title: Development of a RT-PCR ELISA for simultaneous detection of BVDV-1, BVDV-2 and BDV in ruminants and its evaluation on clinical samples.
abstract: The aim of this study was to develop a reverse transcription polymerase chain reaction ELISA (RT-PCR ELISA) for detection of ruminant pestiviruses and to evaluate its diagnostic performance on clinical samples obtained from cattle, sheep and goats. Optimization was carried out by serial dilution of home-made digoxygenin-labelled RT-PCR product standards obtained from pestivirus isolates and pestivirus infected animals. The detection limit of the assay was 10TCID50/ml, similar to virus isolation and real-time RT-PCR but 10-fold higher than RT-PCR. The assay had high analytical specificity along with a good reproducibility. When the assay was evaluated on the samples obtained from animals infected experimentally with BVDV and from the field using virus isolation as standard, it showed a high diagnostic sensitivity (95.9%) and specificity (98.6%) and there was strong agreement (97.5% concordance) between the two tests. However, it displayed an increased diagnostic specificity and sensitivity over RT-PCR. Additionally, when a few samples (n=26) were tested by RT-PCR ELISA and real-time RT-PCR, 100% concordance was obtained between them. Our results showed that RT-PCR ELISA can be a rapid, cost effective and alternative molecular diagnostic test for simultaneous detection of BVDV-1, BVDV-2 and BDV in ruminants in ordinary laboratory settings.
authors.affiliation: National Institute of High Security Animal Diseases (formerly HSADL), Indian Council of Agricultural Research, Anand Nagar, Bhopal, Madhya Pradesh 462021, India.
authors.forename: Pooja
authors.initials: P
authors.lastname: Dubey
authors.affiliation: National Institute of High Security Animal Diseases (formerly HSADL), Indian Council of Agricultural Research, Anand Nagar, Bhopal, Madhya Pradesh 462021, India. Electronic address: mishranir@rediffmail.com.
authors.forename: N
authors.initials: N
authors.lastname: Mishra
authors.affiliation: National Institute of High Security Animal Diseases (formerly HSADL), Indian Council of Agricultural Research, Anand Nagar, Bhopal, Madhya Pradesh 462021, India.
authors.forename: K
authors.initials: K
authors.lastname: Rajukumar
authors.affiliation: National Institute of High Security Animal Diseases (formerly HSADL), Indian Council of Agricultural Research, Anand Nagar, Bhopal, Madhya Pradesh 462021, India.
authors.forename: S P
authors.initials: SP
authors.lastname: Behera
authors.affiliation: National Institute of High Security Animal Diseases (formerly HSADL), Indian Council of Agricultural Research, Anand Nagar, Bhopal, Madhya Pradesh 462021, India.
authors.forename: S
authors.initials: S
authors.lastname: Kalaiyarasu
authors.affiliation: National Institute of High Security Animal Diseases (formerly HSADL), Indian Council of Agricultural Research, Anand Nagar, Bhopal, Madhya Pradesh 462021, India.
authors.forename: R K
authors.initials: RK
authors.lastname: Nema
authors.affiliation: Department of Microbiology, Barkatullah University, Bhopal, India.
authors.forename: A
authors.initials: A
authors.lastname: Prakash
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000821:virology; D030241:Diarrhea Virus 1, Bovine Viral / Q000235:genetics / Q000302:isolation & purification*; D030242:Diarrhea Virus 2, Bovine Viral / Q000235:genetics / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015511:Goat Diseases / Q000175:diagnosis* / Q000821:virology; D006041:Goats; D018182:Pestivirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000821:virology; D014730:Veterinary Medicine / Q000379:methods
keywords: Pestivirus; RT-PCR; RT-PCR ELISA; Real-time RT-PCR; Ruminants; Virus isolation