Application of universal primers for identification of Foot-and-mouth disease virus and Swine vesicular disease virus by PCR and PCR-ELISA.
Two approaches for simultaneous identification of both Foot-and-mouth disease virus (FMDV) and Swine vesicular disease virus (SVDV) are described: (1) a single-step reverse transcription-PCR with three primers and (2) a PCR-ELISA assay with two universal primers for genome amplification and two virus-specific probes for identification. These methods are based on the use of 3D gene universal PCR primers, the structure of which was optimized and refined due to the close relationship between the two viruses belonging to different genera of the Picornaviridae family. In procedure (1), a three-primer PCR containing one universal antisense primer and two virus-specific primers was shown to differentiate between FMDV and SVDV in one reaction, due to the different length of the amplified DNA fragments (600 and 340 base pairs, respectively). In procedure (2), the two viruses were identified by PCR-ELISA, i.e. PCR for the 3D gene followed by two parallel hybridizations with FMDV and SVDV-specific probes in microplate wells and ELISA detection. The application of universal primers could halve the number of PCR experiments in both cases, as compared to the usual virus-specific PCR procedures. Also, we investigated the 3D gene structure of several SVDV strains isolated at different times. No essential changes were detected in the regions coding for conserved motifs of the RNA-dependent RNA polymerase recognized by our universal primers. The multi-primer PCR was successfully tested on 38 FMDV and 15 SVDV strains, and the PCR-ELISA on 32 FMDV and 16 SVDV strains including clinical material from disease cases.
Lomakina
N F
NF
The Ivanovski Institute of Virology, Moscow, Russia.
Fallacara
F
F
Pacciarini
M
M
Amadori
M
M
Lomakin
A I
AI
Timina
A M
AM
Shcherbakova
L O
LO
Drygin
V V
VV
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000235:genetics*; D016375:Antisense Elements (Genetics); D001483:Base Sequence; D017931:DNA Primers / Q000138:chemical synthesis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D029822:Enterovirus B, Human / Q000235:genetics / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D017361:Viral Nonstructural Proteins / Q000235:genetics*
D000956:Antigens, Viral; D016375:Antisense Elements (Genetics); D017931:DNA Primers; D017361:Viral Nonstructural Proteins; C103557:virus-infection-associated antigen, Foot-and-mouth disease virus; D012321:DNA-Directed RNA Polymerases