Evaluation of FTA paper and phenol for storage, extraction and molecular characterization of infectious bursal disease virus.
Infectious bursal disease virus (IBDV) is an important poultry pathogen and is distributed world wide that can cause immune suppression and lesions of the bursa of Fabricius. The main component of the virus, VP2, is not only responsible for the bird's immune response, but is important for the molecular identification of this virus as well. The nucleic acid of the virus must be adequately preserved to be analyzed by reverse-transcriptase PCR (RT-PCR) and sequenced for the molecular characterization of the field strain. Phenol inactivation has been the standard for IBDV tissue collection and international shipment; however, there have been some reports of interference with molecular detection capabilities when using phenol. Phenol is also a hazardous chemical and must be handled and shipped carefully. The ability to use the Flinders Technology Associates filter paper (FTA card) for inactivation of several avian pathogens has been proven previously, however no work has been published on its use in IBDV nucleic acid detection. Bursas from experimentally infected birds was imprinted on FTA cards, and then placed in phenol. Samples were evaluated and compared based on molecular detection capabilities between the two inactivation methods. The nucleic acid of the virus was detected in 85% of the FTA card inactivated samples compared to 71% in the phenol inactivated samples. Sequence analysis was performed on samples inactivated by both methods and no differences were found. When comparing the RNA stability at different temperatures, euthanized IBDV infected birds were held at two different temperatures before sampling. No differences were detected for FTA sampling; however, for tissues in phenol the nucleic acid was only detectable up to 2 h post-mortem in the tissues held at 4 degrees C prior to sampling. These findings indicate that the FTA card is an efficient and reliable alternative collection method for molecular detection and characterization of IBDV.
Purvis
Linda B
LB
Department of Population Health, University of Georgia, Athens, GA 30602, United States.
Villegas
Pedro
P
Perozo
Francisco
F
D000818:Animals; D018175:Birnaviridae Infections / Q000662:veterinary / Q000821:virology; D002060:Bursa of Fabricius / Q000821:virology; D002645:Chickens / Q000821:virology; D007243:Infectious bursal disease virus / Q000235:genetics / Q000302:isolation & purification*; D019800:Phenol; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D013048:Specimen Handling / Q000379:methods*; D038301:Virus Inactivation
D012367:RNA, Viral; D019800:Phenol