Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus.
Neonatal calf diarrhea is a multi-etiology syndrome of cattle and direct detection of the two major agents of the syndrome, group A rotavirus and Bovine coronavirus (BCoV) is hampered by their fastidious growth in cell culture. This study aimed at developing a multiplex semi-nested RT-PCR for simultaneous detection of BCoV (N gene) and group A rotavirus (VP1 gene) with the addition of an internal control (mRNA ND5). The assay was tested in 75 bovine feces samples tested previously for rotavirus using PAGE and for BCoV using nested RT-PCR targeted to RdRp gene. Agreement with reference tests was optimal for BCoV (kappa=0.833) and substantial for rotavirus detection (kappa=0.648). the internal control, ND5 mRNA, was detected successfully in all reactions. Results demonstrated that this multiplex semi-nested RT-PCR was effective in the detection of BCoV and rotavirus, with high sensitivity and specificity for simultaneous detection of both viruses at a lower cost, providing an important tool for studies on the etiology of diarrhea in cattle.
Asano
Karen Miyuki
KM
Department of Preventive Veterinary Medicine and Animal Health, College of Veterinary Medicine, University of São Paulo, Av. Professor Dr. Orlando Marques de Paiva, 87, CEP: 05508-270, Cidade Universitária, São Paulo, SP, Brazil. karen.asano@gmail.com
de Souza
Sibele Pinheiro
SP
de Barros
Iracema Nunes
IN
Ayres
Giselle Razera
GR
Silva
Sheila Oliveira Souza
SO
Richtzenhain
Leonardo José
LJ
Brandão
Paulo E
PE
D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000821:virology; D018352:Coronavirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D017938:Coronavirus, Bovine / Q000302:isolation & purification*; D005243:Feces / Q000821:virology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012401:Rotavirus / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D012367:RNA, Viral