Novel multiplex RT-PCR/RFLP diagnostic test to differentiate low- from high-pathogenic strains and to detect reassortant infectious bursal disease virus.
Three types of infectious bursal disease virus (IBDV) strains are currently circulating worldwide: the low-pathogenic classic and variant strains and the high-pathogenic very virulent strains. There are also natural reassortant viruses that combine genomic segments A and B from different strains and exhibit particular pathogenic characteristics. Detection and characterization of the different IBDVs is extremely critical for improving disease control and performing epidemiologic studies. Here, we present a novel detection and genotyping method based on the simultaneous characterization of both IBDV genomic segments followed by a simple restriction fragment length polymorphism (RFLP) assay. This single restriction enzyme, multiplex reverse transcriptase-PCR/RFLP diagnostic test not only distinguished typical high-pathogenic from low-pathogenic strains but also detected natural reassortant IBDV. The test was based on the detection of single nucleotide polymorphisms (SNP), in both segments, which were strongly linked to the pathogenic phenotype. These SNPs are embedded in highly conserved genomic regions and can be identified with TfiI endonuclease. The application of this methodology in field samples confirmed that the assay is fast, specific, and may be easily adopted by any molecular diagnostic laboratory as an economical and routine method.
Sección Genética Evolutiva, Departamento de Biología Animal, Instituto de Biología, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay.
Martín
M
Hernández
Gonzalo
G
Tomás
Diego
D
Hernández
Pedro
P
Villegas
Alejandro
A
Banda
Leticia
L
Maya
Yanina
Y
Panzera
Ruben
R
Pérez
D000818:Animals; D001483:Base Sequence; D018175:Birnaviridae Infections / Q000175:diagnosis / Q000453:epidemiology / Q000662:veterinary* / Q000821:virology; D002060:Bursa of Fabricius / Q000821:virology*; D002645:Chickens; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D005838:Genotype; D007243:Infectious bursal disease virus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D011201:Poultry Diseases / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment
C075307:endodeoxyribonuclease TfiI; D015252:Deoxyribonucleases, Type II Site-Specific