Detection of foot and mouth disease virus by RT-PCR and microplate hydridization assay using inactivated viral antigens.
A single step RT-PCR was tested for detection of foot and mouth disease virus (FMDV) and immunoenzymatic determination of amplified products in a microplate hybridization assay. Inactivated reference strains (ELISA antigen) of all seven serotypes were used to optimize the test. Oligonucleotide primers were selected from two different genomic regions coding for RNA polymerase and VP1 protein, respectively. The RT-PCR used to amplify the polymerase gene specific RNA detected FMDV strains A, C, O, Asial and SAT1, and the identity of the fragments obtained was confirmed with a specific internal biotin-labelled capture probe. For the amplification of the VP1 genome region, two sets of oligonucleotide primers were used. One primer pair was successfully applied for the detection of serotypes A, C, O and Asial and a second one for serotypes SAT1, SAT2, SAT3. The specific probe enabled the detection of all the amplified products in a PCR ELISA test. By comparison with antigen ELISA, the PCR ELISA method allowed the detection of smaller amounts of FMDV in the inactivated material examined. The application of molecular diagnostic methods to inactivated antigens offers a good alternative procedure for developing and optimizing a sensitive method for detection of FMDV in laboratories that are not allowed to work with viable FMDV.
Barlic-Maganja
D
D
University of Ljubljana, Institute of Microbiology and Parasitology, Virology Unit, Gerbiceva 60, SI-1115 Ljubljana, Slovenia. darja.barlic@vf.uni-lj.si
Grom
J
J
Toplak
I
I
Hostnik
P
P
D000818:Animals; D000956:Antigens, Viral / Q000032:analysis*; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary; D005536:Foot-and-Mouth Disease / Q000175:diagnosis* / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D005814:Genes, Viral; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D013045:Species Specificity
D000956:Antigens, Viral; D036022:Capsid Proteins; D017931:DNA Primers; D012367:RNA, Viral; C442501:VP1 protein, Foot-and-mouth disease virus; D012321:DNA-Directed RNA Polymerases