Development Of A Real-Time Reverse Transcription Pcr Assay For The Detection Of Porcine Kobuvirus

Porcine kobuvirus is suggested to play a role in gastroenteritis in pigs where prompt detection of kobuvirus infection in the field samples is important to limit the spread of disease. A rapid and sensitive assay for this virus is not yet available and therefore the aim of this was to develop a onestep real-time reverse transcription polymerase chain reaction (RT-PCR) assay using a SYBR Green I dye for the detection of Porcine kobuvirus in fecal samples. The assay involved amplifying a cDNA template generated from viral RNA and using in vitro transcribed Porcine kobuvirus RNA to establish a standard curve. The assay was shown to have a detection limit as low as 100 copies/mu l RNA, a reaction efficiency of 91.8%, and a correlation coefficient (R2) of 0.992. The fluorogenic RT-PCR assay was determined to be 10-fold more sensitive than conventional RT-PCR and quantitatively detected kobuvirus RNA levels from 10-fold serial dilutions of titrated viruses without non-specific amplification. Furthermore, melting curve analysis showed no primer-dimers or nonspecific products were produced in the assay. The one-step SYBR Green I RT-PCR developed here is specific, sensitive, and reproducible for the quantification of Porcine kobuvirus in fecal samples. This one-step SYBR Green I RT-PCR strategy may be further optimized for use as a reliable assay for diagnosing and monitoring Porcine kobuvirus infections in pigs.