Development of universal SYBR Green real-time RT-PCR for the rapid detection and quantitation of bovine and porcine toroviruses.
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 x 10(2) BToV and 2.17 x 10(3) PToV copies/reaction (correlation coefficiency=0.99 and 0.97, respectively), whereas those of RT-PCR and nested PCR were 2.54 x 10(5) and 2.54 x 10(4) (BToV) and 2.17 x 10(7) and 2.17 x 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.
Hosmillo
Myra D T
MD
Bio-therapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju, South Korea.
Jeong
Young-Ju
YJ
Kim
Hyun-Jeong
HJ
Collantes
Therese Marie
TM
Alfajaro
Mia Madel
MM
Park
Jun-Gyu
JG
Kim
Ha-Hyun
HH
Kwon
Hyung-Jun
HJ
Park
Su-Jin
SJ
Kang
Mun-Il
MI
Park
Sang-Ik
SI
Cho
Kyoung-Oh
KO
D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D003967:Diarrhea / Q000821:virology; D005243:Feces / Q000821:virology; D005456:Fluorescent Dyes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009930:Organic Chemicals / Q000378:metabolism*; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling / Q000379:methods; D013552:Swine; D017003:Torovirus / Q000302:isolation & purification*; D018176:Torovirus Infections / Q000175:diagnosis / Q000662:veterinary*
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I