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.

Filogenia de vírus da raiva isolados de morcegos frugívoros do gênero Artibeus e relacionados a morcegos hematófagos com base nos genes codificadores da nucleoproteína N e glicoproteína G

“…Por essa razão, para a detecção do RABV por RT-PCR e para sua classificação, o gene N tem sido o alvo consensualmente utilizado (KAMOLVARIN et al, 1993;YAGER, 1995;DE MATTOS et al, 1996;ITO et al, 2001;FAVI et al, 2003;SCAGLIARINI et al, 2003;ROMIJN et al, 2003).…”

A Case Report of Rabies with Long Incubation: Importance in terms of Public Health

“…Reverse transcriptase-PCR is particularly useful for rapid diagnosis in human cases of suspected rabies. This test can be performed on CSF, saliva, or brain tissue [5] . The patient in the present case was diagnosed using RNA extraction and RT-PCR.…”

Electrochemical Biosensors for Virus Detection

“…High percentages (99%) of the registered cases were in Asia and Africa. In order to fight this dangerous disease, many techniques are usually used for diagnostic but are usually complex, time consuming, expensive, difficult to implement, and this is a necessity for developing new detection process [N et al, 1993;Crepin et al, 1998]. Avian Influenza Virus (AIV) infections are a major cause of mortality and rapid identification of the virus has important clinical, economical and epidemiological implications.…”