RT-PCR product was detected in the RNA samples extracted from cells infected with any of the ten other orbiviruses tested in the study. This suggests the specificity of these primers for AHSV and not for those other viruses.
• Through RT-PCR examination of samples with varying concentrations of AHSV-infected cells, it was found that the virus could efficiently be detected even in a small concentration of 1 to 2 cells within a cell monolayer infected with a high virus concentration.
• Overall, the RT-PCR with total RNAs related to the AHSV NS1 gene was identified as a highly specific and sensitive way to detect the presence of AHSV.

Implication

• This finding contributes significantly to the means of detecting the African horse sickness virus.
• It highlights the specificity and sensitivity of the reverse transcription polymerase chain reaction technique in diagnosing a dangerous virus that affects equine populations.