Preservation of rabies virus RNA from brain tissue using glycerine.
The challenge virus standard (CVS) strain and a wild isolate from a Mexican child who died of hematophagous bat (Desmodus rotundus)-transmitted rabies were injected intracerebrally into BALB/c mice. Brains obtained from infected mice were immersed in 80%, 50%, and 40% glycerine/phosphate-buffered saline (PBS). RNA was extracted from brains on days 1, 2, 3, 7, 21, and 60, and reverse transcriptase polymerase chain reaction-restriction fragment length polymorphism (RT-PCR-RFLP) tests were performed for rabies virus characterization. Storage temperature variation was recorded during the preservation period. The RT-PCR-RFLP tests were successfully performed on brain samples preserved in 50% glycerine/PBS, but not in those preserved in 80% or 40% glycerine/PBS. Temperatures ranged from 12 to 33 degrees C and were not harmful, provided that 50% glycerine/PBS was used. We concluded that brain samples obtained and stored under field conditions (i.e. without refrigeration) for up to 60 d can arrive at a reference laboratory in an adequate condition for viral RNA analysis.
Unidad de Investigación Médica en Inmunología, Coordinación de Investigación Médica, Instituto Mexicano del Seguro Social, México DF, México. aaguilas@data.net.mx
Alvaro
A
Aguilar-Setién
Hector
H
Aguila-Tecuatl
Emiliano
E
Tesoro-Cruz
Leticia
L
Ramos-Ramírez
Roberto S
RS
Kretschmer
D000818:Animals; D001921:Brain / Q000821:virology*; D003451:Cryoprotective Agents; D005990:Glycerol; D051379:Mice; D008807:Mice, Inbred BALB C; D012150:Polymorphism, Restriction Fragment Length; D011309:Preservation, Biological / Q000379:methods*; D012367:RNA, Viral / Q000302:isolation & purification*; D011818:Rabies / Q000175:diagnosis*; D011820:Rabies virus / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D003451:Cryoprotective Agents; D012367:RNA, Viral; D005990:Glycerol