A sensitive fluorescence in situ hybridization technique for detection of porcine reproductive and respiratory syndrome virus.
A sensitive fluorescence in situ hybridization (ISH) for detecting porcine reproductive and respiratory syndrome virus (PRRSV) RNA in viral infected tissue was developed using digoxigenin-labeled RNA probes targeted on the nucleocapsid gene of PRRSV. In situ RNA/RNA hybrids were detected with an anti-digoxigenin antibody alkaline phosphatase conjugate and further revealed with Fast Red TR salt/naphthol AS-MX phosphate using a fluorescent microscope. Viral nucleic acid was readily demonstrated within macrophages, known to be the major target of PRRSV. In addition, positively stained cells were found in the salivary gland and skin tissues which have not been reported to contain PRRSV infected cells before. In conclusion, the fluorescence ISH used in this study provides a fast and sensitive means for screening virus-infected tissues in which relatively few cells are affected. This advantage will be especially beneficial for studying viral persistence and for routine diagnosis of PRRSV infection.
Chueh
L L
LL
Department of Veterinary Medicine, National Taiwan University, Taipei, ROC. chuehlin@ccms.ntu.edu.tw
Lee
K H
KH
Jeng
C R
CR
Pang
V F
VF
D000818:Animals; D002460:Cell Line; D004396:Coloring Agents; D003979:Diazonium Compounds; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D019318:Porcine Reproductive and Respiratory Syndrome / Q000473:pathology / Q000821:virology*; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D013552:Swine
D004396:Coloring Agents; D003979:Diazonium Compounds; D012367:RNA, Viral; C027766:Fast Red TA