title: Diagnosis of rabies by polymerase chain reaction with nested primers.
abstract: A simple, sensitive, and specific polymerase chain reaction (PCR) protocol for detection of rabies virus is described. The process consists of sample preparation, reverse transcription, two-step DNA amplification, and detection of the amplified product. RNA was extracted from animal and human brain by phenol-chloroform using guanidinium thiocyanate. Viral RNA was then amplified in a two-step PCR that used two sets of nested primers designed to amplify rabies nucleocapsid (N) sequence. Rabies nucleocapsid sequence was amplified from all brain samples from 95 dogs and 3 humans with rabies confirmed by fluorescent antibody (FAT) and mouse inoculation tests (MIT). Rabies-negative brain samples (110 dogs, 2 humans) were PCR-negative. The process requires < 24 h. Detection of viral RNA was still possible in brain material that was left at room temperature for 72 h. As little as 8 pg of rabies virus RNA could be detected. This technique could have practical applications as a confirmatory test to FAT at busy rabies diagnostic centers.
authors.affiliation: WHO Collaborating Centre for Research on Rabies Pathogenesis and Prevention, Queen Saovabha Memorial Institute, Bangkok, Thailand.
authors.forename: N
authors.initials: N
authors.lastname: Kamolvarin
authors.forename: T
authors.initials: T
authors.lastname: Tirawatnpong
authors.forename: R
authors.initials: R
authors.lastname: Rattanasiwamoke
authors.forename: S
authors.initials: S
authors.lastname: Tirawatnpong
authors.forename: T
authors.initials: T
authors.lastname: Panpanich
authors.forename: T
authors.initials: T
authors.lastname: Hemachudha
mesh_terms: D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000382:microbiology*; D004283:Dog Diseases / Q000175:diagnosis*; D004285:Dogs; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis*; D011818:Rabies / Q000175:diagnosis* / Q000662:veterinary; D011820:Rabies virus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral