Diagnosis of the African horse sickness virus serotype 4 by a one-tube, one manipulation RT-PCR reaction from infected organs.

This study presents a method using a single tube reverse transcription-polymerase chain reaction (RT-PCR) to detect the presence of African horse sickness virus in the spleen tissue of infected horses. The technique can hasten diagnostic confirmation of the virus.

Study Explanation

• The primary goal of this study was to develop a quicker and more efficient method to detect the African horse sickness virus (AHSV) in horses. The virus is often devastating, leading to significant losses in the horse industry.
• The researchers focused their efforts on creating a single tube reverse transcription-polymerase chain reaction (RT-PCR) method. RT-PCR is a highly sensitive technique used to detect and measure the presence of specific genetic material (like DNA or RNA) from any pathogen, including a virus, in a specific tissue sample. In this case, the specific tissues were the spleen tissues of infected horses.
• Double-stranded RNA was extracted from the infected organs of horses and used to synthesize complementary DNA (cDNA), essentially creating a DNA mirror image of the viral genetic material. The two primers selected for this process were specially designed to bind to specific areas on this cDNA sequence, indicating the presence of AHSV.
• The 1179 base pair product that was amplified in this process represents the segment 7 of the AHSV genome which encodes for a specific viral protein VP 7. The amplified product of the RT-PCR was detected through electrophoresis on an agarose gel – a common method to visualize DNA – and specifically stained with ethidium bromide – a fluorescent tag that binds to DNA and allows for clear identification.
• The researchers also hydrolyzed the amplified product with eight different restriction endonucleases, which are enzymes that cut DNA at specific sequences, as an additional step to verify and characterize AHSV.
• The sensitivity of this method, meaning its ability to correctly identify true positive cases of AHSV, is also discussed within the study.

Conclusions

• The researchers conclude that applying the single tube RT-PCR method should help improve both the detection and diagnosis of AHSV infection in horses.
• More specifically, this method should shorten the time required to confirm a clinical diagnosis of AHSV infection, which can be particularly vital in preventing the spread of this debilitating virus, saving valuable time, money, and most importantly, horse lives.