title: Infectious bursal disease viral RNA amplification using RT/PCR from bursa tissue following phenol: chloroform inactivation of the virus.
abstract: The preservation of viable infectious agents for future studies could create complicated logistic problems, and at times it is not feasible. Methods for preserving the genetic integrity of inactivated agents would not only facilitate these studies but would also make it possible to transport inactivated preparations around the world. In this report, the effect of inactivation on the genetic material of infectious bursal disease virus (IBDV) was studied. Tissues from the bursa of Fabricius of birds experimentally infected 3 days earlier with the classic STC strain of IBDV were collected and immediately placed in a solution of phenol:chloroform:isoamyl alcohol (25:24:1) for 24, 48, 72, or 96 hr. Infected bursal tissue not treated with the phenol:chloroform solution and uninfected phenol: chloroform bursal tissue were used as controls. In a separate experiment, bursal tissues collected 5 days following infection of specific-pathogen-free birds with the classic STC or variant 1084-E strain were placed in the phenol:chloroform solution for 2 wk. All bursal samples were tested for viable IBDV following phenol:chloroform treatment. The tissues were washed in phosphate-buffered saline to remove phenol and then homogenized. Viability of the viruses in homogenized bursal tissue was examined by inoculation of embryonated chicken eggs. Viable IBDV was not observed in any phenol:chloroform-treated bursal tissue but was observed in the infected but non-phenol:chloroform-treated control bursa. The reverse transcriptase/polymerase chain reaction (RT/PCR) was used to test the integrity of the viral RNA. Viral RNA from the nontreated control and all infected bursal samples treated with phenol:chloroform solution at all the time points examined were transcribed into DNA, and a 394-bp fragment of the VP2 gene was amplified using specific primers in the PCR. The RT/PCR assay was negative using the phenol:chloroform-treated uninfected bursal tissue. This study clearly demonstrated that phenol:chloroform treatment of infected bursal tissue inactivated the classic and variant IBDV strains tested and preserved the viral RNA for use in the RT/PCR assay.
authors.affiliation: Department of Veterinary Preventive Medicine, Ohio State University, Wooster 44691, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Jackwood
authors.forename: G
authors.initials: G
authors.lastname: Hanes
authors.forename: S H
authors.initials: SH
authors.lastname: Miller
mesh_terms: D000818:Animals; D018175:Birnaviridae Infections / Q000175:diagnosis*; D002642:Chick Embryo; D002645:Chickens; D002725:Chloroform; D014644:Genetic Variation; D007243:Infectious bursal disease virus / Q000235:genetics / Q000302:isolation & purification*; D000439:Pentanols; D019800:Phenol; D010636:Phenols; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics
chemical_list: D000439:Pentanols; D010636:Phenols; D012367:RNA, Viral; D019800:Phenol; D002725:Chloroform; C029683:isopentyl alcohol