A novel simple one-step single-tube RT-duplex PCR method with an internal control for detection of bovine viral diarrhoea virus in bulk milk, blood, and follicular fluid samples.
A simple one-step single-tube RT-PCR method was developed for detection of bovine viral diarrhea virus (BVDV) in bulk milk, blood and follicular fluid samples. A set of universal primers (UTR DL1/4) was designed for RT-PCR to detect BVDV type I and II viruses simultaneously and was tested for efficacy in comparison to published primers for two type strains, 42 field isolates, and 95 diagnostic samples. The sensitivity (100%) and specificity (96.2%) of the RT-PCR assay, with the universal primers for 95 diagnostic samples, were equal to those of virus isolation. An internal control targeting a bovine actin gene was also included in the same reaction tube to monitor RNA preparation and RT-PCR procedure. A total of 632 specimens (378 bulk milk, 140 blood, and 112 follicular samples) were tested in the year 2000 by the RT-duplex PCR assay in parallel with virus isolation. The one-step single-tube RT-duplex PCR with the universal primers UTR DL1/4 was sensitive, specific, less complicated and cost effective for detection of BVDV in various types of specimens.
Kim
Sung G
SG
Animal Health Diagnostic Laboratory, Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine at Cornell, Ithaca, NY 14853, USA. sgk1@cornell.edu
Dubovi
Edward J
EJ
D020121:5' Untranslated Regions; D000818:Animals; D001769:Blood / Q000821:virology*; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000175:diagnosis*; D002417:Cattle; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics*; D005260:Female; D015571:Follicular Fluid / Q000821:virology*; D008892:Milk / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D020121:5' Untranslated Regions; D017931:DNA Primers; D018076:DNA, Complementary