Development of universal SYBR Green real-time RT-PCR for the rapid detection and quantitation of bovine and porcine toroviruses

Hosmillo, Myra D.T.; Jeong, Young-Ju; Kim, Hyun-Jeong; Collantes, Therese Marie; Alfajaro, Mia Madel; Park, Jun-Gyu; Kim, Ha-Hyun; Kwon, Hyung-Jun; Park, Su-Jin; Kang, Mun-Il; Park, Sang-Ik; Cho, Kyoung-Oh

Toroviruses (ToVs) are a group of emerging viruses that cause gastroenteritis in domestic animals and humans. Currently, methods such as real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) have not yet been developed for the rapid detection and quantitation of bovine (BToV) and porcine (PToV) toroviruses. Using BToV and PToV RNA standards generated by in vitro transcription, the detection limit of the SYBR Green real-time RT-PCR assay was 2.54 × 10(2) BToV and 2.17 × 10(3) PToV copies/reaction (correlation coefficiency = 0.99 and 0.97, respectively), whereas those of RT-PCR and nested PCR were 2.54 × 10(5) and 2.54 × 10(4) (BToV) and 2.17 × 10(7) and 2.17 × 10(5) (PToV) cRNA viral copies/reaction, respectively. Archived diarrhea specimens of calves (n = 121) and piglets (n = 86) were subjected to RT-PCR, nested PCR and SYBR Green real-time RT-PCR. By conventional RT-PCR, 1 (0.8%) bovine and 7 (8.1%) porcine samples tested positive to BToV and PToV, respectively. With nested PCR, 13 (10.7%) bovine and 17 (19.8%) porcine samples tested positive. SYBR Green real-time RT-PCR assay detected BToV and PToV in 22 of 121 (18.2%) bovine and 31 of 86 (36.0%) porcine samples. These results indicate that SYBR Green real-time RT-PCR (P < 0.05) is a more sensitive assay, which can be reproduced as a reliable, sensitive, and rapid tool for the detection and quantitation of toroviruses.